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https://jneuroengrehab.biomedcentral.com/track/pdf/10.1186/s12984-018-0477-5
en
User activity recognition system to improve the performance of environmental control interfaces: a pilot study with patients
Journal of neuroengineering and rehabilitation
2,019
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Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation https://doi.org/10.1186/s12984-018-0477-5 RESEARCH (2019) 16:10 Open Access User activity recognition system to improve the performance of environmental control interfaces: a pilot study with patients Arturo Bertomeu-Motos1* , Santiago Ezquerro1 , Juan A. Barios1 , Luis D. Lledó1 , Sergio Domingo2 , Marius Nann4 , Suzanne Martin3 , Surjo R. Soekadar5 and Nicolas Garcia-Aracil1 Abstract Background: Assistive technologies aim to increase quality of life, reduce dependence on care giver and on the long term care system. Several studies have demonstrated the effectiveness in the use of assistive technology for environment control and communication systems. The progress of brain-computer interfaces (BCI) research together with exoskeleton enable a person with motor impairment to interact with new elements in the environment. This paper aims to evaluate the environment control interface (ECI) developed under the AIDE project conditions, a multimodal interface able to analyze and extract relevant information from the environments as well as from the identification of residual abilities, behaviors, and intentions of the user. Methods: This study evaluated the ECI in a simulated scenario using a two screen layout: one with the ECI and the other with a simulated home environment, developed for this purpose. The sensorimotor rhythms and the horizontal oculoversion, acquired through BCI2000, a multipurpose standard BCI platform, were used to online control the ECI after the user training and system calibration. Eight subjects with different neurological diseases and spinal cord injury participated in this study. The subjects performed simulated activities of daily living (ADLs), i.e. actions in the simulated environment as drink, switch on a lamp or raise the bed head, during ten minutes in two different modes, AIDE mode, using a prediction model, to recognize the user intention facilitating the scan, and Manual mode, without a prediction model. Results: The results show that the mean task time spent in the AIDE mode was less than in the Manual, i.e the users were able to perform more tasks in the AIDE mode during the same time. The results showed a statistically significant differences with p < 0.001. Regarding the steps, i.e the number of abstraction levels crossed in the ECI to perform an ADL, the users performed one step in the 90% of the tasks using the AIDE mode and three steps, at least, were necessary in the Manual mode. The user’s intention prediction was performed through conditional random fields (CRF), with a global accuracy about 87%. Conclusions: The environment analysis and the identification of the user’s behaviors can be used to predict the user intention opening a new paradigm in the design of the ECIs. Although the developed ECI was tested only in a simulated home environment, it can be easily adapted to a real environment increasing the user independence at home. Keywords: Environment control interface, Brain-computer interface, User intention prediction, Multimodal system, Brain injury, Spinal-cord injury *Correspondence: abertomeu@umh.es Miguel Hernández University of Elche, Av. Universidad w/n, Ed. Innova, 03202 Elche, Spain Full list of author information is available at the end of the article 1 © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation Background It is estimated that one in six people in the world are diagnosed with a neurological disorder and this number is expected to rise considerable due to extensions of life expectancy [1]. A neurological condition is a damage to the brain, spinal column or nerves due to illness or injury such as spinal cord injury, acquired brain damage, stroke, motor neurons disease and locked in syndrome. Neurological disorders are considered the primary cause of disability in modern society [1, 2]. The debilitating consequences of neurological disorders include communication difficulties, impaired memory, inappropriate behavior, physical disability, restricted independence, social isolation and poor quality of life. Assistive technologies aim to increase quality of life [3–6], reduce dependence on care giver [7] and reduce dependence on the long term care system [8]. Several studies have demonstrated the effectiveness in the use of environment control interfaces (ECI) for environment control or communication through voice commands [9], scan interfaces based on grid structure, eye tracking [10–12] or brain-computer interface (BCI) based on P300 [13], among others. These software platforms actively aid during the Activities of Daily Living (ADL) improving the independence both at home and outside. However, these platforms are based in a manual scan over the different abstraction levels of the ECIs and the scan speed only depends on the users familiarization with the system and the configuration of the grids over the different menus. Thus, introducing the user environment and behavior into this loop will help the navigation agility in the ECIs. On the other hand, The progress of BCI research together with exoskeleton enables a person with motor impairment to interact with new elements of the environment [14, 15]. Thus, this progress will deliver new scenarios to BCI systems out of laboratories and move BCI into the domestic environment. The AIDE project1 aims to create new shared-control paradigm for assistive devices that integrates information from identification of residual abilities, behaviors, emotional state and intentions of the user on one hand and analysis of the environment and context factors on the other hand. In this context, a hybrid BCI model was chosen to control the ECI. It was developed as a fusion between non-invasive electroencephalography (EEG) and electrooculography (EOG) system [16]. The EEG records the sensorimotor rhythms (SMR) called event-related desynchronization (ERD) and event-related synchronization (ERS) during a motor imagery (MI) task [17] whilst the EOG records the horizontal oculoVersion (HOV). This paper aims to evaluate the ECI developed under the AIDE project conditions, a multimodal system developed to assist people with acquired brain damage or neurodegenerative diseases that need a wheelchair and has low (2019) 16:10 Page 2 of 9 or any upper limbs mobility in their ADLs, in a simulated environment able to detect the user intention through the environment analysis and the identification of the user’s behaviors, based on a conditional random fields (CRF) model [18]. Thus, the handling of the interface was studied in two different ways, with and without the prediction of the user’s intention. Users with neurological and muscular diseases and spinal cord injury (SCI) tested the system on a virtual home due to the early phase of the project. Methods This study evaluated the ECI in a simulated scenario under the AIDE project conditions, a multimodal interface able to analyze and extract relevant information from the environments as well as from the identification of residual abilities, behaviors, and intentions of the user. It consisted in a two screen layout: ECI and simulated room, with an EEG and EOG data acquisition system (see Fig. 1). Environment control interface The environment control system used in this experimentation was based in two main software components: GRID3 from Smartbox2 , a commercial augmentative and alternative communication (AAC) solution, and SHX, a specific developed software, presented in Fig 2. The ECI had three different abstraction levels: 1) related with the room (room menu), 2) related to the activities that can be performed in a specific room (activity menu), and 3) related to the actions regarding a specific activity (ADL menu). The jump between two consecutive abstraction levels will be named as step. Levels one and two were specific grid sets, created in GRID3, to be used in the context of the experimentation. They include grids for the different rooms, communication, control a wheelchair and control an exoskeleton arm. In all grids, a color code has been used: red for the alarm; green for communication, computer control and digital leisure; white for wheelchair and arm control (not used in this experimentation); light blue for the rooms; dark blue for environmental control activity. The dark blue cells are referred to environmental control activities and linked Grid3 with the SHX application. The user could scan across the different cells, select one, and then confirm or cancel the selection using the chosen signals (EOG, EEG, eye tracking, etc.). SHX is a custom build solution for environmental control management, level three. It allowed the user to easily configure and select the actions programmed for a specific activity. Different scenes for each of the possible activities in every room were created. Here too, the user could scan across the different cells, select one, and then confirm or cancel the selection using the chosen signals (EOG, EEG, eye tracking, etc.). (2019) 16:10 Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation a Page 3 of 9 b Fig. 1 Experimentation setup. The experimental setup was composed by two screens layout: ECI & Virtual home simulator, and an eye tracking placed in the bottom screen. The users performed tasks in the simulated environment (a) and they also interacted with real elements (b) Data acquisition The acquisition of the brain activity was performed with eight solid-gel electrodes placed according to international 10-20 system placed at F3, C3, Cz, P3, T7 and Mastoid, a reference electrode was placed on C4 and the ground on FpZ. Furthermore, two a electrodes were placed on the outer canthus of the eyes to the EOG signal recording. The EEG/EOG signal was acquired via Bluetooth through the Neuroelectrics amplifier (Enobio, Neuroelectrics, Barcelona, Spain). Skin/electrode resistance was kept below 12 kOhm. b c Fig. 2 Environment Control Interface. The ECI had three different abstraction levels: 1) related with the room, 2) related to the activities that can be performed in a specific room and 3) related to the actions regarding a specific activity. An example menu of each level is shown in (a), (b) and (c) respectively Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation A real-time SMR-based BCI was implemented using BCI2000, a freely distributed software for multipurpose standard BCI platform [19]. EEG and EOG were recorded at a sampling rate of 500 Hz, bandpass filtered at 0.470 Hz and pre-processed using a small Laplacian filter. Based on the maximum values for basal ERD, the ongoing EEG signal associated with the specified SMR rhythm frequency range (11-14 Hz) calculated from C3 electrode, a subject’s individual motor imagery discrimination threshold were set. The EOG discrimination thresholds were calculated regarding the average amplitudes of horizontal saccades. These individual parameters were obtained from the training session and used for later online BCI control [20]. (2019) 16:10 Page 4 of 9 Table 1 Correlation between the ADLs and ADL menu name, in the third abstraction level ADLs ADL menu ADLs ADL menu Open/close fridge Drink or eat Switch on/off air conditioner Air conditioner Open/close microwave Drink or eat Brushing teeth Teeth Eating task Eat Washing face Face Drinking task Drink Raise/lower the bed head Bed Switch on/off Music Entertainment Raise/lower the bed feet Bed Switch on/off PC Entertainment Open/close the blinds Blinds Switch on/off TV TV Switch on/off the Light light Prediction model The proposed ECI combines the environmental information and context factors together with user’s behaviors in order to detect the user intention. Thus, the input information of the prediction model was a sequence of data, the user is moving and looking at the environment, that had to be labeled. In this context, different models were tested (time-delay neural networks, decision trees, hidden markov model (HMM)...) and the CRF model was chosen, showing the best results. The CRF model is a probabilistic model for segmentation and labeling sequence data. This discriminate model takes into account not only the current state but also the previous states to perform its prediction. A conditional model specifies the probabilities of possible label sequences given an observation sequence in contras of the generative models that make very strict independence assumptions on the observation, for instance conditional independence given the labels as HMM [21]. In our case, the inputs of the system were: localization, objects in the environment, object that the user is looking at, temperature of the room, brightness of the room and day time; the output was the ADL menu, i.e. the most probable action that the user wanted to perform that were directly linked with a specific ADL (see Table 1. Participants Eight persons with different neurological pathology and spinal cord injury participated in this study (37 ± 15 years old), their demographic and clinical characteristics are listed in (Table 2). The subjects were evaluated before the experiment with the barthel index [22]. All participants gave informed consent using their standard communication channel prior to participation in the study. The protocol was approved by the Office Research Ethics Northern Ireland - approval granted for project (15/NE/0384). Experimental protocol Subjects were sitting in his/her own wheelchair in front of a table with two screens, as shown in Fig. 1. The screens were used to show the ECI and the virtual home simulator. Subjects used the AIDE multimodal interface, hybrid EEG EOG system, to online control the ECI and preform specific ADLs. Two modes were tested: A) MANUAL mode: the user had to navigate through the three abstraction levels in order to accomplish the task showed in the virtual house. The objects related with the corresponding task were surrounded by a green color in the virtual house environment and the task appeared in the right top corner. B) AIDE mode: in this mode the prediction model was used. The user had to look at the objects related to the specific ADL, showed like in the other mode, and, after the user’s intention prediction, the ECI directly jumped to the corresponding ADL menu. Then the user had to navigate like MANUAL mode. In case of Table 2 Demographic and clinical characteristics of participants ID Patient Sex Age Diagnosis Barthel score 1 Male 32 C4 SCI 4/20 2 Male 22 Duchenne Muscular Dystrophy 6/20 3 Male 55 Brain stem strokes 16.5/20 4 Male 30 C4/C5 SCI 2/20 5 Female 20 C6/C7 SCI 10/20 6 Male 58 Ischemic Stroke 19/20 7 Male 55 Multiple Systems Atrophy 5/20 8 Male 30 C6/C7 SCI 9/10 Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation wrong prediction, the user had to manually go back, the second abstraction level, and complete the corresponding ADL. The observed objects were online detected from the eye tracking Tobbi3 PCEye go, placed on the virtual environment screen, and the rest of the inputs were online simulated. Each subject performed two experimental sessions in two consecutive days. The first session was for training and calibration purpose as well as for the familiarization with the systems to be controlled. This session lasted around 60-80 min. In the last part of this session the user learned how to use the hybrid EEG/EOG interface in order to control the ECI. An example of MI and EOG movements in the training session are shown in Fig. 3c and d, respectively. The second session lasted a maximum of 60 min. The setup and familiarization phase took approximately 15 min (subjects have already tested the system in the first session). They had 10 min to perform a predefined ADLs list in both AIDE and Manual modes (all ADLs can be observed in Table 1). Each ADL can be a single action, it has a visual effect on the house simulator, e.g. swith on a lamp, or an exoskeleton action, the simulator play a short video showing the corresponding action, e.g. drink from a glass. The order of the modes was randomly selected and, before each mode, a baseline of 3 min was acquired. During the break (5 min) and at the end of this session the subjects answered the NASA task load index (tlx) questionnaire [23]. The scan in the ECI was performed through lookingright eye movements (Fig. 3b) implying a forward displacement of the grid marker (Fig. 3a-e). Once the subject stopped at one grid a customized time, chosen in the first session, the ECI ‘switch off ’ the rest of the grids (Fig. 3f ). Then, the next level or the action in the ECI was achieved by on-line ERD detection, like the subjects learned during the first session (Fig. 3a). On the other hand, if the user did not want to click on this specific grid, in the phase Fig. 3f, a looking-left eye movement returns the ECI to the phase Fig. 3a. When the user performs an action, a visual feedback is presented in the virtual home regarding to the action performed (Fig. 3g) and he/she waited for the next task. After both modes, they were instructed to interact with real elements through the ECI and watch a video using objects of a multi-sensory room, as can be observed in Fig. 1b. Results The users performed simulated ADLs during 10 min in a virtual home using an ECI in both Manual and AIDE modes. The number of the performed tasks with respect to the mean time spent per user is presented (2019) 16:10 Page 5 of 9 in Fig. 4a. Furthermore, it has been trained a Support Vector Machine (SVM) model with Gaussian kernel to estimate the boundary between both modes (yellow line in Fig. 4a). It should be noted that statistically significant differences between both modes in terms of number of tasks and mean tasks time is shown (p − value < 0.001 using Wilcoxon test). The steps distribution that the users performed in both modes are shown in Fig. 4c. ADLs manually omitted tasks were excluded from the study due to the subject was blocked during the ECI scan caused by frustration or fatigue over a specific task. On the other hand, the AIDE mode uses a CRF model, previously trained with simulated data using the same virtual home, to predict the user intention. Thus, the confusion matrix of the prediction model regarding the ADL menus is presented in Fig. 4b. In addition, the results obtained through the NASA tlx questionnaire are presented in Fig. 5. Discussion The AIDE project aims to develop a multimodal system in order to help people with neurological diseases wearing a wheelchair. The presented environment allowed the user to navigate through a virtual house and perform several ADLs using a developed ECI. Two modes were estudied, the AIDE mode, that used a CRF model to predict the user intention and ease the ECI scan, and the Manual mode, that needed a complete scan through the ECI to perform a specific ADL. The ECI was online controlled using the AIDE multimodal system based on a combination of EEG and EOG wireless acquisition system [17]. The results presented in Fig. 4a show the mean time per task spent in the AIDE mode is less than in the Manual mode being able to perform more tasks in the same time, both modes show statistically significant differences (p < 0.001). Furthermore, both modes can be easily classified by training a SVM model with Gaussian kernel, the boundary is presented with a yellow line in Fig. 4a. On the other hand, Fig. 4c shows the difference between both groups in terms of steps, i.e. the ECI abstraction levels that the user had to cross in order to perform a specific ADL. It must be noticed that in the AIDE mode, the users performed one step in the 90% of the tasks and three steps, at least, were necessary in the Manual mode. Regarding the AIDE mode, three steps were necessary only when the CRF model realized a wrong prediction and, therefore, the user had to return to the activity menu and select the proper ADL menu. Although a bad prediction is sometimes performed, the multimodal system helps in terms of location, i.e. the activity that the system predicts is always related with the room where the user is, facilitating the navigation. Perform five or seven steps in the Manual mode implies that a wrong abstraction level was selected, Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation (2019) 16:10 Page 6 of 9 a c b d Fig. 3 Multimodal system processing for one ADL in AIDE mode. The user had to perform different actions in order to execute the corresponding ADLs, in this example, the user had to switch on the TV, phases a-g show the behavior of both screens during the task. EEG (a) and EOG (b) signals were acquired to online control the ECI in order to perform ADLs in a virtual house. When the task started (vertical purple line), the scan through the ECI was performed by EOG activity detection [orange line in (b)], i.e. when HOV activity exceeded the threshold [indicated by the orange dashed line in (b)] the grid marker moved forward (phases a-e). Once the subject stopped at one grid, a task confirmation was needed [indicated by the vertical black line] and the ECI ‘switched off’ the rest of the grids indicating this purpose (phase f). The confirmation was performed by the detection of SMR-ERD [indicated by red line in (a)] and the action was done, so the ADL finished (vertical dotted purple line). This ADL was performed in one step, i.e. the user only needed to navigate through the last abstraction level to complete the task. Before the experimentation, the user was trained in motor imagery (c) and EOG movement (d) to the set up the control system with the personalized parameters Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation (2019) 16:10 Page 7 of 9 a b c Fig. 4 Environment control interface performance. The users performed two different trials with the same goal: complete as many tasks as possible in 10 min by using the Manual mode and the AIDE mode (a). Both modes have been used to train a SVM model with Gaussian kernel and find the boundary between them [yellow line in (a)]. The interaction with the ECI was measured by steps that a user had to perform in order to complete the tasks (c). Furthermore, in AIDE mode the user’s intention prediction was performed through CRF model and, therefore, the confusion matrix of the model is obtained (b) due to user confusion or lack of practice, and the user had to go back in the ECI. The AIDE mode uses a CRF model to perform the predictions about the user’s intention. The model uses the information of the virtual home and the object that the user is looking at, acquired thought an eye tracking device. The model was previously trained with simulated data using the same virtual house environment. Thus, Fig. 4b shows the confusion matrix of the prediction model, regarding the ADL menu, with a global accuracy about 87%. The CRF model, as it takes into account not only the current state but also the previous states to perform its prediction, it could fail in the prediction of task with common features. Therefore, the ADLs related to the Drink menu are sometimes predicted as the ADLs related with the Eat menu, in this case around the 50% of the trials. In addition, TV and the Teeth menus are rarely selected as Entertainment and Face menu, respectively, by the prediction model. After each mode, the subjects answered the NASA tlx questionnaire in order to assess the workload between the modes, showed in Fig. 5. Unexpectedly, it has not statistically significant differences, so we can say that the users do not notice subjective differences between both modes in terms of workload. It can be explained because it was the first time that the users handle the complex multimodal control system (EEG+EOG) with this ECI. We assume that, observing the results presented in Fig 4, the workload should decrease, at least, in the AIDE mode. Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation (2019) 16:10 Page 8 of 9 Fig. 5 NASA task load index. The subjects answered the NASA tlx questionnaire after each mode Conclusion The presented ECI allowed the users to perform simulated ADLs with a multimodal control system. The platform was tested in two different scenarios: Manual and AIDE mode. The first one was presented as a simple ECI where the user had to achieve the corresponding ADL. The second mode used a CRF model to predict the user’s intention through the environment analysis and identification of the user’s behaviors. We conclude that, even the users do not perceive subjective differences between both modes in terms of workload, the AIDE mode helps the user to perform mode ADLs, spending less time per task, showing statistically significant differences with respect to the Manual mode. This effect is caused by the user’s intention prediction as the ECI jumps directly to the last abstraction level of the ECI. The environment analysis and the identification of the user’s behaviors can be used to predict the user intention and will allow to speed up the ECIs scan opening a new paradigm in the design of these interfaces. Although the developed ECI was tested only in a simulated home environment, it can be easily adapted to a real environment increasing the user independence at home. Endnotes 1 http://aideproject.umh.es/ 2 http://www.thinksmartbox.com 3 http://www.tobii.com Abbreviations AAC: Augmentative and alternative communication; ADL: Activity of daily living; BCI: Brain computer interface; CRF: Conditional random field; ECI: Environment control interface; EEG: Electroencephalography; EOG: Electrooculography; HOV: Horizontal oculoversion; MI: Motor Imagery; SMR-ERD: Sensorimotor rhythm event-related desynchronization; SVM: Support vector machine; tlx: task load index Acknowledgments The authors thank the subjects for their participation. In addition, we acknowledge the great support from CEDAR Foundation, Belfast, UK and the Biomedical Research Networking Center (CIBER). Funding This work has been supported by the AIDE project through the grant agreement no. 645322 of the European Commission and by the Ministry of Economy and Competitiveness through the project DPI2015-70415-C2-2-R. Availability of data and materials Data and materials can be made available upon request to the corresponding author. Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation (2019) 16:10 Authors’ contributions ABM, SM, SRS and NGA designed the study. ABM, SE, JAB, LDL and SD collect the data. SD developed the environment control interface. LDL designed the virtual home environment. ABM, SE and JAB interpreted and analyze the data. ABM, SE created the figures. ABM, SE, JAB, LDL and SD wrote the manuscript. SM, NGA and SRS deeply revised the manuscript. ABM, SE, JAB, LDL, SE, MN, SM, SRS and NGA edited the manuscript. All authors read and approved the final manuscript. 10. Lupu RG, Ungureanu F, Siriteanu V. Eye tracking mouse for human computer interaction. In: E-Health and Bioengineering Conference (EHB), 2013. IEEE; 2013. p. 1–4. https://doi.org/10.1109/EHB.2013.6707244. 11. Adjouadi M, Sesin A, Ayala M, Cabrerizo M. 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North Holland: Elsevier; 1988. p. 139–83. https://doi.org/10.1016/S01664115(08)62386-9. Ethics approval and consent to participate This study was conducted according to the declaration of Helsinki and had ethical approval by the Office Research Ethics Northern Ireland (June 2016) – approval granted for AIDE project (15/NE/0384). All subjects gave written informed consent prior to data collection. Consent for publication Participants gave written informed consent to data treatment in this research study and permission to publish anonymous data and results. Competing interests Sergio Domingo is a software developer at BJ Adaptaciones, the developer of SHX software. Sergio Domingo declares no competing interests. The rest of the authors declare no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 Miguel Hernández University of Elche, Av. Universidad w/n, Ed. Innova, 03202 Elche, Spain. 2 BJ Adaptaciones, St Mare de Déu del Coll, 70, 08023 Barcelona, Spain. 3 The Cedar Foundation, 1 Upper Lisburn Road, BT10 0GW Belfast, UK. 4 University Hospital of Tuebingen, Applied Neurotechnology Lab, Calwerstr. 14, D-72076 Tübingen, Germany. 5 Clinical Neurotechnology Laboratory, Neuroscience Research Center (NWFZ), Charité University Medicine Berlin, Charitéplatz 1, 10117 Berlin, Germany. Received: 25 July 2018 Accepted: 18 November 2018 References 1. World Health Organization. Neurological Disorders: Public Health Challenges. Geneva: World Health Organization; 2006. http://www.who. int/iris/handle/10665/43605. 2. Winstein CJ, Stein J, Arena R, Bates B, Cherney LR, Cramer SC, Deruyter F, Eng JJ, Fisher B, Harvey RL, et al. Guidelines for adult stroke rehabilitation and recovery: a guideline for healthcare professionals from the american heart association/american stroke association. Stroke. 2016;47(6):98–169. 3. Sellers EW, Vaughan TM, Wolpaw JR. 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HIGHLIGHT visualization on the CRISPR-Explorer browser. In the Browse mode, potential gRNA candidates are color-coded based on their specificities and efficiencies to facilitate selection, and detailed information for an individual gRNA design can be displayed in a pop-up. In the Batch mode, the user can input a gene, a gene list, a genome coordinate, or a list of genome coordinates. The output of the “Batch Design Exporter” is an interactive table that allows the user to rank and/or filter his/her gRNA designs. The table can be exported in a text file format. www.jbmethods.org POL Scient How to cite this article: Chen K, Jin Y, Lin YC. CRISPR Explorer: A fa Biol Methods 2016;3(4):e56. DOI: 10.14440/jbm.2016.138 CRISPR-Explorer facilitates the design of high-quality gRNA for the low- and high-throughput needs of researchers due to its ease of use, comprehensiveness, and instantaneous feedback. The advantages of our web application over other existing web applications are: (1) fast: gRNA design results based on published empirical studies are generated instantly, thus removing wait times; (2) intuitive: the user can visualize the high-quality gRNAs with detailed design information through an interactive genome browser; and (3) flexible: the user can define his/her own parameters for gRNA selection and various input formats are recognized. The scoring schemes that are used include the specificity score and the efficiency score. Targeting multiple regions can be achieved simultaneously through Batch Design. Guide RNA design results can be ranked and filtered on the browser before export. The user will select the genome of interest for genome editing, the desired gRNA length, and the cognate promoter (U6 or T7) that drives the gRNA expression. After selecting these options, the user will have the choice to input a gene name (official gene symbol, Ensembl ID, or Refseq ID), genome coordinates, or a DNA sequence for gRNA p a i m l i g f f s t a t m CRISPR-Explorer facilitates the design of high-quality gRNA for the low- and high-throughput needs of researchers due to its ease of use, comprehensiveness, and instantaneous feedback. CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing Kenian Chen, Yi Jin, Yin C. Lin* Baylor Institute for Immunology Research, Dallas, TX 75246, USA Baylor Institute for Immunology Research, Dallas, TX 75246, USA *Corresponding author: Yin C. Lin, Email: Yin.Lin@BSWHealth.org Competing interests: The authors have declared that no competing interests exist. Abbreviations used: gRNA, guide RNA; PAM, protospacer adjacent motif; DSBs, double-stranded breaks; NHEJ, non-homologous end-joining; HDR, homology-directed Abbreviations used: gRNA, guide RNA; PAM, protospacer adjacent motif; DSBs, double-stranded breaks; NHEJ, non-homologous end-joining; HDR, homology-directed Abbreviations used: gRNA, guide RNA; PAM, protospacer adjacent motif; DSBs, double-stranded breaks; NHEJ, non-homologo Received June 8, 2016; Revision received August 31 2016; Accepted August 31 2016; Published October 10, 2016 Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 Resource ABSTRACT The RNA-guided CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeat-CRISPR-associated 9) system has become a revolutionary technology for targeted genome engineering. The critical step of this technology requires the design of a highly specific and efficient guide RNA (gRNA) that will guide the Cas9 nuclease to the complementary DNA target sequence. CRISPR-Explorer is a new and user-friendly web server for selecting optimal CRISPR sites. It implements the latest scoring schemes of gRNA specificity and efficiency based on published empirical studies. The gRNA design results are generated instantly, thus removing wait times. The user can visualize the high-quality gRNAs with detailed design information through an interactive genome browser. Furthermore, the user can define and specify the parameters for gRNA selection in the Batch Design mode, which recognizes various input formats. CRISPR Explorer is freely accessible at: http://crisprexplorer.org. Keywords: CRISPR-Cas9, guide RNA design, open-source web application HIGHLIGHT The advantages of our web application over other existing web applications are: (1) fast: gRNA design results based on published empirical studies are generated instantly, thus removing wait times; (2) intuitive: the user can visualize the high-quality gRNAs with detailed design information through an interactive genome browser; and (3) flexible: the user can define his/her own parameters for gRNA selection and various input formats are recognized. The scoring schemes that are used include the specificity score and the efficiency score. Targeting multiple regions can be achieved simultaneously through Batch Design. Guide RNA design results can be ranked and filtered on the browser before export. The user will select the genome of interest for genome editing, the desired gRNA length, and the cognate promoter (U6 or T7) that drives the gRNA expression. After selecting these options, the user will have the choice to input a gene name (official gene symbol, Ensembl ID, or Refseq ID), genome coordinates, or a DNA sequence for gRNA CRISPR-Cas is an RNA-guided nuclease system that allows ef­ ficient perturbation of gene functions and is increasingly being used for genome-wide functional screens [1-3]. The type II CRISPR-Cas system from Streptococcus pyogenes Cas9 (SpCas9) has been adapted to target a specific DNA sequence for Cas9 nuclease cleavage using a programmable guide RNA (gRNA) [4-7]. Targeting is mediated by the first 20 nucleotides at the 5’-end of the gRNA, which are comple­ mentary to the target DNA sequence, followed by a 3 bp protospacer www.jbmethods.org 1 POL Scientific How to cite this article: Chen K, Jin Y, Lin YC. CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing. J Biol Methods 2016;3(4):e56. DOI: 10.14440/jbm.2016.138 How to cite this article: Chen K, Jin Y, Lin YC. CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing. J Biol Methods 2016;3(4):e56. DOI: 10.14440/jbm.2016.138 1 www.jbmethods.org Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 Resource adjacent motif (PAM) sequence. Cas9 nuclease can be directed by a programmable gRNA to induce DNA double-stranded breaks (DSBs) at specific locations in the genome. In mammalian cells, genome ed­ iting typically occurs through the repair of the Cas9-induced DSB by the error-prone non-homologous end-joining (NHEJ) mechanism, which introduces variable-length insertion/deletion (indel) mutations, or through homology-directed (HDR) repair in the presence of an exogenous DNA template. HIGHLIGHT The sequence model predicts that the editing efficiency is influenced by the DNA target sequence as well as the flanking sequences [13]. Indeed, such a sequence model has been experimentally validated, and gRNA efficiency can be calculated based on this model [13]. Therefore, to generate a highly specific and efficient gRNA design, we believe that it is important to consider both the specificity and efficiency of the gRNA. To date, a number of web-based applications have been developed to select highly specific genome-editing sites [9,12,14,15]. Typically, these applications require the target sequence or gRNA as input. The potential CRISPR sites on the target sequence are identified, and this is followed by the searching of potential off-target sites throughout the reference genome. The wait time depends on the computation time for each target site calculation. The wait time increases as the number of input sequences increases, which can result in a long wait time. The output is often presented on a plain web page that provides a basic display of the positions of the CRISPR sites that match the input sequence. A few applications use the UCSC Genome Browser to display results that are not interactive. Most of these applications only consider the specificity of To achieve fast query speed, the results were sorted and indexed using the Tabix program [19]. We adopted the WashU Epigenome Browser [20] as the presentation framework for display customization and linking to the gRNA database. The batch design exporter that we implemented is based on the Angular UI Grid (http://ui-grid.info/) JavaScript framework, which provides an interactive output. PROCEDURE Input The CRISPR-Explorer homepage contains 3 panels. See Figure 1. 1. ‘Basic Options’ panel. Choose the genome that you want to edit and which promoter you are planning to use. Currently, only Cas9 PAM (-NGG) is supported because it is the most widely used and has a more mature scoring scheme based on empirical studies. However, we will include other PAMs (such as Cpf1) in the future when possible. By default, repeat regions are excluded for off-target searching. However, the user has the option to include repeat regions during the gRNA design. 2. ‘Browse gRNAs’ panel. Browse gRNAs for your gene of interest by entering the gene name, the genome coordinate, or a DNA sequence from the genome. The input format for the gene name can be the official gene symbol, Ensembl gene name, or refGene name. HIGHLIGHT As for the calculation of the truncated (18 bp) gRNA, the first two nucleotide positions and their experimentally determined effects were removed from the calculation of the specificity score. The efficiency score was calculated using the SSC program [13]. g p , p y [ ] Numerous studies have shown that CRISPR-Cas9 not only targets its intended on-target sites, but also certain off-target sites in the ge­ nome that share sequence similarity with the on-target sites [9-12]. This off-target effect is attributed to the ability of Cas9 to recognize a non-canonical PAM sequence and tolerate the nucleotide mismatch between gRNA and its target sequence. To achieve productive genome editing, the efficiency of Cas9-mediated modification is also critical. The sequence model predicts that the editing efficiency is influenced by the DNA target sequence as well as the flanking sequences [13]. Indeed, such a sequence model has been experimentally validated, and gRNA efficiency can be calculated based on this model [13]. Therefore, to generate a highly specific and efficient gRNA design, we believe that it is important to consider both the specificity and efficiency of the gRNA. To date, a number of web-based applications have been developed to select highly specific genome-editing sites [9,12,14,15]. Typically, these applications require the target sequence or gRNA as input. The potential CRISPR sites on the target sequence are identified, and this is followed by the searching of potential off-target sites throughout the reference genome. The wait time depends on the computation time for each target site calculation. The wait time increases as the number of input sequences increases, which can result in a long wait time. The output is often presented on a plain web page that provides a basic display of the positions of the CRISPR sites that match the input sequence. A few applications use the UCSC Genome Browser to display results that are not interactive. Most of these applications only consider the specificity of Numerous studies have shown that CRISPR-Cas9 not only targets its intended on-target sites, but also certain off-target sites in the ge­ nome that share sequence similarity with the on-target sites [9-12]. This off-target effect is attributed to the ability of Cas9 to recognize a non-canonical PAM sequence and tolerate the nucleotide mismatch between gRNA and its target sequence. To achieve productive genome editing, the efficiency of Cas9-mediated modification is also critical. HIGHLIGHT To identify potential off-targets for each possible gRNA, we used the “all-mapper” of the Genome Multitool (GEM) mapper, which gives all alignments of a specific short sequence with a user-defined number of mismatches [17]. The number of off-target alignments in the reference genome for a specific CRISPR site grows rapidly as the number of mismatches increases, and therefore, up to 4 mismatches in the 20 bp gRNA were allowed when searching for off-target sites. Three mismatches were used for the truncated (18 bp) gRNA since previous studies showed that a truncated gRNA with more than 3 mismatches usually does not have a detectable off-target effect [18]. To calculate the specificity score (or aggregate scores of single hits), we adopted the algorithm that was developed by the Zhang F group [12] and incorporated their experimentally determined effect on targeting of each mismatch position (M= [0, 0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583]). As for the calculation of the truncated (18 bp) gRNA, the first two nucleotide positions and their experimentally determined effects were removed from the calculation of the specificity score. The efficiency score was calculated using the SSC program [13]. gi [ ] Briefly, the whole reference genomes of human (hg19) and mouse (mm10) for every possible CRISPR site (e.g. 5’(N~20)-NGG, 5’(N~20)- NAG sites, etc.) were scanned. To identify potential off-targets for each possible gRNA, we used the “all-mapper” of the Genome Multitool (GEM) mapper, which gives all alignments of a specific short sequence with a user-defined number of mismatches [17]. The number of off-target alignments in the reference genome for a specific CRISPR site grows rapidly as the number of mismatches increases, and therefore, up to 4 mismatches in the 20 bp gRNA were allowed when searching for off-target sites. Three mismatches were used for the truncated (18 bp) gRNA since previous studies showed that a truncated gRNA with more than 3 mismatches usually does not have a detectable off-target effect [18]. To calculate the specificity score (or aggregate scores of single hits), we adopted the algorithm that was developed by the Zhang F group [12] and incorporated their experimentally determined effect on targeting of each mismatch position (M= [0, 0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583]). HIGHLIGHT If you know the genomic location of the region, you can directly query the browser with the genome coordinate (either in bed format or the following format: 2. ‘Browse gRNAs’ panel. Browse gRNAs for your gene of interest by entering the gene name, the genome coordinate, or a DNA sequence from the genome. The input format for the gene name can be the official gene symbol, Ensembl gene name, or refGene name. If you know the genomic location of the region, you can directly query the browser with the genome coordinate (either in bed format or the following format: J Biol Methods | 2016 | Vol. 3(4) | e56 POL Scientific HIGHLIGHT An indel mutation at the spliced coding exon of the target gene frequently results in a coding frameshift and the initiation of nonsense-mediated decay of the gene transcript, which causes gene inactivation. In addition to its utility in genome editing, by removing the Cas9 nuclease activity, catalytically inactive Cas9 (dCas9) proteins that are fused to a co-activation or co-repression domain can be guided by gRNA to a specific DNA sequence to activate or repress gene transcription, respectively [8]. the gRNA, but not the efficiency. One application, CRISPR-ERA, does consider the specificity and efficiency for gRNA design but uses an ad hoc scoring scheme [14]. Finally, most of the existing web applications lack an intuitive and convenient way for browsing and batch design. the gRNA, but not the efficiency. One application, CRISPR-ERA, does consider the specificity and efficiency for gRNA design but uses an ad hoc scoring scheme [14]. Finally, most of the existing web applications lack an intuitive and convenient way for browsing and batch design. Here, our goal was to develop a guide RNA design web server that eliminates the above issues and offers the most up-to-date and proven scoring schemes for gRNA specificity and efficiency. CRISPR-Explorer is fast, intuitive, and flexible. It is particularly useful for genome-wide functional screening and high-throughput screening using the CRIS­ PR-Cas9 technology. The CRISPR-Cas system is a rapidly moving technology. We will continue to update the scoring schemes as new knowledge and data become available, and we expect to incorporate the scoring schemes for new Cas nucleases that are identified in the future [16]. Here, our goal was to develop a guide RNA design web server that eliminates the above issues and offers the most up-to-date and proven scoring schemes for gRNA specificity and efficiency. CRISPR-Explorer is fast, intuitive, and flexible. It is particularly useful for genome-wide functional screening and high-throughput screening using the CRIS­ PR-Cas9 technology. The CRISPR-Cas system is a rapidly moving technology. We will continue to update the scoring schemes as new knowledge and data become available, and we expect to incorporate the scoring schemes for new Cas nucleases that are identified in the future [16]. Briefly, the whole reference genomes of human (hg19) and mouse (mm10) for every possible CRISPR site (e.g. 5’(N~20)-NGG, 5’(N~20)- NAG sites, etc.) were scanned. L Scienti 2 J Biol Methods | 2016 | Vol. 3(4) | e56 2 POL Scientific Resource Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 The ‘SSC score > 0’ option selects gRNAs that have efficiency scores that are greater than zero [13]. The ‘Mismatch > 1’ option selects for gRNAs that have more than 1 mismatch with the most similar sequence in the whole genome. Output The two main display options in CRISPR-Explorer are the interactive browser (Fig. 2 and 3) and a table (Fig. 4) 4. After the ‘Browse’ button inside the ‘Browse gRNAs’ panel is clicked, the browser launches (Fig. 2). The gRNAs are color-coded based on different specificity and efficiency score cutoffs. The legend on the bot­ tom left-hand side defines the color codes. Red gRNAs have the highest specificity and efficiency scores, and green gRNAs have the lowest specificity and efficiency scores. By rolling the cursor over the gRNA bar, brief information about that gRNA is displayed. To get detailed information about a gRNA, clicking on the gRNA leads to a pop-up window that shows the gRNA’s genome coordinate, strand-specificity, length, closest mismatch, specificity and efficiency scores, sequence, and detailed off-target information (#OT) (Fig. 2). A click on the ‘Details’ link in this pop-up will lead to a page with detailed information about the off-target sequences of the selected gRNA. The table that is found on this page is interactive and contains the following information: off-target sequences with the mismatches in upper case letters (‘Off-target se­ quence’), genome coordinate with strand specificity that is indicated by “+” or “-” (‘Location’), number of mismatches (‘Mismatch’), off-target score (‘Score’), and the name of the gene in which the exons are hit by the off-target mismatch (‘Hit Exon’). 5. To change the gRNA display on the browser, first right-click on the browser track and then click on ‘Config­ ure’. One can choose different filters (‘Closes Mismatch’, ‘Specificity’, or ‘Efficiency’) to limit the gRNAs that are displayed on the browser (Fig. 3). For example, in Figure 3, the selection of ‘Specificity’ with the scoring range between 50 and 100 restricts the display of gRNAs to those whose scores fall within this range. 5. To change the gRNA display on the browser, first right-click on the browser track and then click on ‘Config­ ure’. One can choose different filters (‘Closes Mismatch’, ‘Specificity’, or ‘Efficiency’) to limit the gRNAs that are displayed on the browser (Fig. 3). Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 Figure 1. A screen shot of the CRISPR-Explorer homepage that shows the three input panels: “Basic Options”, “Browse gRNAs”, and “Batch Design Exporter”. Figure 1. A screen shot of the CRISPR-Explorer homepage that shows the three input panels: “Basic Options”, “Browse gRNAs”, and “Batch Design Exporter”. chrX:NNNNN-NNNNN). When the target sequence (with a limit of 20000 bases) is supplied as input, the Blat program (an alignment tool) from the UCSC Genome Browser is used to map the sequence to the genome location. The speed of this part depends on the UCSC Blat server. A guide RNA ID that is generated from Batch Design Exporter can be used to access the detailed information about the gRNA. chrX:NNNNN-NNNNN). When the target sequence (with a limit of 20000 bases) is supplied as input, the Blat program (an alignment tool) from the UCSC Genome Browser is used to map the sequence to the genome location. The speed of this part depends on the UCSC Blat server. A guide RNA ID that is generated from Batch Design Exporter can be used to access the detailed information about the gRNA. 3. 3. ‘Batch Design Exporter’ panel. Multiple genome coordinates or gene entries can be entered simultaneously. Currently, a maximum number of 100 entries is allowed per run to prevent server overload. You can also reduce the output and get a faster response by applying pre-defined filters. The ‘Exonic guide only’ option selects gRNAs that fall within the exonic regions. RefGene provides the gene structure to define the exonic regions. The ‘MIT score ≥ 50’ option selects gRNAs that have specificity scores greater than or equal to 50 [12]. The ‘SSC score > 0’ option selects gRNAs that have efficiency scores that are greater than zero [13]. The ‘Mismatch > 1’ option selects for gRNAs that have more than 1 mismatch with the most similar sequence in the whole genome. 3. ‘Batch Design Exporter’ panel. Multiple genome coordinates or gene entries can be entered simultaneously. Currently, a maximum number of 100 entries is allowed per run to prevent server overload. You can also reduce the output and get a faster response by applying pre-defined filters. The ‘Exonic guide only’ option selects gRNAs that fall within the exonic regions. RefGene provides the gene structure to define the exonic regions. The ‘MIT score ≥ 50’ option selects gRNAs that have specificity scores greater than or equal to 50 [12]. Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 For example, in Figure 3, the selection of ‘Specificity’ with the scoring range between 50 and 100 restricts the display of gRNAs to those whose scores fall within this range. 6. After the ‘Submit’ button inside the ‘Batch Design Exporter’ panel is clicked, an interactive table launches (Fig. 4). When a gene name is used as input, the output will contain information about the exon (ranked in 6. After the ‘Submit’ button inside the ‘Batch Design Exporter’ panel is clicked, an interactive table launches (Fig. 4). When a gene name is used as input, the output will contain information about the exon (ranked in Resource Resource Figure 2. An example of the browser track dis­ play of “Browse gRNA”. The human IL1A gene was used as an input. By clicking on the gRNA, the pop-up box reveals detailed information about it. The “Details” link directs the user to off-target sequence information. Figure 2. An example of the browser track dis­ play of “Browse gRNA”. The human IL1A gene was used as an input. By clicking on the gRNA, the pop-up box reveals detailed information about it. The “Details” link directs the user to off-target sequence information. Figure 2. An example of the browser track dis­ play of “Browse gRNA”. The human IL1A gene was used as an input. By clicking on the gRNA, the pop-up box reveals detailed information about it. The “Details” link directs the user to off-target sequence information. Figure 3. An example of the browser track dis­ play of “Browse gRNA”. The human IL1A gene was used as an input. Selecting “Configure” after right clicking on the track allows the user to choose the gRNAs that are displayed based on closest mismatch, specificity and efficiency scores. Figure 3. An example of the browser track dis­ play of “Browse gRNA”. The human IL1A gene was used as an input. Selecting “Configure” after right clicking on the track allows the user to choose the gRNAs that are displayed based on closest mismatch, specificity and efficiency scores. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied before the generation of this table. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied before the generation of this table. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied before the generation of this table. composite gene model) in which the target site falls (‘HitExon’ column). If a transcript name is use as input (e.g. Ensembl transcript name), the ‘HitExon’ will rank by that transcript. Genome coordinates is the ideal input format for gRNA design in unannotated genomic regions. When genome coordinates are supplied as input, the ‘HitExon’ information is not reported. J Biol Methods | 2016 | Vol. 3(4) | e56 3 3 POL Scientific Resource Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 Figure 2. An example of the browser track dis­ play of “Browse gRNA”. The human IL1A gene was used as an input. By clicking on the gRNA, the pop-up box reveals detailed information about it. The “Details” link directs the user to off-target sequence information. Figure 3. An example of the browser track dis­ play of “Browse gRNA”. The human IL1A gene was used as an input. Selecting “Configure” after right clicking on the track allows the user to choose the gRNAs that are displayed based on closest mismatch, specificity and efficiency scores. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied before the generation of this table. Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 J Biol Methods | 2016 | Vol. 3(4) | e56 Resource composite gene model) in which the target site falls (‘HitExon’ column). If a transcript name is use as input (e.g. Ensembl transcript name), the ‘HitExon’ will rank by that transcript. Genome coordinates is the ideal input format for gRNA design in unannotated genomic regions. When genome coordinates are supplied as input, the ‘HitExon’ information is not reported. 7. 7. The interactive table generated by the ‘Batch Design Exporter’ contains the following information: gRNA sequence (‘Guide RNA’), genome coordinates (‘Location’), strand specificity (‘Strand’), target’s name (‘Target’), exon number where the gRNA falls (‘HitExon’), number of mismatches (‘MM’), specificity score (‘SpecMIT’), efficiency score (‘EffiSSC’), exonic or not (‘Exon’), number of potential off-targets that is exonic (‘OtExon’), number of mismatches contained by the potential off-targets that fall in exons (‘OtEx­ onDist’), number of potential off-targets in the whole genome (‘OT’), number of mismatches contained by the potential off-targets that fall in the genome (‘OtDist’), and the link to the detailed information about the off-target sequences (‘GuideID’) (Fig. 4). The description in the ‘OtExonDist’ column (for example, 0:0:0:0:4 7. The interactive table generated by the ‘Batch Design Exporter’ contains the following information: gRNA sequence (‘Guide RNA’), genome coordinates (‘Location’), strand specificity (‘Strand’), target’s name (‘Target’), exon number where the gRNA falls (‘HitExon’), number of mismatches (‘MM’), specificity score (‘SpecMIT’), efficiency score (‘EffiSSC’), exonic or not (‘Exon’), number of potential off-targets that is exonic (‘OtExon’), number of mismatches contained by the potential off-targets that fall in exons (‘OtEx­ onDist’), number of potential off-targets in the whole genome (‘OT’), number of mismatches contained by the potential off-targets that fall in the genome (‘OtDist’), and the link to the detailed information about the off-target sequences (‘GuideID’) (Fig. 4). The description in the ‘OtExonDist’ column (for example, 0:0:0:0:4 J Biol Methods | 2016 | Vol. 3(4) | e56 POL Scientific L Sc ent J Biol Methods | 2016 | Vol. 3(4) | e56 References 1. 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PMID: 23849981 Please contact the Webmaster, Kenian.Chen@BSWHealth.org, for any requests, comments, and bug reporting. We expect to maintain this web application for at least two years and may provide updates that include the newly identified CRISPR systems that target RNA. 9. Bae S, Park J, Kim J (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30: 1473-1475. doi: 10.1093/bioinformatics/btu048. PMID: 24463181 10. Cho SW, Kim S, Kim JM, Kim J (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31: 230-232. doi: 10.1038/nbt.2507. PMID: 23360966 Acknowledgments 11. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, et al. (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31: 822-826. doi: 10.1038/nbt.2623. PMID: 23792628 The authors thank Carson Harrod for critical reading of the manu­ script. This work has been supported by the Baylor Charles A. Sammons Cancer Center and the Baylor Institute for Immunology Research (Yin C. Lin). 12. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31: 827- 832. doi: 10.1038/nbt.2647. PMID: 23873081 13. Xu H, Xiao T, Chen C, Li W, Meyer CA, et al. (2015) Sequence determinants of improved CRISPR sgRNA design. Genome Res 25: 1147-1157. doi: 10.1101/ gr.191452.115. PMID: 26063738 POL Scientific POL Scientific Journal of Biological Methods | 2016 | Vol. 3(4) | e56 DOI: 10.14440/jbm.2016.138 Resource in Fig. 4, first row) means that all of the predicted targets have 4 mismatches. In this example, none of the predicted off-targets have zero, one, two or three mismatches. in Fig. 4, first row) means that all of the predicted targets have 4 mismatches. In this example, none of the predicted off-targets have zero, one, two or three mismatches. 8. Each column of the interactive table can be ranked alphabetically or numerically from lowest to highest or vice versa. This can be achieved by clicking on the right of the individual header of the first row of the table where an up or down arrow is. 8. Each column of the interactive table can be ranked alphabetically or numerically from lowest to highest or vice versa. This can be achieved by clicking on the right of the individual header of the first row of the table where an up or down arrow is. 9. Different features can be selected for the ‘Guide RNA’, ‘Location’, ‘Strand’, ‘Target’, ‘Exon’, ‘OtExon’, ‘OtExonDist’, ‘OT’, and ‘OtDist’ columns. The filtering functions (equals/less/or greater) are available for the ‘HitExon’, ‘MM’, ‘SpecMIT’, and ‘EffiSSC’ columns. This can be achieved by rolling the cursor to the left of the individual column header. Then, click on the ‘Ξ’ icon that appears and the filtering option will be revealed. 10. After filtering and ranking, the personally customized table can be exported as a ‘.csv’ file by clicking the “Export to CSV” button at the bottom of the table. The user can keep the default file name or type in the file name inside the textbox that is above the “Export to CSV” button. 10. After filtering and ranking, the personally customized table can be exported as a ‘.csv’ file by clicking the “Export to CSV” button at the bottom of the table. The user can keep the default file name or type in the file name inside the textbox that is above the “Export to CSV” button. References Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821. doi: 10.1126/science.1225829. PMID: 22745249 19. Li H (2011) Tabix: fast retrieval of sequence features from generic TAB- delimited files. 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https://ecommons.aku.edu/cgi/viewcontent.cgi?article=1372&context=eastafrica_fhs_mc_obstet_gynaecol
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Intimate partner violence against adolescents and young women in sub-Saharan Africa: who is most vulnerable?
Reproductive health
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eCommons@AKU eCommons@AKU eCommons@AKU eCommons@AKU eCommons@AKU eCommons@AKU Medical College, East Africa Medical College, East Africa Obstetrics and Gynaecology, East Africa Authors Authors Yohannes Dibaba Wado, Martin K. Mutua, Abdu Mohiddin, Macellina Y. Ijadunola, Cheikh Faye, Carolina V. N. Coll, Aluisio J. D. Barros, and Caroline W. Kabiru Intimate partner violence against adolescents and young women Intimate partner violence against adolescents and young women in sub-Saharan Africa: who is most vulnerable? in sub-Saharan Africa: who is most vulnerable? See next page for additional authors Follow this and additional works at: https://ecommons.aku.edu/eastafrica_fhs_mc_obstet_gynaecol Part of the Obstetrics and Gynecology Commons Abstract Background:  Intimate partner violence (IPV) is a global public health and human rights issue that affects millions of women and girls. While disaggregated national statistics are crucial to assess inequalities, little evidence exists on inequalities in exposure to violence against adolescents and young women (AYW). The aim of this study was to deter- mine inequalities in physical or sexual IPV against AYW and beliefs about gender based violence (GBV) in sub-Saharan Africa (SSA). Methods:  We used data from the most recent Demographic and Health Surveys (DHS) conducted in 27 countries in SSA. Only data from surveys conducted after 2010 were included. Our analysis focused on married or cohabiting AYW aged 15–24 years and compared inequalities in physical or sexual IPV by place of residence, education and wealth. We also examined IPV variations by AYW’s beliefs about GBV and the association of country characteristics such as gender inequality with IPV prevalence. Results:  The proportion of AYW reporting IPV in the year before the survey ranged from 6.5% in Comoros to 43.3% in Gabon, with a median of 25.2%. Overall, reported IPV levels were higher in countries in the Central Africa region than other sub-regions. Although the prevalence of IPV varied by place of residence, education and wealth, there was no clear pattern of inequalities. In many countries with high prevalence of IPV, a higher proportion of AYW from rural areas, with lower education and from the poorest wealth quintile reported IPV. In almost all countries, a greater pro- portion of AYW who approved wife beating for any reason reported IPV compared to their counterparts who disap- proved wife beating. Reporting of IPV was weakly correlated with the Gender Inequality Index and other societal level variables but was moderately positively correlated with adult alcohol consumption (r = 0.48) and negative attitudes towards GBV (r = 0.38). Conclusion:  IPV is pervasive among AYW, with substantial variation across and within countries reflecting the role of contextual and structural factors in shaping the vulnerability to IPV. The lack of consistent patterns of inequalities by the stratifiers within countries shows that IPV against women and girls cuts across socio-economic boundaries sug- gesting the need for comprehensive and multi-sectoral approaches to preventing and responding to IPV. Keywords:  Physical or sexual violence, Attitudes, SSA, Adolescents and young women, Equiplot Authors Authors Wado et al. Reprod Health 2021, 18(Suppl 1):119 https://doi.org/10.1186/s12978-021-01077-z Open Access Open Access © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​ mmons​.org/publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Intimate partner violence against adolescents and young women in sub‑Saharan Africa: who is most vulnerable? Carolina V. N. Coll4, Aluisio J. D. Barros4  and Caroline W. Kabiru1 Plain English summary Intimate partner violence (IPV) is a global public health and human rights issue that affects millions of women and girls. Disaggregated national data are needed to *Correspondence: ywado@aphrc.org 1 African Population and Health Research Center, Nairobi, Kenya Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​ mmons​.org/publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 2 of 13 assess inequalities in exposure to violence against adoles- cents and young women (AYW). and violated because they have less bargaining power within their relationships [10, 11]. Data from the WHO violence against women surveys show that globally 30% of adolescent girls (aged 15 to 19  years) have experi- enced physical and/or sexual violence by an intimate partner in their lifetime [3]. Moreover, one study that used Demographic and Health Survey (DHS) data from 30 developing countries estimated that 28% of ado- lescents (15–19 years) and 29% of young women (20– 24  years) had experienced physical or sexual intimate partner violence (IPV) [12]. In this study, we examined inequalities in physical or sexual IPV against AYW in sub-Saharan Africa (SSA) using data from the most recent national surveys from 27 countries. Our analyses focused on married or cohabit- ing adolescents and young women aged 15–24 years and compared inequalities in IPV by place of residence, edu- cation and wealth. We also examined IPV variations by AYW’s beliefs and country characteristics such as gender inequality.h Much research on the determinants of IPV against women has been informed by an ecological framework that outlines multiple factors operating at different lev- els—individual, relationship, community, and societal levels—that explain why some groups of people are at greater risk [13–15]. For instance, socioeconomic ine- qualities and socio-cultural norms such as those around male dominance over women contribute to the high prevalence of GBV in SSA [12, 16]. Evidence from a sys- tematic review also shows that individual characteristics such as age, age difference with the partner, and educa- tion level are risk factors of GBV [17]. Moreover, there are various contextual and country-specific drivers of violence in SSA. There is a strong link between pov- erty and violence among young women with those from poor households and communities being at greater risk [18]. Low education, exposure to violence in childhood, unequal power in intimate relationships, and attitudes and norms accepting violence and gender inequality also increase the risk of experiencing IPV and sexual violence [13]. The percentage of AYW reporting IPV in the year before the survey ranged from 6.5% in Comoros to 43.3% in Gabon. Overall, IPV levels were higher in countries in the Central Africa region than other sub-regions. IPV levels varied by place of residence, education and wealth although there was no clear pattern of inequalities. A higher percentage of AYW from rural areas, with lower education and from the poorest households reported IPV. In almost all countries, a higher percentage of AYW who approved wife beating reported IPV compared to their counterparts who disapproved wife beating. IPV is pervasive with substantial variations between and within countries reflecting the role of contextual and structural factors in shaping the vulnerability to IPV. There is a need for comprehensive and multi-sectoral approaches to preventing and responding to IPV against AYW. Background Education was coded into two categories (primary or less, and secondary or more) based on the distribu- tion of AYW’s education. In the majority of the coun- tries included in this analysis, only a small proportion of AYW had no formal education. Age was recoded into two categories: adolescents (15–19  years) and young women (age 20–24  years). Age difference with the partner was categorized into two groups based on the distribution of the data (a difference of more than 5 years vs. a difference of 5 years or less). Demographic and Health Surveys are an important source of data to study cross-national and regional ine- qualities in exposure to IPV because they are nation- ally-representative and use standardized tools that follow ethical and safety recommendations for research on domestic violence against women [24]. Such cross- national and regional comparisons will enable the identi- fication of groups of AYW that are most affected. As the DHS are conducted about every 5 years, they are valuable in monitoring the progress and effectiveness of inter- ventions targeting the protection and empowerment of AYW to prevent violence. Population-based surveys that highlight differences in IPV by wealth index, residence and education and other individual- and community- level determinants of violence against AYW are useful for informing the design and targeting of interventions. In this study, we drew on DHS data to examine inequali- ties in IPV against AYW in SSA and identify groups that experience the highest levels of violence in different contexts. f The main outcome variable was intimate partner vio- lence (physical, sexual and physical or sexual violence) against AYW in the past year. The current prevalence of IPV was defined as the percentage of currently married or cohabiting AYW who reported having experienced at least one act of IPV in the 12 months before the sur- vey. In the DHS, violence information is obtained from ever-married and cohabiting respondents on violence committed by their current and former spouses/partners and by others. Physical IPV is measured using a short- ened, modified version of the Conflict Tactics Scale [26] which asks the respondent if she has ever been—pushed, shaken, slapped, punched with a fist or something that could hurt, kicked, dragged or beaten up, choked, burned on purpose or had something thrown at her [27]. Background Violence against women and girls is a global public health and human rights issue that affects millions of women and girls. According to the World Health Organization approximately one-third of women globally have expe- rienced some form of violence (physical or sexual) from a partner or non-partner in their lifetime [1]. While the effects of gender based violence (GBV) on the physical, mental health and social well-being of women and girls are relatively well-documented [2–4], its health conse- quences continue to be unabated due to the persistent high prevalence. For instance, young women in sub- Saharan Africa (SSA) continue to carry the brunt of high HIV infection due to sexual violence, poverty and social norms around marriage, gender inequalities and harm- ful traditional practices that reinforce unequal power dynamics with young women particularly disadvantaged [5–8]. With the Sustainable Development Goals (SDGs), the international community committed to the achieve- ment of gender equality and elimination of all forms of violence against women and girls by 2030. Promoting gender equality, preventing violence against women and girls (SDG, goal 5) and ensuring responsive and inclusive societies (SDG target 16.1) are far-reaching goals in the SDGs to ensure gender equity [19]. While the relation- ship between gender and violence is complex, evidence indicates that gender inequalities increase the risk of violence by men against women and inhibit the ability of those affected to seek protection [9, 20]. SDG target 17.18 also calls for disaggregated national statistics by income, rural–urban residence, gender and other vari- ables to assess inequalities. Yet, little evidence exists on inequalities in exposure to violence against AYW in SSA. In this regard, the agenda to “leave no one behind” and Countdown to 2030 are well-timed to provide inequality data for the purpose of designing effective interventions to improved gender equity and address violence against AYW in SSA countries [21] since existing evidence of Gender inequalities increase the risk of violence against women and girls and inhibit the ability of those affected to seek protection [9, 10]. Adolescent girls and young women (AYW), particularly those married to older men, and/or married as children or adolescents, may be disproportionately at risk of being exploited Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 3 of 13 interventions are skewed towards high income countries [20, 22, 23]. and the fifth quintile represents the wealthiest 20% [25]. Methods D t We used data from the most recent DHS with the vio- lence against women module. We limited our analyses to data from 27 countries in SSA whose most recent survey was conducted after 2010. Eight countries with surveys were excluded because the violence module was not applied (Guinea, Lesotho, Madagascar, Maurita- nia and Guinea) or only had data from national surveys carried out before 2010 (Ghana, Liberia and Sao Tome). We also included similar number of countries with at least one round of surveys since 2010 for the analysis of AYW’s attitudes towards wife beating. The study popula- tion included married or cohabiting adolescent girls aged 15–19  years and young women aged 20–24  years. We used the United Nations Population Division grouping of countries in SSA into four sub-regions: Central Africa, Eastern Africa, Southern Africa and Western Africa. Table 1 presents the countries included in the study by region and year of survey.hi The DHS collects various proxy indicators of women’s empowerment including attitudes towards wife beating, also named beliefs towards gender based violence. As attitudes towards violence is one of the key predictors of exposure to GBV [15, 28], we examine variation in atti- tudes towards wife beating by the stratifiers. In the DHS, all respondents are asked a series of questions to assess their attitudes to wife-beating. The questions ask whether a husband is justified in hitting or beating his wife in any of the following scenarios; if she goes out without tell- ing him; if she neglects the children; if she argues with him; if she refuses to have sex with him and if she burns the food. A single composite variable ‘disagreement with wife-beating’ was constructed by grouping women into two categories: women who endorse at least one reason for wife-beating and women who reject all reasons of wife-beating. Unlike the IPV, which is administered to a sub-sample of households, the attitudes towards violence data is collected from all women. The equity stratifiers used in this analysis included household wealth quintiles, rural–urban residence, woman’s education, age and age difference with the partner. Accordingly, inequality in this analysis refers to differences in the outcome indicator (IPV against AYW) between two or more sub-groups. In the DHS, the wealth index is coded into five quantiles, however, in our analyses, we compared the two extreme catego- ries (the first quintile vs. the fifth quintile). Background Sexual violence was assessed using the following items: physi- cally forced to have unwanted sex, or forced into other unwanted sexual acts. An affirmative answer to one or more of the items listed in the Conflict Tactics Scale constitutes evidence of physical and sexual violence (see DHS reports, www.dhspr​ogram​.org). We also looked at the prevalence of a combination of the two types of vio- lence (physical or sexual).h Methods D t We analyzed survey data for each country at the national level, to compare the prevalence of IPV (physi- cal or sexual violence) among AYW by the equity strati- fiers—wealth quintiles, education, place of residence, and age. We also examined how attitudes towards wife beating, one of the major predictors of IPV, vary by the stratifiers. Data presented are weighted using the domes- tic violence weight, to adjust for within country sample selection and nonresponse. We use equiplots and charts to visualize inequalities in IPV against AYW by the strati- fiers. Equiplots are used to present intervention cover- age by groups, making it possible to visualize both the level of coverage in each group and the distance between groups, which represents absolute inequality [34]. We also use other relative measures of inequality such as ratios to demonstrate how one sub-group differs from the In addition, we performed an ecological analysis and used multiple data sets to examine the correlation between country IPV prevalence with key societal char- acteristic: Gross National Income per capita extracted from the World Bank [29]; Gender Inequality Index (GII) an index that measures gender inequalities in reproduc- tive health, empowerment and labor force participation, from the UNDP [30]; Fragile States Index—a measure of state fragility and instability—extracted from the 2019 Fragile States Index annual report [31]; urbanization levels from PRB’s world population data sheet [32]; edu- cational attainment and AYW’s beliefs about GBV both extracted from the DHS; and adult male alcohol con- sumption per capita extracted from the WHO data base [33]. Methods D t The first quintile represents the poorest 20% in the population Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 4 of 13 Table 1  List of countries by year of DHS surveys for the intimate partner violence analysis Region Country IPV data Most recent survey Eastern and Southern Africa Eastern Africa Rwanda 2014 2014 Malawi 2015 2015 Kenya 2014 2014 Ethiopia 2016 2016 Zambia 2013 2013 Burundi 2016 2016 Tanzania 2015 2015 Uganda 2016 2016 Mozambique 2015 2015 Comoros 2012 2012 Southern Africa Zimbabwe 2016 2016 Namibia 2013 2013 South Africa 2016 2016 West and Central Africa West Africa Mali 2018 2018 Senegal 2017 2017 Burkina Faso 2010 2010 Sierra Leone 2013 2013 Nigeria 2018 2018 Togo 2013 2013 Cote d’ivoire 2011 2011 Benin 2017 2017 Gambia 2013 2013 Central Africa Congo Brazzaville 2011 Gabon 2012 2012 Cameroon 2011 2011 Angola 2015 2015 Congo DRC 2013 2013 Chad 2014 2014 Table 1  List of countries by year of DHS surveys for the intimate partner violence analysis Table 1  List of countries by year of DHS surveys for the intimate partner violence analysis West and Central Africa Central Africa prevalence of IPV at country level by the stratifiers. The median and the interquartile range were used to sum- marize IPV prevalence at regional and sub-regional levels. We analyzed survey data for each country at the national level, to compare the prevalence of IPV (physi- cal or sexual violence) among AYW by the equity strati- fiers—wealth quintiles, education, place of residence, and age. We also examined how attitudes towards wife beating, one of the major predictors of IPV, vary by the stratifiers. Data presented are weighted using the domes- tic violence weight, to adjust for within country sample selection and nonresponse. We use equiplots and charts to visualize inequalities in IPV against AYW by the strati- fiers. Equiplots are used to present intervention cover- age by groups, making it possible to visualize both the level of coverage in each group and the distance between groups, which represents absolute inequality [34]. We also use other relative measures of inequality such as ratios to demonstrate how one sub-group differs from the prevalence of IPV at country level by the stratifiers. The median and the interquartile range were used to sum- marize IPV prevalence at regional and sub-regional levels. West and Central Africa Violence against adolescents and young women Across the 27 countries, the reported prevalence of phys- ical or sexual IPV ranged from 6.5% in Comoros (2012) to 43.3% in Gabon (2012). It varied widely between the countries. The median prevalence of combined physical or sexual IPV against AYW was 25.2%. The prevalence was highest in Central Africa (39.8%), followed by South- ern Africa (28.4%) respectively (Fig. 1; Additional file 1: Table S1). In 5 of the 27 countries (Gabon, Burundi, DRC Congo, Cameroon, and Sierra Leone) more than 35% of AYW reported experiencing either physical or sex- ual IPV in the 12 months before the survey. Within the sub-regions, large inequalities between countries were observed in Eastern Africa. In East Africa, the prevalence of physical or sexual IPV varied from 36.4% in Burundi to only 6.5% in Comoros (Table 2). A close examination of the prevalence of IPV by the different stratifiers showed no clear overall pattern and little within-country variations in AYW’s experience of physical or sexual IPV in the 12  months preceding the survey (see Additional file  1: Table  S1). For instance, there is no clear pattern in rural–urban inequalities in the prevalence of physical or sexual IPV. In the major- ity of countries (15 of the 27) the prevalence was higher in urban areas although the differences were statistically significant in only three of the 17 countries; Mozam- bique, Angola, and Cote d’Ivoire (Fig. 2). The prevalence of physical IPV varied from 5.2% in Comoros (2012) to 38.8% in Gabon (2012). The preva- lence of physical IPV was higher in the Central Africa region where AYW in three of the five countries (Congo DRC, Cameroon and Gabon) reported a prevalence of over 30%. The reported prevalence of sexual IPV ranged from 1.5% in Burkina Faso (2010) to 23.9% in DRC Congo In 11 of the 27 countries, a higher proportion of AYW from rural areas experienced physical or sexual IPV in the past year with differences being statistically signifi- cant in three countries (Burundi, Ethiopia and Uganda). The pattern of inequality by education was similarly mixed. In 14 out of 27 countries, the prevalence of IPV was higher among AYW with primary education, and the differences were significant in four countries (Uganda, Burundi, Tanzania and Malawi). Data analysis Data were analyzed using STATA software version 14 (StataCorp, 2015). We used proportions to estimate Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 5 of 13 (2013). The prevalence of sexual IPV was highest in Cen- tral Africa (16.1%), followed by Southern Africa (10.4%), Eastern Africa (10.1%) and West Africa (7.7%) respec- tively (Fig. 1). Outside Central Africa (DRC Congo, Gabon and Cameroon) more than one sixth of AYW in countries such as Burundi, Uganda and Malawi reported sexual violence (Table 2). (2013). The prevalence of sexual IPV was highest in Cen- tral Africa (16.1%), followed by Southern Africa (10.4%), Eastern Africa (10.1%) and West Africa (7.7%) respec- tively (Fig. 1). Outside Central Africa (DRC Congo, Gabon and Cameroon) more than one sixth of AYW in countries such as Burundi, Uganda and Malawi reported sexual violence (Table 2). other. Moreover, proportions of key outcome indicators are presented with their confidence intervals in tables. Accordingly, significant differences between sub-groups are determined based on non-overlapping confidence intervals. To assess the association of IPV with selected societal determinants (at the country level) we used scatter plots, Pearson correlation coefficient and linear regression analyses to test associations with IPV preva- lence at the 5% level. For some of the indicators, we did log transformations to reduce the influence of outliers. There is a high correlation between the prevalence of sexual IPV and physical IPV in SSA (Pearson’s correla- tion, coefficient, r = 0.66), and several countries with high physical IPV (DRC, Gabon, Burundi, Uganda) also reported high levels of sexual IPV. In a few countries, for instance in Burundi and Malawi, AYW reported nearly equal or higher level of sexual IPV than physical IPV. On the other hand, there are a few countries with very low sexual IPV overall; AYW in Burkina Faso, Comoros, Mozambique and Gambia reported sexual IPV preva- lence of about 2% or less (Table 2). Violence against adolescents and young women Physical or sexual IPV prevalence was higher among AYW with secondary and above education in 11 countries but the differences were not statistically significant except in Mozambique (Addi- tional file 1: Table S1). 0 5 10 15 20 25 30 35 40 45 50 IPV (%) Median Central Eastern Southern West Fig. 1  Median prevalence of physical or sexual intimate partner violence against AYW by region, SSA i The patterns by wealth quintile shows that in about three-fifth of countries, a higher proportion of AYW from the poorest wealth quintile reported physical or sexual IPV although the differences were significant for only four countries (Uganda, Tanzania, Rwanda, and Gabon). In nine countries, the reported prevalence of physical or sexual IPV was higher among AYW from the richest wealth quintile. However, the difference was statistically significant in four countries; Mozambique, Senegal, Sierra Leone, and Cameroon (Additional file 1: Table S1). Fig. 1  Median prevalence of physical or sexual intimate partner violence against AYW by region, SSA Wado et al. Violence against adolescents and young women However, in many coun- tries, IPV prevalence was higher among AYW whose age difference with the partner was less than 5 years and the differences were statistically Significant in three coun- tries; Cameroon, Burundi, Angola (Additional file  1: Table S1). i The prevalence of physical or sexual IPV varied mark- edly by attitudes towards wife beating. As shown in Fig. 3, a higher proportion of AYW who approved wife- beating for any reason reported physical or sexual IPV than their counterparts who disapproved wife beating for any reason. The differences were significant at the 5% level in 15 of the 27 countries and remarkable dis- parities were seen in countries such as Namibia, Mali, Violence against adolescents and young women Reprod Health 2021, 18(Suppl 1):119 Page 6 of 13 Table 2  Percentage of adolescents and young women 15–24 years who experienced intimate partner violence, national DHS surveys, 2010–2018 (in parenthesis 95% confidence interval) Country Physical and or sexual IPV Physical IPV Sexual IPV N Gabon 43.4 [36.7,50.3] 38.8 [32.3,45.6] 16.1 [11.6,21.9] 573 Congo DRC 40.9 [36.8,45.1] 32.6 [29.1,36.4] 23.9 [20.0,28.3] 1136 Cameroon 39.8 [36.1,43.5] 33.4 [29.7,37.4] 16.3 [13.6,19.5] 943 Sierra Leone 36.7 [32.2,41.4] 32.9 [28.7,37.4] 8.6 [6.3,11.7] 681 Burundi 36.4 [33.4,39.6] 23.6 [20.9,26.5] 23.2 [20.6,26.1] 1010 Uganda 32.5 [29.6,35.6] 23.9 [21.5,26.4] 18.3 [16.1,20.7] 1675 Tanzania 32.4 [29.2,35.7] 28.9 [25.9,32.0] 11.6 [9.7,13.8] 1491 Zambia 31.8 [29.0,34.8] 26.2 [23.5,29.0] 14.9 [13.0,17.0] 1694 Angola 30.6 [27.2,34.2] 28.5 [25.3,31.9] 9.8 [8.1,11.8] 2086 Namibia 29.1 [20.9,38.9] 26.7 [19.2,36.0] 10.1 [5.5,17.9] 125 Zimbabwe 28.4 [25.2,31.9] 21.5 [18.7,24.6] 12.9 [10.7,15.5] 1081 Malawi 26.1 [23.3,29.1] 15.8 [13.6,18.4] 16.9 [14.5,19.6] 1265 Côte d’Ivoire 25.3 [22.2,28.8] 24.4 [21.3,27.7] 5.9 [4.2,8.1] 960 Kenya 25.2 [21.1,29.8] 21.1 [17.2,25.6] 9.1 [6.5,12.6] 676 Mali 24.7 [20.6,29.2] 20.3 [17.0,24.1] 12.9 [10.3,16.1] 847 Rwanda 23.9 [18.9,29.8] 19.1 [14.7,24.4] 8.4 [5.5,12.8] 219 Ethiopia 21.7 [17.8,26.1] 18.7 [14.9,23.1] 8.4 [5.8,12.0] 814 Chad 18.2 [14.9,22.2] 14.2 [11.5,17.3] 8.7 [6.4,11.9] 948 Nigeria 17.0 [14.8,19.4] 12.8 [10.9,14.9] 8.4 [6.7,10.4] 1530 South Africa 15.8 [9.8,24.5] 12.2 [7.2,19.8] 8.0 [3.6,16.8] 185 Togo 15.1 [12.5,18.0] 11.9 [9.6,14.5] 7.1 [5.3,9.4] 785 Benin 14.2 [11.5,17.3] 9.1 [7.0,11.7] 7.7 [5.7,10.3] 832 Senegal 14.1 [10.3,19.0] 10.4 [6.9,15.2] 8.2 [5.4,12.2] 504 Mozambique 12.4 [9.6,16.0] 11.8 [9.0,15.3] 2.1 [1.2,3.5] 661 Burkina Faso 8.4 [7.1,9.9] 7.8 [6.6,9.1] 1.5 [1.0,2.2] 2475 Gambia 7.1 [5.2,9.6] 5.3 [3.7,7.4] 2.2 [1.2,4.0] 869 Comoros 6.5 [4.4,9.6] 5.2 [3.5,7.7] 2.0 [1.0,4.1] 479 We also examined inequalities in physical or sexual IPV by AYW’s attitudes towards wife beating, age difference between the partner and AYW’s age. Overall, the preva- lence of IPV was higher among young women than ado- lescents, although the differences were significant in only four countries (Nigeria, Tanzania, Angola and Burkina Faso). In the four countries (Zimbabwe, Malawi, Benin and Senegal) where a greater proportion of adolescents than young women reported IPV, the differences were not statistically significant. Angola, Burundi and Uganda. In Namibia for instance, the proportion of AYW who reported physical or sexual IPV varied from 13.3% among those who disapproved wife-beating to 50.2% among those who accepted wife- beating. The prevalence of physical or sexual violence did not vary significantly by the age difference between the respondent and her partner. Societal characteristics and reporting IPV While there were observable variations between coun- tries, the within countries variation by the stratifiers was relatively inconsistent. Key questions emerging from this observation are: what explains the large Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 7 of 13 Fig. 2  Percentages of AYW reporting physical or sexual intimate partner violence by stratifiers, SSA the 27 countries in the analysis. Moreover, wide within- country inequalities in attitudes towards wife-beating existed by place of residence, wealth quintile and educa- tion. In almost all the 27 countries, a higher proportion of AYW residing in urban areas rejected wife-beating com- pared to their rural counterparts and the differences were statistically significant for 18 countries. Wider rural– urban differences of over 20 or more percentage points were observed in countries such as Ethiopia, Nigeria, Gambia and Namibia (Fig. 5, Additional file 3: Table S3). variation between countries? We examined the corre- lation between reporting IPV at the country level and selected societal characteristics—Gender Inequality Index (GII), educational attainment (proportion of AYW with secondary and above education), GNP per capita, urbanization levels, adult male alcohol consumption per capita and the prevalence of negative attitudes towards wife beating. All bivariate analyses showed no or weak associations between the societal characteristics and IPV among AYW. Moderate or weak correlations were observed with adult male alcohol consumption per capita (Pearson correlation coefficient, r = 0.48) and the preva- lence of negative attitudes towards wife beating (Pearson correlation coefficient, r = 0.38). There was no or very weak association with the GII, GNP per capita, Fragile States Index, and level of education of young people in the society (see Fig. 4 and Additional file 2: Table S2). i The disparity by wealth quintile was also remarkable. In 17 of the 27 countries, the proportion of AYW who rejected wife-beating was significantly higher among the richest quintile compared to the poorest. Large dispari- ties by wealth were observed in Ghana, Nigeria, Angola, Ethiopia, Namibia, Zambia and Senegal. Inequalities by education were also notable with a higher proportion of AYW with secondary education rejecting wife-beating. The differences between AYW with secondary education and those with primary or lower education were statis- tically significant for 17 countries (see Additional file 3: Table S3). Attitude towards wife beatingh The median proportion of AYW who rejected all rea- sons of wife-beating ranged from 17.9% in Mali (2018) to 92.5% (2016) in South Africa with a median of 47.7% in Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 8 of 13 Fig. 3  Percentages of AYW reporting physical or sexual IPV by empowerment measures, SSA r=0.3831 r=0.4809 Fig. 4  Scatterplot matrix of the correlation between the societal determinant and IPV Fig. 3  Percentages of AYW reporting physical or sexual IPV by empowerment measures, SSA Fig. 3  Percentages of AYW reporting physical or sexual IPV by empowerment measures, SSA r=0.3831 r=0.4809 Fig. 4  Scatterplot matrix of the correlation between the societal determinant and IPV Fig. 4 Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 9 of 13 Fig. 5  Percentage of adolescents and young adult women who reject wife-beating by stratifiers observable regional and between-country variation, the reporting of IPV by AYW was not strongly corre- lated with societal characteristics such as the Gender Inequality Index, GNP per capita, Fragile States Index, or aggregate levels of AYW educational attainment at the national level. It was moderately correlated with male adult alcohol consumption per capita and approval of wife beating at the national level. The poor correla- tion with Gender Inequality Index is unexpected. Some studies have reported a moderate correlation between the index and IPV among women aged 15–49 in low and middle income countries [36, 37]. Our study shows that conventional indicators of socio-economic develop- ment may not explain large inter-country differences in reported IPV against AYW in the region (Fig. 5). Discussion As documented by several studies [15, 16] social norms play a significant role with a large pro- portion of AYW from the poorest households viewing spousal violence as a normal and justified occurrence in marriage. Traditional beliefs that men have a right to control women and that increase vulnerability to violence are more common among the poorest, less educated and rural adolescents in SSA [9, 20, 37, 43]. Consistent with the findings of our ecological analysis, the association between partner alcohol use and increased risk of IPV victimization at the individual level has been reported by various studies [44, 45]. These findings have implications for the targeting of violence prevention interventions aimed at promoting more equitable gender norms.i respectively. In particular, in many countries with a very high prevalence of IPV (Gabon, DRC Congo, Uganda, Burundi and others) a higher proportion of AYW from the poorest wealth quintile experienced IPV compared to their counterparts. Previous studies have highlighted associations between low education, poverty and violence among young women and noted that young women from poor households may have low decision-making abili- ties, resources and empowerment, which increases their vulnerability to violence [12, 38]. Thus, interventions that broaden women’s access to economic resources and opportunities may help empower women and help reduce the risk of IPV. The finding that in countries like Angola, Cote d’ivoire, Mali, Mozambique and Burkina Faso, more educated, urban and wealthier AYW were more likely to report IPV is noteworthy. These AYW may challenge the traditional status quo and may be considered more “empowered”. Their male partners may therefore resort to using vio- lence to maintain a dominant position in contexts where male dominance is normative [8, 15]. “Empowered” AYW may also be more willing to disclose IPV [39]. However, these inconsistent patterns also suggest that violence against women and girls is pervasive across all socio-eco- nomic backgrounds [3, 20, 23].fl While the regional differences in reporting may reflect important cultural, political, or religious differences [40, 41] differential reporting by women of different socio-economic groups within a country is also possi- ble depending on cultural and social norms that under- lie the acceptance of violence in each settings [37, 40]. The culture of silence that affects the reporting and what constitutes violence varies across cultures and can make comparability difficult. Discussion Findings from these analyses indicate that IPV against AYW is a pervasive problem in SSA with large between and within-country variations. Across the 27 countries included in our analyses, more than one in four AYW reported physical or sexual IPV in the 12 months before the surveys. National prevalence of physical or sexual IPV varied from 6.5% in Comoros to 43.3% in Gabon. Over- all, the prevalence of physical or sexual IPV was higher in Central Africa region compared to other sub-regions. In countries such as Gabon, Cameroon, Sierra Leone and Congo DRC over one-third of AYW reported experienc- ing physical or sexual IPV in the past year. Many of these countries are in conflict or post conflict situations that might have exacerbated pre-existing patterns of violence against women and girls as conflicts can result in higher levels of violence against women and girls, including arbi- trary killings, torture, sexual violence and forced mar- riage [35].f While the between countries variation in the preva- lence of IPV is large, the analysis demonstrated that the within country inequalities by the stratifiers are not con- sistent. However, in close to half of the countries, AYW residing in rural areas, with lower education and those from the poorest wealth quintile experienced more IPV than their counter-parts from urban areas, with higher education and those from the richest wealth quintiles, Previous studies show that differences in contextual and structural factors may explain some of the differ- ences observed in IPV prevalence between countries [12, 20, 23]. Our analysis, however, showed that despite Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 10 of 13 Page 10 of 13 specific and vary among and within countries [14, 42]. The disaggregated analysis showed greater inequality in IPV by AYW’s attitudes towards wife beating, with lower IPV levels among those who reject wife beating for any reason compared to those who accept wife beating. In most countries, trend analysis of DHS data shows that attitudes towards wife beating are changing. However, we found that approval of wife beating is more common among rural dwellers, those from poor households, and those with lower levels of education. Similar findings were recently reported in a publication describing the most vulnerable groups in low and middle-income coun- tries that high IPV prevalence among those who accept wife beating [37]. Discussion Evidences show that levels of IPV may be under reported due to fear of retaliation by part- ners, shame and stigma, lack of awareness of available services or access to such services among other reasons [2, 40]. Interestingly, as approval of wife beating is asso- ciated with IPV prevalence, it is possible that AYW who accept wife beating are more willing to disclose experi- ence of IPV than their counterparts who disapprove wife beating. However, while the magnitude of under- reporting is unknown, the DHS violence against women module uses validated and standardized questions that are implemented following WHO recommendations of studies on violence against women and girls to improve data quality, protect the safety of respondents and enable comparability across countries. Overall, the findings demonstrate that IPV is pervasive among AYW, with substantial variation between coun- tries and regions reflecting the role of contextual and structural factors in shaping vulnerability to IPV. The lack of consistent pattern of inequalities by the stratifiers within countries shows that IPV against women and girls cuts across socio-economic boundaries suggesting the need for comprehensive and multi-sectoral approaches to preventing and responding to IPV in line with the ecological framework [46, 47]. Moreover, the observed variation by attitudes towards wife-beating shows that promoting gender equitable norms from early childhood through multi-sectoral strategies (including school-based interventions that address gender norms and attitudes from younger ages, and community interventions that can empower women such as microfinance schemes; and media interventions to increase public awareness) can help in reducing violence against women and girls [9, 20, 48]. However, the effectiveness of IPV mitigation actions and care services in SSA remain to be evaluated. Globally, efforts to prevent and respond to cases of violence against women and girls have increased in the last few decades. Many countries in SSA have adopted laws and policies addressing different forms of violence, including rape, child sexual abuse, and domestic and/or intimate partner violence. However, the implementation The ecological model, which is most widely used for understanding the causes of violence, proposes that vio- lence is a result of factors operating at various levels. While there is limited research on community and soci- etal influences, many of the factors identified are context Wado et al. Availability of data and materials The dataset used for the current study is available for free from https​://dhspr​ ogram​.com/data/avail​able-datas​ets.cfm and can be provided up on request by the corresponding author. Consent for publication Not required. Consent for publication Not required. Discussion Reprod Health 2021, 18(Suppl 1):119 Page 11 of 13 Page 11 of 13 of these laws is hampered by weak institutional capaci- ties and limited reporting by victims of violence [30, 48]. Nonetheless, there is an increasing availability of data and lessons from programmatic responses that can be used to scale up prevention and response mechanisms to attain the SDG goal of eliminating all forms of violence against women and girls by 2030. Funding The Countdown to 2030 for Women’s, Children’s and Adolescents’ Health is the recipient of an investment of the Bill & Melinda Gates Foundation. This study also benefitted from funding of Canada Partnership for Women’s and Children’s Health. The contents of this research article only reflect the authors’ opinions but do not show interest/s of either of the organizations involved in the funding. Acknowledgements The authors gratefully acknowledge Ties Boerma for the review and com- ments he provided on the various drafts of the manuscript. g g y This study is not without limitations. It is important to note that rates of IPV may be under-reported due to cultural and social norms that underlie the acceptance of violence. Although the DHS violence against women module is implemented following WHO recommenda- tions of research, the fact that the module is implemented within a wide range of health modules means that women are likely to under report due to social desirability bias [49]. Evidence from countries with two or more sur- veys with the module (e.g., Nigeria, Malawi, Kenya and Rwanda) also shows that prevalence of IPV against AYW between consecutive surveys were not consistent indica- tive of reporting issues. Moreover, as these modules are implemented in a sub-sample of households and individ- uals, the sample of married adolescents is relatively small which may have resulted in wide confidence intervals for some parameters. But, we have excluded surveys with less than 30 observations from the analysis. Abbreviations AYW​: Adolescents and young women; DHS: Demographic and Health Surveys; GBV: Gender based violence; IPV: Intimate partner violence; SDGs: Sustainable Development Goals; SSA: Sub-Saharan Africa; GII: Gender Inequality Index; GNP: Gross National Product. Competing interests h h d l h p g The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. About this supplement This articles has been published as part of Reproductive Health Volume 18 Supplement 1 2021: ASRH for all in SSA: are inequalities reducing?. The full contents of the supplement are available at https​://repro​ducti​ve-healt​h-journ​ al.biome​dcent​ral.com/artic​les/suppl​ement​s/volum​e-18-suppl​ement​-1. Supplementary Information The online version contains supplementary material available at https​://doi. org/10.1186/s1297​8-021-01077​-z. The online version contains supplem org/10.1186/s1297​8-021-01077​-z. Authors’ contributions YDW, MK, AM, MC and CF conceptualized the study. MK and YDW conducted data analysis and interpretation. AM and MYI performed literature review. YDW wrote the initial draft of the manuscript. CK, CC, AB and DM participated in interpretation of findings and revised the manuscript. All authors read and approved the final manuscript. Author details 1 1 African Population and Health Research Center, Nairobi, Kenya. 2 Aga Khan University, Nairobi, Kenya. 3 Department of Epidemiology and Medical Sta- tistics, University of Ibadan, Ibadan, Nigeria. 4 International Centre for Equity in Health, Universidade Federal de Pelotas, Pelotas, RS, Brazil. Received: 10 January 2021 Accepted: 11 January 2021 Published: 17 June 2021 Ethics approval and consent to participate Overall, the findings demonstrate that IPV is pervasive among AYW, with substantial variation between coun- tries and regions reflecting the role of contextual and structural factors in shaping vulnerability to IPV. The lack of consistent pattern of inequalities by the strati- fiers within countries shows that IPV against AYW cuts across socio-economic boundaries suggesting the need for comprehensive and multi-sectoral approaches to preventing and responding to IPV. The between country variation however is poorly measured by conventional development indicators. On the other hand, the observed variation by attitudes towards wife-beating show that promoting gender equitable norms from early childhood helps in reducing violence against women and girls. Ethical approval for the DHS is obtained from national ethics review boards through the national institutions involved in the survey and from ICF Interna- tional. The description of consent process is available from https​://www.dhspr​ ogram​.com/What-We-Do/Prote​cting​-the-Priva​cy-of-DHS-Surve​y-Respo​ndent​ s.cfm. 2004/11/20). 29. WorldBank. GNI per capita, Atlas method (current US$), World Bank national accounts data, and OECD National Accounts data files. The World Bank 2019; Accessed 7 Feb 2020. 7. Shannon K, Leiter K, Phaladze N, Hlanze Z, Tsai AC, et al. Gender inequity norms are associated with increased male-perpetrated rape and sexual risks for HIV infection in Botswana and Swaziland. PLoS ONE. 2012;7:e28739. 30. UNDP. Violence against women, a cause and consequence of inequality, United Nations Development Programme, 2019 Accessed on 21 Jan 2019. Accessed 7 Feb 2020. 8. Jewkes RK, Dunkle K, Nduna M, Shai N. 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Mathur S, Okal J, Musheke M, Pilgrim N, Kishor S, et al. High rates of sexual violence by both intimate and non-intimate partners experienced by adolescent girls and young women in Kenya and Zambia: find- ings around violence and other negative health outcomes. PLoS ONE. 2018;13(9):e0203929. 27. Kishor S, Kierston J. Profiling domestic violence—a multi-country study. Calverton: ORC Macro.; 2004. https​://dhspr​ogram​.com/pubs/pdf/od31/ od31.pdf. 5. Jewkes R, Morrell R. Gender and sexuality: emerging perspectives from the heterosexual epidemic in South Africa and implications for HIV risk and prevention. J Int AIDS Soc. 2010;13:6 (Epub 2010/02/26). 28. Dasgupta S. Attitudes about wife-beating and incidence of domestic violence in India: an instrumental variables analysis. J Fam Econ Issues. 2019;40(4):647–57. p p 6. Pettifor AE, Measham DM, Rees HV, Padian NS. Sexual power and HIV risk, South Africa. Emerg Infect Dis. 2004;10(11):1996–2004 (Epub 2004/11/20). Page 13 of 13 Wado et al. Reprod Health 2021, 18(Suppl 1):119 the international conference on population and development. J Adolesc Health. 2019;65(6S):S16–40 (Epub 2019/11/26). 49. Adedimeji AA, Omololu FO, Odutolu O. HIV risk perception and constraints to protective behaviour among young slum dwellers in Ibadan, Nigeria. J Health Popul Nutr. 2007;25(2):146–57 (Epub 2007/11/08). Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Wado et al. Reprod Health 2021, 18(Suppl 1):119 Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 13 of 13 Page 13 of 13 2019/07/03). The political, research, programmatic, and social responses to adolescent sexual and reproductive health and rights in the 25 years since 24. WHO. Putting women’s safety first: ethical and safety recommendations for research on domestic violence against women. Geneva: World Health Organization; 1999. Report No, 1999 Contract No.: WHO/EIP/GPE/99.2, Accessed 5 Feb 2020. 24. WHO. Putting women’s safety first: ethical and safety recommendations for research on domestic violence against women. Geneva: World Health Organization; 1999. Report No, 1999 Contract No.: WHO/EIP/GPE/99.2, Accessed 5 Feb 2020. Wado et al. Reprod Health 2021, 18(Suppl 1):119 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W4361263986
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Supplementary Figure 4 from Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate &lt;i&gt;mir-34a, mir-34b,&lt;/i&gt; and &lt;i&gt;mir-34c&lt;/i&gt; Expression, and Induce Senescence
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30 40 50 7 days 14 days t Senescence NT Nutlin-3a Nutlin-3b 0 10 20 Percent 30 40 50 7 days 14 days t Senescence NT Nutlin-3a Nutlin-3b 0 10 20 Percent
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interactions of model cationic drug with newly synthesized starch derivatives
ADMET & DMPK
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Keywords potato starch, adsorption, interaction, methylene blue 1Department of Physical Chemistry and Biophysics, Wrocław Medical University, Borowska 211A, 50-556 Wrocław, Poland 2Department of Food Storage and Technology, Faculty of Biotechnology and Food Science, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37, 51-630 Wrocław, Poland *Corresponding Author: E-mail: witold.musial@umw.edu.pl; Tel.: + 48-717-840-228 Received: June 26 2023; Revised: September 8 2023; Published: September 20 2023 *Corresponding Author: E-mail: witold.musial@umw.edu.pl; Tel.: + 48-717-840-228 *Corresponding Author: E-mail: witold.musial@umw.edu.pl; Tel.: + 48-717-840-228 Received: June 26, 2023; Revised: September 8, 2023; Published: September 20, 2023 Received: June 26, 2023; Revised: September 8, 2023; Published: September 20, 2023 Abstract Background and purpose: The aim of the work was to compare the interactions of three newly synthesized non-toxic starch derivatives, with varied anionic and non-ionic functional groups with methylene blue (MB) as a model cationic drug, and selection of starch derivative with highest affinity to the MB. Experimental approach: The native potato starch (SN), modified via acetylation (SM1), esterification and crosslinking (SM2) and crosslinking (SM3), was evaluated in MB adsorption studies and assessed by FTIR, PXRD, and DSC. Key results: The adsorption of MB on SM2 and SM3 matched the BET isotherm model, which confirmed physisorption on the low-porous surface. In the case of SM1, adsorption took place via electrostatic attraction between the heterogeneous adsorbent surface and the adsorbate, as demonstrated by the Freundlich plot. The FTIR confirmed vibrations assigned to N=C stretching bonds at 1600 cm-1 in the case of MB adsorbed on the SN and SM2. The most intense PXRD peaks belonged to SN and the least to SM2. In the DSC study, the thermal stability via ΔT was assessed, with SM2 of lowest ΔT value (179.8 °C). Conclusion: SM2 presented the best adsorption capacity, followed by SM3 and the weakest SM1. The interactions were confirmed in the adsorption studies and may reflect applications of the modified starches as drug carriers. In the FTIR study, a probable interaction between the OH- groups of SM2 and N+ of MB was revealed. The most amorphous struc- ture was shown for SM2, which was correlated with the lowest thermal stability provided by the DSC study. ©2023 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). doi: https://doi.org/10.5599/admet.1950 Original scientific paper ADMET & DMPK 11(3) (2023) 387-407; https://doi.org/10.5599/admet.1950 ADMET & DMPK 11(3) (2023) 387-407; https://doi.org/10.5599/admet.1950 Introduction There are numerous newly developed carriers for therapeutic substances [1-3]. They are often completely new synthetic polymers [4,5]. Among polymers of plant origin, there are natural examples of potential carriers [6-10]. Wheat, maize and potato starch are available in large quantities from natural sources [11-13]. So far, starch has been modified by Utomo, Odeniyi, Singh et al. [14-16], among others. Previous publications have presented new methods for obtaining a number of starch derivatives [17-19] and proposed the potential use of some of them, including acetylated starch [20,21] and starch citrate [22]. The figure below shows the structural models of the modified potato starches created by the team from the University of Environmental and Life Science (Wrocław, Poland) (Figures 1a,b,c). doi: https://doi.org/10.5599/admet.1950 387 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. a b c d Figure 1. Structural models of the adsorbates as: a - starch acetate (SM1), b - starch citrate (SM2), c - starch diphosphate (SM3) and the d - adsorbent methylene blue tautomers (MB). b a d c c Figure 1. Structural models of the adsorbates as: a - starch acetate (SM1), b - starch citrate (SM2), c - starch diphosphate (SM3) and the d - adsorbent methylene blue tautomers (MB). Some drugs have a 1,4-thiazine ring, which is included in the structure of methylene blue (MB). The study that used the similarity between MB and other molecules was performed on acridine and phenothiazine derivatives for anti-prion therapy [23]. Selected drugs include the 1,4-thiazine ring: phenothiazine, which has antiparasitic, antiseptic and antioxidant effects [24], promethazine, which has antihistamine and anti-allergic effects [25], chlorpromazine and [26] thioridazine - as psychotropic drugs. The ionic interaction between the promethazine and Eudragit® was ascribed to the electrostatic interaction between the drug and ammonium groups in the polymer backbone [27]. The interactions of chlorpromazine with cyclodextrin were realized via ionic bonds between the ionized amine group of chlorpromazine and anionic group of cyclodextrin sulfate [28]. Another study, which exploited cyclic voltammetry, interpreted the interactions of chlorpromazine and thioridazine with bovine serum albumin nanoparticles as a result of hydrophobic interaction [29]. Therefore, research has been undertaken on MB as a model drug representing the structural features of some active pharmaceutical ingredients (API). These studies may determine whether similar drugs could adsorb on modified starch. Preparation of citrate starch (SM2) Native potato starch (SN) was esterified with citric acid. 10 g of citric acid per 100 g of starch dry matter was dissolved in 90 mL of water and thoroughly mixed. The resulting starch paste was left for 12 h at room temperature and then dried in an air dryer (Memmert, Germany) at 50 °C for 12 h. The starches were calcined for 3 h at 100 °C. The sample was rinsed three times with ethyl alcohol with a concentration of 95 mL of ethanol per 100 mL of solution, and each time, the solution was poured over the sediment. The washed precipitate was dried in an air dryer at 30 °C for 12 h. The obtained citrate starch was hardened at a temperature 2 degrees below the gelatinization temperature (determined by DSC), dried in an air dryer at 30 °C, ground in a laboratory mill and passed through a sieve with a mesh size of 400 μm [17]. Preparation of diphosphate starch (SM3) Native potato starch (SN) (94.6 wt.%), sodium trimetaphosphate (1.2 wt.%), sodium carbonate (2.1 wt.%) and sodium chloride (2.1 wt.%) were introduced into the reaction vessel, 500 mL of distilled water at 45 °C was added. The mixture was adjusted to pH 10.5 with a 3 % NaOH solution. It was kept at 45 °C for 30 minutes with constant stirring, then neutralized with 8 % HCl to pH 6.5-6.8. The cross-linked starch was washed several times with distilled water on a funnel under vacuum and dried for 48 h at 25 °C. After obtaining starch diphosphate, it was hardened for 24 h at 2 degrees below the gelatinization temperature, dried in an air dryer at 30 °C, milled and sieved through a sieve with a mesh size of 200 μm. Preparation of the modified starches The thermal and chemical modifications of native potato starch were conducted and resulted in acetylation (SM1), esterification and crosslinking (SM2), and crosslinking (SM3). Preparation of acetylated starch (SM1) The fractions of starch particles with an average volume moment diameter D [3,4] equal to 39.1 and 61.7 µm were separated from the native potato starch (SN) by measuring the volume diameter (Malvern laser particle size analyzer). Larger starch particles were acetylated with acetic anhydride in the amount of 13 cm3 /100 g of starch. After drying the acetylated starch, an initial gelatinization temperature of 49.17 °C was determined by differential scanning calorimeter Flash DSC (Mettler Toledo, Poland). The acetylated starch has been stirred for 24 h at 48 °C. It was then washed three times with five-liter portions of distilled water and separated from the slurry using the Contifuge Stratos (Heraeus, Germany) flow centrifuge. The obtained starch was dried for 24 h in an air dryer at 30 °C. Materials The following materials were employed in the study: methylene blue (MB, Reko, Dzierżoniów, Poland) - a model active pharmaceutical ingredient (API), native potato starch (SN) (PPZ, Niechlów, Poland), modified potato starches (starch acetate - SM1, starch citrate - SM2 and starch diphosphate - SM3) prepared according to below-described methods. Introduction To date, studies have analyzed the adsorption of MB and other dyes on activated carbon, minerals of natural origin such as perlite, clay and its derivatives, bentonite [30,31], and agricultural solid wastes [32]. In summary, the important group that led to the intermolecular activity may be the ammonia group, which specifically interacts with anionic functionals. Because of the wide potential application of modified potato starch [33-36], we decided to compare the adsorption capacity of selected starch derivatives, characterized by a distinct negative charge of carboxyl and phosphate groups, or a characteristic ester group, towards methylene blue as a cationic substance. The aim of the work was the evaluation of the prospective pharmaceutical applicability of three newly synthesized non-toxic starch derivatives, assessed via analysis of interactions of the starches, modified by varied acidic and non-ionic functional groups, with methylene blue, as a model cationic drug. The main question was the selection of the starch derivative with the highest affinity to the MB molecule. The possible interactions were evaluated in the terms of spectroscopic structural studies, as well as thermal assessments. The interactions, which may lead to drug release prolongation, were practically confirmed in the adsorption studies, which may reflect use of the above-mentioned modified starches as drug carriers for topical application, with retardant and prolonging effect against drug release process. 388 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives Model cationic drug and newly synthesized starch derivatives Powder X-ray diffraction (PXRD) analysis Powder X-ray diffraction (PXRD) analysis The analysis of starch was supplemented by X-ray diffraction measurements (PXRD). The powder PXRD data were recorded on a Bruker D2 PHASER diffractometer (Bruker AXS, Karlsruhe, Germany) with a Lynxeye detector using Cu Kα radiation (0.15418 nm). All samples were measured at 295 K with 3.0 mm slit and 1.0 mm shutter. Diffractograms were obtained between 7.5° and 40° (2) (step size of 0.02° (2) and 0.25 s per step). The X-ray generator operated at 30 kV and 10 mA. The PXRD patterns were processed using the software Diffrac.Eva V 3.2. (Bruker AXS). The percentage of crystallinity and amorphousness of the tested starch samples in relation to SN was determined. Spectroscopic evaluation Fourier transform infrared spectroscopy (FTIR) Fourier-transform infrared spectroscopy (FTIR) and attenuated total reflectance (ATR) appetizer (Nicolet 380 FTIR, Thermo Scientific, Waltham, MA, USA) with OMNIC ™ software were used to determine possible interactions between MB particles and starch. The formulations of MB adsorbed on every 50 mg of starch (SN, SM1, SM2 and SM3) were dried at 40 °C and compared with a physical mixture of MB and starch in a weight ratio of 1:10 and with the standards of the pure substances. The spectra of powders were recorded at wavelengths of 400 to 4000 cm-1 at 32 scans per sample and a resolution of 4 cm−1. Determination of the potential number of functional groups of modified starches Based on the recipe for preparing individual modified starches, the mass values of substrates attached to native starch (SN), such as acetic anhydride, trimethaphosphate and citric acid, were converted to moles. The converted values per gram of SN are given in Table 1. doi: https://doi.org/10.5599/admet.1950 389 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. Table 1. Number of moles of individual functional groups of modified starches per 1 g of SN. Modified starch Concentration, mmol g-1 Carbonyl groups Carboxyl groups Phosphate groups SM1 2.74 - - SM2 - 1.56 - SM3 - - 0.12 Spectroscopic evaluation Table 1. Number of moles of individual functional groups of modified starches per 1 g of SN. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC 214 Polyma, Netzsch, Selb, Germany) was performed to investigate the samples of dried adsorbed MB on 50 mg starches (SN, SM1, SM2, SM3), their physical mixtures of 1:10 w/w and pure ingredients. The grated samples of 3 to 5 mg were explored in aluminium pans with lids under a nitrogen atmosphere, with a flow rate of 50 mL min-1. The thermograms were recorded at a constant heating rate of 5 °C min-1 in the temperature range from 0 to 350 °C. Determination of the effect of MB adsorption on the pH of starch Determination of the effect of MB adsorption on the pH of starch The effect of MB adsorption on starch pH was investigated. The suspensions of pure starch in distilled water and suspensions of starch with adsorbed MB were prepared according to adsorption conditions (50 mg starch per 40 mL of water). pH measurements were taken after 1 and 72 h at 22±2 °C. Each measurement was taken three times. An ANOVA test with α = 0.05 was performed for these measurements. Evaluation of the adsorption kinetics of MB on the modified starches 1/ F s = n tq k C 1 a n = F ln b k = ( ) m L s L s = 1 q k C q k C + m 1 a q = m L 1 b q k = ( ) ( ) ( ) ( ) ( ) ( ) m s BET s s s s BET L BET L BET s = 1- 1- + q C k q C k C k C k     ( ) ( ) ( ) ( ) BET L BET L BET s m BET s 2- = k k k a q k ( ) ( ) ( ) BET s BET L m BET s -2 = k k b k q ( ) m BET s 1 = c k q 2 2 1 = + t t t q k q q The values of q and qt were calculated by the equations (1) and (2): 1/ F s = n tq k C 1 a n = F ln b k = ( ) m L s L s = 1 q k C q k C + m 1 a q = m L 1 b q k = ( ) ( ) ( ) ( ) ( ) ( ) m s BET s s s s BET L BET L BET s = 1- 1- + q C k q C k C k C k     ( ) ( ) ( ) ( ) BET L BET L BET s m BET s 2- = k k k a q k ( ) ( ) ( ) BET s BET L m BET s -2 = k k b k q ( ) m BET s 1 = c k q 2 2 1 = + t t t q k q q The values of q and q were calculated by the equations (1) and (2): The values of q and qt were calculated by the equations (1) and (2): ( ) 0 s C -C = V q m (1) ( ) 0 C -C = t t V q m (2) (2) m where C0 is initial concentration of the solution (mg L-1), Ct is concentration of the solution in time t (mg L-1), V is volume of solution (L) and m is adsorbent mass (g). doi: https://doi.org/10.5599/admet.1950 Evaluation of the adsorption kinetics of MB on the modified starches Evaluation of the adsorption kinetics of MB on the modified starches The absorbance study was performed using four 50 mL conical flasks (A, B, C, D) as a series. 40 mL 6 mg L-1 MB was added for each flask. The SN, SM1, SM2 and SM3 starch probes of 2, 5, 10, 25, 50, 125, 250, 500, 750 and 1000 mg were placed in the flasks during the series, respectively. The flasks were put on the orbital shaker (at 100 rpm) at 22±0.5 °C. Four measurements of absorbance were proceeded based on the pharmacopoeial method by sampling the MB solution volumes of 3 mL in every 5, 10, 15, and 20 minutes , and than in equal periods up to 4 h. The solution was returned to the flasks. Analysis of taken samples was done by the spectrophotometer UV/VIS Jasco V-530 (Tokyo, Japan) at 663 nm, according to the available bibliography [37-39], and compared to the absorption spectrum of MB. A standard curve based on three series of measurements with five concentration points from 0.5 to 5.0 mg L-1 was prepared. The results were examined according to pseudo-zero-order kinetics and first-order kinetics. ANOVA statistical test was performed for independent groups at the significance level α = 0.05. Freundlich, Langmuir and BET isotherm models were taken for analysis of adsorption processes (Table 2). BET isotherm equation was adapted to 390 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives form BET isotherm for liquid phase adsorption [40], where kBET(s) applies to the equilibrium constant of adsorption of the first layer and kBET(L) refers to the equilibrium constant of adsorption of upper layers. Table 2. Isotherm and kinetics models applied for evaluation of obtained data, Cs - concentration of substance in the solution in the equilibrium state (e.s.), qt - adsorbed quantity of adsorbate in time, q -adsorbed quantity of adsorbate in the e.s., qm - maximum monolayer capacity, kF, kL and kBET - adsorption equilibrium constants, k1, k2 - equilibrium rate constants, 1/n - constant, t - time, a - slope, b - intercept, r2 - regression coefficient. Evaluation of the adsorption kinetics of MB on the modified starches where C0 is initial concentration of the solution (mg L-1), Ct is concentration of the solution in time t (mg L-1), V is volume of solution (L) and m is adsorbent mass (g). Equation (3) was used for the adsoprption (%) calculation: Equation (3) was used for the adsoprption (%) calculation: 0 0 Adsorption 100 t m m m   − =     0 0 Adsorption 100 t m m m   − =     0 0 Adsorption 100 t m m m   − =     (3) (3) where m0 - the initial mass of the adsorbate, mt - is the adsorbate mass after time t. Determination of the effect of MB adsorption on the pH of starch Results The results of the starch synthesis procedures yielded products with structures that conformed to the patterns (Figure 1 in the Introduction), and the appearance was as shown in Figures 2A and 2B. The molar ratio of functional groups added to the modified starches was converted per 1 g of native starch. Spectroscopic evaluations The molar ratio of functional groups added to the modified starches was converted per 1 g of native starch. Spectroscopic evaluations The molar ratio of functional groups added to the modified starches was converted per 1 g of native starch. Spectroscopic evaluations FTIR spectroscopy FTIR spectroscopy The FTIR spectrum of pure MB showed a broad peak from 3219 to 3364 cm-1, indicating an O-H bending bond. The sharp peaks at 3050 cm-1 and in the range of 817 to 884 cm-1 were responsible for the presence of C-H or =C-H bonds in the aromatic ring. Similarly, a sharp peak around 2700 cm-1 may have occurred due to the C-H stretching bonds [41]. doi: https://doi.org/10.5599/admet.1950 391 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. Figure 2. Images of actual appearance (above) and images magnified 10 times with a stereoscopic microscope (SMZ-171-TLED, Motic, Hongkong, China) (below) of A - SM1, B - SM2, C - SM3; petri dish diameter = 10 cm. Figure 2. Images of actual appearance (above) and images magnified 10 times with a stereoscopic microscope (SMZ-171-TLED, Motic, Hongkong, China) (below) of A - SM1, B - SM2, C - SM3; petri dish diameter = 10 cm. Characteristic peaks interacting at 1596 cm-1 might have been assigned to N=C stretching bonds. The peaks in the range 1420-1490 cm-1 most likely belonged to the stretching C=C-C bonds in the aromatic ring. Vibration from 1340 to 1360 cm-1 revealed the presence of C-N stretching bonds in aromatic tertiary amine [41] and peaks from 1059 to 1064 cm-1 and 661 cm-1 are related to C-S-C stretching bonds in heterocycle [42]. The pure starches FTIR spectra indicated a broad peak of O-H stretching bond at about 3300 cm-1, C-O stretching vibration at the range 1185-1347 cm-1, especially the strong peak at 1240 cm-1 for SM1 (Figure 3 b), C-O stretching bond at 930 cm-1 [43] and O-H bending bond at 988 cm-1 [44] (Figure 3). 92 a b c d Figure 3. The FTIR spectra of methylene blue (MB), the pure starches (a) SN, (b) SM1, (c) SM2 and (d) SM3, the physical mixture and the experimental formulation of the starches with adsorbed MB, respectively. b d b a c d c d Figure 3. The FTIR spectra of methylene blue (MB), the pure starches (a) SN, (b) SM1, (c) SM2 and (d) SM3, the physical mixture and the experimental formulation of the starches with adsorbed MB, respectively. FTIR spectroscopy 392 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives The plot of the spectrum of adsorbed MB on the starch (red line) mostly followed the plot of the spectrum of the respective starch in its pure state (green line). However, there were locations where individual peaks were in excess. The peaks assigned to N=C stretching bonds at about 1600 cm-1 appeared in the case of MB adsorbed on the SN and SM2, however, to a very slight extent (Figure 3 a,c). The peak indicating the presence of the C=O bond of the acetyl group at 1730 cm-1 [45] was spotted on the SM1 spectra (Figure 3b). PXRD Diffractograms of modified starches were compared to those of SN (Figure 4). The peaks with the highest intensity were in the 2 range from 17.2 to 17.4°. The most intense belonged to SN and the least to SM2. The proportion of crystallinity and amorphousness for the tested samples was determined using the amorphous subtraction method [46,47] and presented in Table 3. Table 3. Composition of crystallinity and amorphousness for SM1, SM2 and SM3 samples compared to SN (n = 3). Starch types SN SM1 SM2 SM3 Crystallinity, % 14.3 ± 0.80 9.8 ± 0.14 3.2 ± 0.26 5.7 ± 0.75 Amorphousness, % 85.7 ± 0.80 90.2 ± 0.14 96.8 ± 0.26 94.3 ± 0.75 2 / ° Figure 4. The diffractograms of acetylated starch (SM1), starch citrate (SM2) and starch diphosphate (SM3) compared to native starch (SN). Table 3. Composition of crystallinity and amorphousness for SM1, SM2 and SM3 samples compared to SN (n = 3). Starch types SN SM1 SM2 SM3 Crystallinity, % 14.3 ± 0.80 9.8 ± 0.14 3.2 ± 0.26 5.7 ± 0.75 Amorphousness, % 85.7 ± 0.80 90.2 ± 0.14 96.8 ± 0.26 94.3 ± 0.75 Table 3. Composition of crystallinity and amorphousness for SM1, SM2 and SM3 samples compared to SN (n = 3). Starch types SN SM1 SM2 SM3 Crystallinity, % 14.3 ± 0.80 9.8 ± 0.14 3.2 ± 0.26 5.7 ± 0.75 Amorphousness, % 85.7 ± 0.80 90.2 ± 0.14 96.8 ± 0.26 94.3 ± 0.75 2 / ° Figure 4. The diffractograms of acetylated starch (SM1), starch citrate (SM2) and starch diphosphate (SM3) compared to native starch (SN). Figure 4. The diffractograms of acetylated starch (SM1), starch citrate (SM2) and starch diphosphate (SM3) compared to native starch (SN). Thermal properties DSC analysis results shown in Figure 5 compared pure MB and evaluated starches with adsorbed MB and physical mixtures of these components. The thermograms showed the results of heat flow measurements from 0 to 350 °C taken in two cycles, with a cooling interval in between. The exothermic direction has been marked with an appropriate arrow (exo). The results of DSC testing of native and modified starches under two heating cycles are shown in Figure 6. The glass transition midpoint temperatures Tg, the difference in the heat capacity between the transition from glass to the liquid state ΔCp and ΔT - difference between onset crystallization temperature and Tg were analyzed using Proteus 7.0 software (Netzsch, Selb, Germany). The results are shown in the Supplementary material, Table S1. doi: https://doi.org/10.5599/admet.1950 393 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. bryń et al. ADMET & DMPK 11(3) (2023) 387-407 a b c d e f g h Figure 5. The DSC thermograms of methylene blue (MB), the pure starches (SN, SM1, SM2 and SM3), physical mixtures, and the experimental formulations of the starches with adsorbed MB. The subfigures (a,b,c,d) represent thermograms of the first heating cycle and (e,f,g,h) thermograms of the second heating cycle. a b a c e d d c f h f e e h g h g Figure 5. The DSC thermograms of methylene blue (MB), the pure starches (SN, SM1, SM2 and SM3), physical mixtures, and the experimental formulations of the starches with adsorbed MB. The subfigures (a,b,c,d) represent thermograms of the first heating cycle and (e,f,g,h) thermograms of the second heating cycle. 394 Model cationic drug and newly synthesized starch derivatives ADMET & DMPK 11(3) (2023) 387-407 b a b Figure 6. The DSC thermograms of the starches (SN, SM1, SM2 and SM3). The subfigure (a) represents thermograms of the first heating cycle and (b) the second heating cycle. a a Figure 6. The DSC thermograms of the starches (SN, SM1, SM2 and SM3). The subfigure (a) represents thermograms of the first heating cycle and (b) the second heating cycle. doi: https://doi.org/10.5599/admet.1950 Adsorption tests The graphs showing the course of MB adsorption on starch showed the lowest saturation for SN and the highest for SM2 for 2 mg starch samples. It was 7.60 mg g-1 for SN and 22.86 mg g-1 for SM2 (Figure 7). The graphs showing the course of MB adsorption on starch showed the lowest saturation for SN and the highest for SM2 for 2 mg starch samples. It was 7.60 mg g-1 for SN and 22.86 mg g-1 for SM2 (Figure 7). The graphs showing the course of MB adsorption on starch showed the lowest saturation for SN and the highest for SM2 for 2 mg starch samples. It was 7.60 mg g-1 for SN and 22.86 mg g-1 for SM2 (Figure 7). b d a b c d Figure 7. The plot of MB adsorption on the starch samples with time: (a) SN, (b) SM1, (c) SM2, (d) SM3; ● - 2 mg, ○ -5 mg, ■ - 10 mg, □ - 25 mg, ▲ - 50 mg, Δ - 125 mg, ♦ - 250 mg, ◊ - 500 mg, - - 750 mg, + - 1000 mg, n = 4, at 22±0.5 °C and 6.0 mg L-1 MB solution with pH 4.26. b a a c d d c Figure 7. The plot of MB adsorption on the starch samples with time: (a) SN, (b) SM1, (c) SM2, (d) SM3; ● - 2 mg, ○ -5 mg, ■ - 10 mg, □ - 25 mg, ▲ - 50 mg, Δ - 125 mg, ♦ - 250 mg, ◊ - 500 mg, - - 750 mg, + - 1000 mg, n = 4, at 22±0.5 °C and 6.0 mg L-1 MB solution with pH 4.26. The amount of adsorbed MB increased with the enhancement of the mass of starch samples. The parameter values, adjusted to the tested kinetics models, based on the correlation factor, are given in the Supplementary material, Table S2. Table 4 exhibits the parameters adapted to the chosen isotherm models. The samples of the starches were divided into two categories dependent on the mass; “A” was between 2 and 125 mg and “B” was between 250 and 1000 mg. Freundlich isotherm described in a satisfactory way adsorption equilibriums of doi: https://doi.org/10.5599/admet.1950 395 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. MB adsorbed on the SN A, SM1, SM2 and SM3 B. Determination of the effect of MB adsorption on the pH of starch The pH of starch suspensions with adsorbed MB compared to that of pure starch suspensions was higher for SM2 and lower for SN and SM1. In addition, there was a proportional increase in pH values after 72 h (Figure 8). a b Figure 8. Comparison of pH values of starch suspensions with adsorbed MB to pure starch suspensions after (a) 1 h and (b) 72 h at 22±2 °C (n = 3). NosignificantdifferenceswereobservedbetweenthepHvaluesof SN-MBandSM1-MBafter1h(Figure8a) a Figure 8. Comparison of pH values of starch suspensions with adsorbed MB to pure starch suspensions after (a) 1 h and (b) 72 h at 22±2 °C (n = 3). No significant differences were observed between the pH values of SN-MB and SM1-MB after 1 h (Figure 8a). Adsorption tests The Freundlich exponent 1/n between 0.5723 and 0.9221 for SN “A” and SM1 “B” indicated favorable adsorption for which 1 ≤ 1/n. The adsorption on SM2 “B” had the largest value of kF. The adsorption on SM1 “A” had the lowest kF-value. In the case of the Langmuir isotherm, only SM2 “B” and SM3 “B” gave a satisfactory value of r2. However, in this case, the values of qm and kL were negative. The largest r2 values for the BET isotherm belonged to SM2 “B” and SM3. Table 4. Parameters of selected isotherm models for adsorption of 6 mg L-1 MB solution on SN, SM1, SM2 and SM3 starches. A - applies to 2 to 125 mg, B - applies to 250 to 1000 mg of the adsorbents (n = 4). Isotherm model Parameters SN SM1 SM2 SM3 A B A B A B A B Freundlich 1/n 0.5723 1.0254 4.8442 0.9221 2.4503 3.5491 2.4769 1.5960 kF / L g-1 2.1632 0.9702 0.0010 0.3727 0.3964 7.7983 0.1407 0.5681 r2 0.9071 0.8096 0.8467 0.8834 0.8954 0.9162 0.9083 0.9981 Langmuir kL / L g-1 0.3020 -0.0712 -0.1593 0.0488 -0.1671 -1.2145 -0.1317 -0.2738 qm / mg g-1 8.9767 -12.594 -0.3920 7.9177 -4.7619 -0.4387 -3.4807 -1.5094 r2 0.6793 0.0771 0.5560 0.0836 0.7378 0.9220 0.8701 0.9684 BET kBET(s) / L mg-1 1.4106 4.7971 0.5151 0.7223 0.0216 5.1912 1.8188 0.7352 kBET(L) / L mg-1 0.0821 0.6768 0.0741 0.1666 0.1399 1.2943 0.1539 0.2591 qm / mg g-1 3.3822 0.0318 0.0290 0.3175 27.676 0.2730 1.4069 0.4535 r2 0.7331 0.3582 0.6907 0.2395 0.7706 0.9334 0.9434 0.9813 meters of selected isotherm models for adsorption of 6 mg L-1 MB solution on SN, SM1, SM2 and SM3 applies to 2 to 125 mg, B - applies to 250 to 1000 mg of the adsorbents (n = 4). Discussion Spectroscopic evaluations Spectroscopic evaluations DSC The DSC thermograms of pure MB in the first heating cycle displayed an endothermic peak at about 80 °C responsible for the dehydration (Figure 5 a-d). The thermogram of the second heating cycle no longer showed this peak, while an endothermic peak in the 100 °C range appeared (Figure 5 e-h). MB forms several hydrates. In addition, it reacts with an aluminum container for DSC [51]. This endothermic peak may indicate a transition from one form of MB hydrate to another. In the thermogram of the first heating cycle, it was most likely obscured by the broad peak of dehydration. These speculations are confirmed by the shifted exothermic peaks occurring at 171 and 233 °C in the first heating cycle (Figure 5 a-d) and 201 °C in the second cycle (Figure 5 e-h). They testify to the decomposition of MB [52]. Omer et al. [53] indicated the occurrence of oxidation of MB at 260 °C. A difference was observed between the spectrum of adsorbed MB and physical mixed MB. An exothermic peak appeared, indicating the presence of MB in the physical mixture with SN (177.6 °C), SM1 (175.9 °C), SM2 (174.0 °C) and SM3 (177.2 °C) (Figure 5 a,b,c,d respectively). No MB peak was observed in MB adsorbed at SN and SM1 (Figure 5 a,b), which may indicate the absence or complete decomposition of MB. For MB adsorbed on SM2 and SM3, the course of these peaks was flattened at 174.4 and 176.7 °C, respectively, which may indicate the interaction of MB with these starches (Figure 5 c,d). Glass transition temperature Tg value readings showed the lowest Tg for SM2 (47.2 °C) and the highest for SM3 (55.9 °C) (Table S1). Hydrogen bonds are among the factors affecting Tg [54]. Taylor et al. [55] observed significantly lower Tg of sucrose compared to other disaccharides. This was due to the less extensive hydrogen bonding network in the amorphous state. Similarly, Zhou et al. [56] demonstrated the dependence of Tg on hydrogen bonds occurring between polymer and copolymer particles (maleimide-isobutene alternating copolymers). A network of hydrogen bonds of carboxyl groups resulted in a higher Tg value than hydrogen bonds between hydroxyl groups. The lowest Tg occurred in the absence of hydrogen bonds between polymer molecules. PXRD According to Table 3, the SN was the most crystalline, and SM2 the least. The crystallinity of SM increased according to the following pattern, SM2<SM3<SM1<SN, which suggests that the implementation of ionic groups resulted in the highest amorphicity, whereas the non-ionic acetyl groups in the lowest extent influenced the increase of amorphicity. This remark agrees with the results of Bhargav et al. [49], who assessed the combination of PVA with NaI. As the salt content of the polymer increased, the DSC diffraction peaks became less intense. They suggested high susceptibility of the amorphous structure of the polymer to ionic diffusivity of ionic particles. Similarly, Hodge et al. [50] found a correlation between peak intensity and the degree of crystallinity. They observed that the addition of water resulted in a decrease in XRD peak intensity and an increase in PVA amorphousness. Model cationic drug and newly synthesized starch derivatives Model cationic drug and newly synthesized starch derivatives FTIR spectroscopy FTIR spectroscopy Due to the appearance of peaks at 1599 cm-1 assigned to the C=N bond on the FTIR spectrum of samples of adsorbed MB on SN and SM2, we postulate the existence of an interaction between the N+ ion derived from MB and the OH- group belonging to starch, which probably leads to enhanced adsorption of the dye on the polymer. This observation coincides with the mechanism of MB adsorption on black olive stone carried out by Al-Ghouti et al. [48]. 396 ADMET & DMPK 11(3) (2023) 387-407 DSC In our case, the holders of the hydroxyl groups were SN and SM1 (Tg 52.2 and 51.15 °C respectively) (Table S1) and the carboxyl groups were present in the SM2 molecule. In addition, phosphate groups appeared in the SM3 molecule. High Tg values for SM3 may suggest the self-association of these starch molecules, whose Tg decreased to 53.0 °C after bond formation with MB [55]. In the case of SN, Tg values increased after MB adsorption (54.7 °C), what may indicate low self-association of SN particles in the pure state. In the present work, the values of ΔT were assessed to determine the thermal stability of the tested samples. The higher the value of ΔT, the better the thermal stability. The value for SN was the highest (221.07 °C), demonstrating the highest thermal stability of this starch. At the same time, its highest percentage of crystallinity was confirmed (Table 3). On the contrary, SM2 showed the lowest value of ΔT (179.8 °C) - the lowest thermal stability. Keshk et al. [43] demonstrated the dependence of thermal doi: https://doi.org/10.5599/admet.1950 397 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. stability on ΔT-values, which correlated with the degree of crystallinity of corn starch-cellulose mixtures, depending on the mixture ratio. They found an inversely proportional relationship between ΔT values and ΔCp values. Our ΔCp studies confirmed such a relationship outside of the SM2 samples having the lowest ΔCp value (0.465 J g-1·K-1) according to the lowest ΔT (Table S1). This phenomenon may be supported by the observation of Liu et al. [57]. They studied various starches whose structure was more or less amorphous. According to them, amorphousness gives the compounds a liquid-like structure. More amorphous compounds have a less ordered structure than those with a crystalline structure. They showed that the process of lowering the temperature below Tg leads to the reorganization of the structure. In our study, SM1, SM2 and SM3 starches were hardened at 2 degrees below Tg. Moreover, the research is confirmed by PXRD studies, where SM2 had the highest degree of amorphousness (Table 3). The thermograms of the second heating cycle can show the structure of the compound when structural reorganization occurs after the cooling cycle. In the case of SM2 there was an endothermic peak at 215 °C, from which an increase in heat flow is observed. DSC The thermograms of adsorbed MB on starches are characterized by different temperatures of the start of the exothermic process. The increase in heat flow started fastest for SM2_MB (adsorbed) at 157 °C and slowest for SM3_MB (adsorbed) at 163 °C (Figures 5g and 5h, respectively). This endothermic event might be attributed to reduced hydrogen bonding as well as the interference of molecular organization due to the interaction between SM2, SM3 and MB. For SN_MB (adsorbed) and SM1_MB (adsorbed), no increase in heat flow was observed (Figures 5e and 5f). Adsorption tests 398 Model cationic drug and newly synthesized starch derivatives ADMET & DMPK 11(3) (2023) 387-407 Figure 9. Isotherms of MB adsorbed on the starches: SN, SM1, SM2 and SM3 at 22±0.5 °C and 6.0 mg L-1 MB solution with pH 4.26 (n = 4). Figure 9. Isotherms of MB adsorbed on the starches: SN, SM1, SM2 and SM3 at 22±0.5 °C and 6.0 mg L-1 MB solution with pH 4.26 (n = 4). Table 5. Maximum adsorption capacities (qm) of different starches and other polysaccharides toward MB. Type of adsorbent qm / mg g-1 Ref. Hydrogel poly(sodium methacrylate) with wheat starch and eggshell 1.95 [66] Crosslinked porous corn starch 9.46 [67] Starch cryogel of a rice flour and a tapioca starch 34.84 [68] Honeycomb biomass adsorbent based on oxidized corn starch-gelatin 1551.5 [69] Hydrogel poly(acrylamide) with cassava starch 1917 [70] Hydrogel poly(acrylic acid) with potato starch crosslinked with N,N’-methylene-bisacrylamide 2967.66 [71] Xanthate modified magnetic chitosan 197.8 [72] Chitosan/activated charcoal 500 [73] Cellulose-grafted poly 4-hydroxybenzoic acid magnetic nanohybrid 7.5 [74] Hydrogel of hydroxypropyl cellulose (HPC) composited with graphene oxide 6.59 [75] Native potato starch 7.60 This work Acetylated potato starch 8.57 This work Diphosphate potato starch 16.10 This work Citrate potato starch 22.86 This work The Freundlich model describes multilayer adsorption on a surface, with an inhomogeneous distribution of binding sites and, therefore, an inhomogeneous energy distribution. The constant kF expresses the maximum adsorption on the surface of the adsorbent. The BET isotherm describes the phenomenon of physisorption on a non-microporous surface. The constant kBET is related to the adsorption energy. Bhattacharyya et al. [36] found graphene oxide-potato starch almost perfectly fitted to the Freundlich isotherm model. Dipa et al. [37] adapted MB adsorption on kaolinite to the Langmuir model. Bestani et al. [62] studied MB adsorption on carbon-activated leaves, which they fitted to the Langmuir and Freundlich models. El Qada et al. [31] matched MB adsorption on activated carbon to Redlich-Peterson and Langmuir isotherm models. The Redlich-Peterson model is a compilation of the elements in the equations of the Langmuir and the Freundlich isotherms. Langmuir isotherm mostly applies to chemisorption on a monolayer. Activated carbon is highly porous and is a well- known adsorbent used in industry as a filter [77]. The dependence of the isotherm model on the structure of the adsorbent can also be supported by the study of Ma et al. Adsorption tests MB adsorption on starch predominantly matched a pseudo-second-order model. A study by Lin et al. [58] of MB adsorption on activated carbon indicated a better pseudo-second-order fit to both linear and nonlinear models. Similarly, Ma et al. [45] matched the adsorption of heavy metal ions on modified potato starch to the pseudo-second-order model. In our study, the highest k2 values obtained for SN, SM3, SM2 and SM1 were 1.9623, 1.5367, 0.9240, 0.1848 g mg-1·min-1, respectively, and occurred at a weight of 1000 mg for SN and SM1 and 2.0 mg for SM2 and SM3 (Table S2). This suggests that MB adsorbed fastest on SN, but this was dependent on the surface area of the adsorbent: the larger the surface area, the faster the saturation. In the case of SM2 and SM3, MB adsorbed fastest already on the smallest starch particle, suggesting a high affinity of the adsorbate to the adsorbent. The slowest adsorption was observed in SM1. The q values in Table S2 differ from qm obtained according to Langmuir and BET isotherms. Moreover, some of the Langmuir adsorption constants (qm and KL) showed negative values (Table 4). Langmuir isotherm is known to get different parameters according to the calculation method [59-61], therefore, we used the course of the isotherm of MB on the starches to establish qm values (Figure 9). Methylene blue is a substance that has been thoroughly studied by scientists because of its harmfulness to the environment. As a dye used in industry, it leaks into water waste in large quantities. Their purification is a major challenge for researchers. Many study substances that are adsorbents for MB and can serve as filters [32,61-65]. Some of these include starches and other polysaccharides. Table 5 shows the qm values of example adsorbents for MB. The sigmoidal course of the SM2 and SM3 starch isotherms falls into type IV according to the IUPAC classification. SN and SM1 isotherms are more aligned with type VI [76]. Type IV represents single and multilayer adsorption plus capillary condensation. Type VI illustrates that the adsorption isotherm can have one or more steps. In our study, the adsorptions on SN and SM1 starches best fitted to the Freundlich isotherm model. On the other hand, for SM2 and SM3 starches, the adsorption courses more closely matched the BET isotherm (Table 4). J. Kobryń et al. J. Kobryń et al. ADMET & DMPK 11(3) (2023) 387-407 Determination of the effect of MB adsorption on the pH of starch Determination of the effect of MB adsorption on the pH of starch Determination of the effect of MB adsorption on the pH of starch The measured pH and pKa values of the acids from the literature enabled us to estimate the number of moles of functional groups derived from the molecules and finally present at the modified starches. The calculated values are gathered in Table 6. Table 6. Concentrations of functional groups located on 50 mg of modified starches recalculated on the basis of parameter values of the Henderson-Hasselbalch equation [78] (n = 3). SM1 SM2 SM3 pH after 1h 6.44 ± 0.05 5.19 ± 0.09 5.98 ± 0.07 pKa1* 4.76 3.13 2.16 pKa2* - 4.76 7.21 pKa3* - 6.40 12.32 Concentration of functional groups, mmol mg-1 6.07 ± 1.64 ×10-7 7.07 ± 2.82 × 10-6 1.90 ± 0.62 × 10-8 *according to database in LibreTexts™ chemistry [79] Table 6. Concentrations of functional groups located on 50 mg of modified starches recalculated on the parameter values of the Henderson-Hasselbalch equation [78] (n = 3). The initial assumptions of the ratio of the number of millimoles of functional groups on the modified starches used to react with the native starch during its modification was 23:13:1 for SM1:SM2:SM3, respectively (Table 1). The millimole ratio of the same groups, calculated from the pH values of the modified starch suspensions after 1 h, was 1:13:0.03. This shows that the activity of acyl and phosphate groups decreased 23 and 33 times compared to carboxyl groups, respectively. MB solution with pH of 4.26 resulted in the acidification of SN and SM1 suspensions. It did not change the pH for SM3. The increase in the pH value of the suspension of adsorbed MB on SM2 starch may indicate the most effective binding of MB to the carboxyl groups of SM2 (Figure 9). Adsorption tests [45], who used potato starch modified with high temperature to increase its porosity. Adsorption of heavy metal ions on such modified starch showed good adherence to the Langmuir model. In the case of our experiment, only SM2 was subjected to high-temperature modification at 100 °C. This could most likely increase its adsorption properties. doi: https://doi.org/10.5599/admet.1950 399 Effect of type of functional groups of modified starches on interactions with MB The MB adsorbed on the 1000 mg of the starch decreased according to the following pattern: SN > SM2 > SM3 > SM1 (Figure 7). The hydroxyl, carboxyl and phosphate groups of starch favored MB binding to the polymer to the greatest extent, while the acetyl groups had the greatest effect on the reduction of adsorption capacity. The rationale for the decreased sorption capacity could be the blocking of starch functional groups in the structure of starch polymer or the specific activity of functional groups towards MB. Dipa et al. [37] observed the effect of NaOH-derived hydroxyl groups on the increase of MB adsorption on kaolinite. However, these were groups added to kaolinite. In our case, only the carboxyl, phosphate and acyl groups originated from acids and were added during the starch modification process. Physisorption may be taken into account in the case of SN and SM3. It differs for SM2 and SM1, where acid groups can affect MB adsorption. Similar conclusions were issued by Huang et al. [80] after studying the adsorption of Pb2+ ions on activated carbon with additional functional groups. Pb2+ ions were adsorbed according to three mechanisms: (1) in mesopores, (2) via bonds between -OH and C=O and Pb2+, (3) H+ from the acidic carboxyl and phosphate groups is replaced by the Pb2+ ion. An additional element worth highlighting is the effect of the method of preparation of modified starches on their structure. Two of our SM2 and SM3 starches were prepared using the crosslinking method, which provides increased starch stabilization by forming bridges between hydroxyl groups and anhydroglucoses [17] found crosslinking reactions caused a decrease in solubility in water and a decrease in phase transition. Shen et al. [82] used corn starch and citric acid, malic acid, succinic acid and 1,2,3,4-butanetetra- carboxylic acid (BTCA) as cross-linkers. They observed that the acid with more carboxyl groups (citric acid and BTCA) showed higher crosslinking degrees and enhanced mechanical properties than acids with two carboxylic groups. Moreover, as they are used in food modification, poly-carboxylic acids are in the category 400 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives of safe chemicals [83]. In the case of SM1 and SM3, the chemically modified starches with a low degree of substitution (DS) are permitted for use in the food industry and are denoted with the symbol “E” followed by an appropriate number. Conclusions Citrate starch (SM2) presented the best adsorption capacity, followed by phosphate starch (SM3), and acetylated starch (SM1) showed the weakest adsorption capacity, according to the adsorption results. MB adsorption on SM2 and SM3 followed the BET isotherm model, which shows the physical adsorption of the cationic dye on the low-porous surface. In the case of SM1, MB adsorption probably took place via electrostatic attraction between the heterogeneous adsorbent surface and the adsorbate molecules, as indicated by the Freundlich adsorption model. Spectral methods confirmed the adsorption capacity of SM2. In the FTIR study, a probable interaction between the OH- groups of SM2 starch and N+ of MB was revealed on the basis of characteristic vibration of the C=N bond in the adsorbent derived from MB at 1600 cm-1. The DSC results showed the lowest thermal stability of SM2 correlated with its amorphous structure. Moreover, according to DSC, the possibility of interaction between SM2 or SM3 and MB was demonstrated via the existence of an exothermic peak belonging to MB in samples of these two adsorbents. The appearance of endothermic peaks in the case of SM2 and SM3 samples with adsorbed MB in the second heating cycle confirmed the interaction between the adsorbents and the adsorbates. The effect of the manufacturing method of the modified starches on their durability was noted. SM2 and SM3 starches were prepared using the crosslinking method, which provided increased starch stabilization. The above presented properties and, in addition, the "safe chemistry" and slightly acidic pH of the tested starches make them useful, especially citrate starch, as carriers for biologically active substances containing cationic groups, in topical skin applications. The present research confirms our previous studies on lidocaine hydrochloride. The modified starches may be the potential carriers in the form of hydrogels, similar to the hydrocolloid dressings. Author contributions: Conceptualization, J.Kobryń and W.Musiał; methodology, J.Kobryń, W.Musiał and T.Zięba; formal analysis, J.Kobryń, M.Rzepczyńska and W.Musiał; investigation, J.Kobryń, W.Musiał, M.Rzepczyńska and T.Zięba; resources, W.Musiał; data curation, J.Kobryń and W.Musiał; writing—original draft preparation, J.Kobryń and W.Musiał; writing—review and editing, J.Kobryń, W.Musiał and T.Zięba; visualization, J.Kobryń, W.Musiał and T.Zięba; supervision, W.Musiał; project administration, W.Musiał; funding acquisition, W.Musiał. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Wroclaw Medical University, grant No SUBZ.D060.23.025. Effect of type of functional groups of modified starches on interactions with MB Among others, acetylated starch (E1420) and di-starch phosphate (E1412) belong to this group [84]. Conclusions Acknowledgement: The authors gratefully acknowledge the Laboratory of Elemental Analysis and Structural Research at the Faculty of Pharmacy at Wroclaw Medical University for allowing access to DSC, FTIR and PXRD instruments. Conflict of interest: The authors declare no conflict of interest. doi: https://doi.org/10.5599/admet.1950 401 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. References [1] Z.N. Salman, I. Al-Ani, K.M. Al Azzam, B.J. M. Majeed, H.H. Abdallah and El-S. Negim. Enhancement of apixaban's solubility and dissolution rate by inclusion complex (β-cyclodextrin and hydroxypropyl β- cyclodextrin) and computational calculation of their inclusion complexes. ADMET and DMPK (2023). http://doi.org/10.5599/admet.1885. [1] Z.N. Salman, I. Al-Ani, K.M. Al Azzam, B.J. M. Majeed, H.H. Abdallah and El-S. Negim. 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ISBN 978-1-118-66955-6. http://doi.org/10.1002/9781119044901. doi: https://doi.org/10.5599/admet.1950 407 ©2023 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/) doi: https://doi.org/10.5599/admet.1950 407 ©2023 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/) ©2023 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/) doi: https://doi.org/10.5599/admet.1950 407
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Individual differences in the influence of task-irrelevant Pavlovian cues on human behavior
Frontiers in behavioral neuroscience
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Keywords: Pavlovian-to-instrumental transfer, cue-controlled behavior, Sign-Tracker, Goal-Tracker, reinforcement learning Gregory B. Bissonette, University of Maryland, USA Reviewed by: Jeremy J. Clark, University of Washington, USA Donna J. Calu, National Institute on Drug Abuse, USA *Correspondence: Sara Garofalo, Department of Psychiatry, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0SR, UK garofalosara56@gmail.com Individual differences in the influence of task-irrelevant Pavlovian cues on human behavior Sara Garofalo 1,2,3* and Giuseppe di Pellegrino 1 1 Center for Studies and Research in Cognitive Neuroscience, Department of Psychology, University of Bologna, Cesena, Italy, 2 Department of Psychiatry, University of Cambridge, Cambridge, UK, 3 Behavioural and Clinical Neuroscience Institute, Department of Psychology, University of Cambridge, Cambridge, UK Pavlovian-to-instrumental transfer (PIT) refers to the process of a Pavlovian reward- paired cue acquiring incentive motivational proprieties that drive choices. It represents a crucial phenomenon for understanding cue-controlled behavior, and it has both adaptive and maladaptive implications (i.e., drug-taking). In animals, individual differences in the degree to which such cues bias performance have been identified in two types of individuals that exhibit distinct Conditioned Responses (CR) during Pavlovian conditioning: Sign-Trackers (ST) and Goal-Trackers (GT). Using an appetitive PIT procedure with a monetary reward, the present study investigated, for the first time, the extent to which such individual differences might affect the influence of reward- paired cues in humans. In a first task, participants learned an instrumental response leading to reward; then, in a second task, a visual Pavlovian cue was associated with the same reward; finally, in a third task, PIT was tested by measuring the preference for the reward-paired instrumental response when the task-irrelevant reward-paired cue was presented, in the absence of the reward itself. In ST individuals, but not in GT individuals, reward-related cues biased behavior, resulting in an increased likelihood to perform the instrumental response independently paired with the same reward when presented with the task-irrelevant reward-paired cue, even if the reward itself was no longer available (i.e., stronger PIT effect). This finding has important implications for developing individualized treatment for maladaptive behaviors, such as addiction. Introduction Goal-directed behavior can be variably influenced by external and internal factors which impact the values and priorities assigned to rewards and goals (Doya, 2008). One of the most simple and effective mechanisms for influencing choice is reinforcement learning. Reinforcement learning allows animals to connect spatially and/or temporally related events in order to predict future events. Given the complexity of the animal’s environment, learning that an arbitrary cue (e.g., a sound) is predictive of a certain goal (e.g., obtain a reward, such as food), allows the animal to learn a flexible response that facilitates achievement of the goal itself. In most cases such cue-controlled behavior is adaptive; for example it helps one obtain food when hungry (Perks and Clifton, 1997; Holmes et al., 2010). However, an inflexible association can lead to perseverance in the same choice Received: 07 April 2015 Accepted: 08 June 2015 Published: 24 June 2015 Edited by: Gregory B. Bissonette, University of Maryland, USA ORIGINAL RESEARCH published: 24 June 2015 doi: 10.3389/fnbeh.2015.00163 Citation: PIT has been mainly studied in non-human animals (Rescorla and Solomon, 1967; Lovibond, 1981; Colwill and Rescorla, 1988; Balleine, 1994; Rescorla, 1994a, 1997, 2000; Delamater, 1995, 1996; Holland et al., 2002; Corbit and Balleine, 2003; Holland and Gallagher, 2003; Holland, 2004; Delamater and Holland, 2008; for review, see Dickinson and Balleine, 1994, 2002; Holmes et al., 2010), but some recent studies have also reported this effect in humans (Paredes-Olay et al., 2002; Hogarth et al., 2007, 2010, 2013a,b; Bray et al., 2008; Allman et al., 2010; Nadler et al., 2011; Prévost et al., 2012; Lovibond and Colagiuri, 2013). An important, but still neglected, aspect in the human literature about PIT concerns individual differences In the engage the location of reward delivery (the Goal), even if it is not yet available. The first CR has been categorized as Sign-Tracking behavior, while the second CR has been categorized as Goal- Tracking behavior. ST and GT can be conceived of as different learning styles, expressed through a specific CR during Pavlovian learning. ST behavior is thought to arise from the attribution of incentive salience to Pavlovian reward-paired cues, which consequently become a powerful source of motivation for future behavior (Flagel et al., 2011). In ST, incentive stimuli become attractive, eliciting approach towards them and promoting potentially maladaptive cue-controlled behaviors; ST individuals, indeed, are generally more vulnerable to addiction and relapse (Tomie et al., 1998; Flagel et al., 2008; Robinson and Flagel, 2009). The ST and GT profiles do not seem to be limited to the CR expressed, but are also associated with differences in traits such as impulsivity; ST individuals are characterized by higher levels of impulsive behavior compared to GT individuals (Tomie et al., 2000; Flagel et al., 2009). A deeper investigation into individual differences in attributing incentive salience to reward-paired stimuli would thus be important for understanding and reducing the propensity to develop maladaptive behaviors. The aim of the present study was to investigate individual differences in human PIT. Specifically, the present study explored, for the first time in humans, whether individual differences in the propensity to approach and engage a Sign (cue-predicting reward) or a Goal (reward) are predictive of cue-controlled behavior. To this end, a typical PIT experimental design was used, comprising three tasks. Citation: Garofalo S and di Pellegrino G (2015) Individual differences in the influence of task-irrelevant Pavlovian cues on human behavior. Front. Behav. Neurosci. 9:163. doi: 10.3389/fnbeh.2015.00163 Garofalo S and di Pellegrino G (2015) Individual differences in the influence of task-irrelevant Pavlovian cues on human behavior. Front. Behav. Neurosci. 9:163. doi: 10.3389/fnbeh.2015.00163 June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 1 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino even if the goal itself is no longer available, or has negative long- term consequences (Holmes et al., 2010). For example, a cue associated with drugs can induce relapse even when the drug is not voluntary sought, and a sign associated with food can induce craving in the absence of hunger, leading to compulsive over-eating (Volkow et al., 2008). These biases on voluntary choice are also implemented in marketing strategies, such as advertisements, to influence consumer behavior (Smeets and Barnes-Holmes, 2003; Bray et al., 2008; de Wit and Dickinson, 2009). Cue-controlled behaviors have been interpreted as the endpoint of an initial intentional seeking behavior (of a reward), which leads to habitual, and ultimately compulsive, conduct characterized by a loss of control over behavior (Everitt and Robbins, 2005). This interesting framework proposes that the transition from intentional volition to habit and compulsion can be explained by interactions between Pavlovian and instrumental learning processes: a reward acts as an instrumental reinforcer by enhancing actions that are able to produce it, while Pavlovian learning confers incentive salience to cues (Conditioned Stimuli or CS) closely associated with the reward (Everitt and Robbins, 2005). Such cues can elicit craving and motivation towards the associated reward, thus biasing choice. Well-known evidence of this effect can be found in the so-called Pavlovian-to- Instrumental Transfer (PIT) effect (Estes, 1943, 1948). PIT captures the ability of a Pavlovian cue (i.e., a CS associated with a reward) to increase the likelihood of an instrumental response independently paired with the same (specific-PIT), or a similar (general-PIT), reward (Rescorla and Solomon, 1967; de Wit and Dickinson, 2009; Holmes et al., 2010). This effect emerges without any formal association between Pavlovian and instrumental contingencies, and even when the reward itself is no longer available (Talmi et al., 2008). Citation: In the first phase, participants performed an Instrumental Conditioning task, in which they were presented with two possible choices, one paired with an actual monetary win (Rewarded Choice) and the other paired with a neutral outcome (Unrewarded Choice). In a subsequent session, participants performed a Pavlovian Conditioning task, during which they learned to associate a specific visual cue with an actual monetary win (CS+), and another visual cue with a neutral outcome (CS−). During this phase, eye-movements were recorded and subsequently analyzed in order to identify the expressed CR and characterize participants as ST or GT. Mirroring previous studies conducted in animals (Boakes, 1977; Flagel et al., 2007, 2008, 2011; Saunders and Robinson, 2013), in which the CR is identified based on the amount of approaching behavior expressed during CS presentation, in the present study ST and GT participants were distinguished based on a learned oculomotor CR. Specifically, it was measured the tendency to direct contiguous eye-gazes toward the location where the visual CS (Sign) or the reward (Goal) would be presented. Finally, PIT was tested in an extinction phase (without any rewards), during which participants had to choose between the same two options given during instrumental conditioning, while presented with the task-irrelevant CS. In this final phase, PIT would be observed if presentation of the CS+, compared to the CS−, enhanced instrumental responses to the choice rewarded during instrumental conditioning (Congruent Choice), relative to the previously unrewarded choice (Incongruent Choice). If consistent with animal literature, this effect should be stronger An important, but still neglected, aspect in the human literature about PIT concerns individual differences. In the animal literature, the extent to which a Pavlovian cue becomes attractive and exerts a biasing effect varies between individuals. In particular, Sign-Trackers (ST) and Goal-Trackers (GT) have been shown to have different learning styles, consisting of a tendency to attribute more or less incentive salience to Pavlovian reward-associated cues. In a typical Pavlovian conditioning paradigm, a CS (e.g., lever presentation) is paired with a reward (e.g., food pellet), which is delivered in a different spatial position. In such a situation, two different Conditioned Responses (CR; i.e., learned responses to a previously neutral stimulus) might be expressed. Frontiers in Behavioral Neuroscience | www.frontiersin.org Citation: Some animals approach and engage the CS (the Sign) itself and, only after its termination, reach the location of reward delivery; other animals, upon CS presentation, immediately June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 2 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino in ST individuals than in GT individuals, possibly indicating a stronger biasing effect of Pavlovian cues over behavior in the first group relative to the second. FIGURE 1 | Graphical illustration of the three tasks: Instrumental Conditioning Task (Panel A), Pavlovian Conditioning Task (Panel B); Pavlovian-to-Instrumental Transfer (PIT) Task (Panel C). Stimuli and Procedure The whole experiment consisted of three tasks. The same visual background was used in all three tasks. Four black squares (4 cm2) were displayed on a 17-inch color monitor with a black background. The squares were highlighted by a white frame and positioned as follows: top center, bottom center, right center, left center. Two black-and-white fractal images (balanced for luminance, complexity and color saturation) were used as Pavlovian cues (CS) and presented within the top center square. An image of a 10 euro cent coin was used as the reward, and a light-yellow circle (equally sized) was used as the neutral outcome (no-reward). Both these visual cues appeared within the bottom center square (Figure 1). A computer running Presentation software (Neurobehavioral Systems, Albany, CA, USA) controlled stimulus presentation. On arrival, participants were comfortably seated in a silent room and their position was centered relative to the screen, at a viewing distance of 60 cm from the eye-tracker and 75 cm from the screen. The eye-tracker was positioned under the screen, and was centered relative to both the screen and the participant. Eye- movements and behavioral responses were collected throughout the experiment and stored for offline analysis. Participants were asked to remain as still as possible to avoid confounding effects on eye-movements. The whole experiment was conducted in a dark room to facilitate eye-movement recording. The experimental session began with calibration of the eye-tracker device, during which the participant fixated nine specific points on the computer screen. The experimental session followed the standard paradigm for testing PIT. It was composed of three tasks administered in succession: an Instrumental Conditioning task, in which participants learned a response-contingent reward; a Pavlovian Conditioning task, in which participants learned a cue- contingent reward; and a PIT task, during which the influence of irrelevant Pavlovian cues on instrumental responding was tested. In each task, participants were required to pay attention to the screen and follow the instructions reported at the beginning of the task. A few example trials were always performed and, if necessary, further clarifications were given before beginning each task. At the end of the experimental FIGURE 1 | Graphical illustration of the three tasks: Instrumental Conditioning Task (Panel A), Pavlovian Conditioning Task (Panel B); Pavlovian-to-Instrumental Transfer (PIT) Task (Panel C). Stimuli and Procedure FIGURE 1 | Graphical illustration of the three tasks: Instrumental Conditioning Task (Panel A), Pavlovian Conditioning Task (Panel B); Pavlovian-to-Instrumental Transfer (PIT) Task (Panel C). session, participants completed the Barratt Impulsiveness Scale (BIS-11; Patton et al., 1995). Previous studies on animals reported an association between Sign-Tracking behavior and reduced impulse control (Flagel et al., 2011). Thus, this measure allowed further investigation into the differences between ST and GT individuals. Participants Forty-five volunteers (27 female; 2 left-handed; mean age = 24.87, sd = 2.5; mean education = 17.53, sd = 1.5) with no history of neurological diseases were recruited from the student population at the University of Bologna. All participants gave written informed consent to take part in the experiment and received payment corresponding to the amount earned during the tasks. The study was conducted in accordance with institutional guidelines and the 1964 Declaration of Helsinki. It was approved by the Ethics Committee for Psychological Research at the University of Bologna. Frontiers in Behavioral Neuroscience | www.frontiersin.org Eye Tracking E t Eye movements were recorded in a dimly lit room using a Pan/Tilt optic eye-tracker (Eye-Track ASL-6000) which registers real-time gaze at 50 Hz. Data acquired during the Pavlovian Conditioning task were analyzed offline using EyeNal Analysis Software (ASL). Dwell time during the 5 s of CS presentation was then measured for two specific areas of interest (AOI): ‘‘Sign’’, corresponding to the 4 cm square at the top center, plus a 1 cm margin; ‘‘Goal’’, corresponding to the 4 cm square at the bottom center, plus a 1 cm margin. Dwell time was defined as the amount of time during which a series of contiguous fixations remained within the same AOI. The Pavlovian speeded reaction time response described above (‘‘press the button upon patch presentation’’) has been successfully used in previous studies Talmi et al. (2008) and was introduced to obtain a behavioral measure of Pavlovian conditioning. The main reason for using a speeded response was to mirror PIT studies on animals, in which Pavlovian conditioning is measured by a behavior performed to gain the reward (e.g., latency of the first nose-poke or frequency of nose- pokes; Dickinson et al., 2000; Holland, 2004; Corbit and Balleine, 2005). The rationale here is to observe a faster reaction times when a reward was predicted (CS+ condition) than when a neutral outcome was predicted (CS−condition). To avoid a possible instrumental influence on the task, participants were explicitly told that, in this task, the reward was not contingent on their response. It was demonstrated that, if no answer was given, the patch would disappear anyway after 1.5 s, revealing the outcome. Importantly, this speeded reaction time response Sign-Tracker and Goal-Tracker Categorization Participants were categorized as ST or GT based on the oculomotor CR expressed during the Pavlovian Conditioning task. Previous studies used approaching and engaging behaviors during Pavlovian Conditioning to identify ST and GT. In these studies, the numbers of contacts with the Sign (i.e., lever) and the Goal (i.e., food tray) were compared to obtain an index of behavior, and divide the subjects into ST (i.e., high probability to engage the lever) and GT (i.e., high probability to engage the food-tray) individuals (Flagel et al., 2007, 2008, 2011; Robinson and Flagel, 2009; Saunders and Robinson, 2013; Robinson et al., 2014). Pavlovian-to-Instrumental Transfer (PIT) Task Participants received exactly the same instructions as in the Instrumental Conditioning phase requiring them to choose between the right and left white squares. The task was identical to the Instrumental Conditioning task, except in two aspects: first, the task-irrelevant Pavlovian CS were presented sequentially within the top square, changing every 30 s, the task was completely performed in extinction, so all choices always lead to no-reward. (Figure 1C). Extinction is a standard procedure for assessing PIT, both in human and animal research, since it allows one to test the influence of Pavlovian cues on instrumental responding without the confounding effects of the reward (Rescorla, 1994a,b; Corbit et al., 2001; Bray et al., 2008; Talmi et al., 2008). Indeed, the rationale here is to test the ability of a task-irrelevant Pavlovian cue to drive choices (presumably, towards the response previously associated with a reward) even if the reward is not available anymore. The PIT task lasted about 6 min, during which subjects were free to perform as many choices as they wished, with no time pressure. Instrumental Conditioning Task Participants were instructed to choose between two squares to gain a reward. One square was paired with an actual monetary Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 3 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino win (Rewarded Choice), while the other was paired with a neutral outcome (Unrewarded Choice). The right and left squares were presented in white and indicated as possible choices to be selected by a mouse click. The mouse pointer was centrally positioned before each choice, in order to not encourage a specific choice. Only one square was associated with a reward following a partial reinforcement schedule, so that between one reward and the next a variable interval between 4 and 12 s was always associated with no-reward. After each choice, a corresponding neutral image (light-yellow circle) or reward image (10 euro cents coin) appeared for 1 s in the bottom square (Figure 1A). Participants were aware that they would receive an actual payment corresponding to the amount of coins collected during the task. The association between square and outcome was counterbalanced across subjects. The rationale of this task was to make participants learn an association between a specific response (left or right square) and the reward; thus, participants would get a higher frequency of Rewarded Choices if they learned the correct association. The task lasted about 6 min, during which subjects were free to perform as many choices as they wished, with no time pressure. allowed us to obtain a measure of the learning rate that is independent from ST/GT behavior. To identify ST and GT CR, eye-movements were recorded in order to evaluate contiguous eye-gazes directed toward the ‘‘Sign’’ (top center square) and the ‘‘Goal’’ (bottom center square). Mirroring animal studies, these two CR were subsequently used to distinguish participants as ST or GT, depending on the tendency to direct eye-gaze toward the Sign or the Goal during the 5 s of CS presentation (Flagel et al., 2011). Pavlovian Conditioning Task In each trial, one of two possible visual cues (fractal images) appeared for 5 s within the top square, followed by a white patch within the bottom square. Upon presentation of the patch, participants were instructed to press the left-Ctrl button on the keyboard as quickly as possible to remove the patch and discover the outcome hidden below. To perform this button press, participants did not need to remove their gaze from the screen. The outcome was then presented for 1 s. One fractal was associated with a reward (10 euro cent coin) on 80% of trials (CS+), while the other fractal was associated with no-reward (light-yellow circle) on all trials (CS−; Figure 1B). The task consisted of 40 trials (20 per condition) with a variable inter-trial- interval between 0.5 and 4 s. Participants were aware that they would receive an actual payment corresponding to the amount of coins collected during the task. The association between visual cue and outcome was counterbalanced across subjects. The whole task lasted around 6 min. Frontiers in Behavioral Neuroscience | www.frontiersin.org Eye Tracking E t This method was adapted in the present experiment by calculating contiguous eye-gazes (Dwell Time) toward the cue (Sign) and the reward (Goal) AOI, during CS Frontiers in Behavioral Neuroscience | www.frontiersin.org Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 4 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino presentation (see above). ST behavior has been defined as a CR to approach and engage ‘‘the cue or sign that indicates impending reward delivery’’; while GT behavior has been defined as a tendency to ‘‘engage the location of unconditioned cue delivery, even though it is not available until conditioned cue termination’’ (Flagel et al., 2011). Thus, a learned oculomotor CR towards the location of the Sign or the Goal is a practical method for distinguishing between ST and GT individuals. On this basis, an eye-gaze index was created based on the Dwell Time spent on the Sign and Goal locations. An individual dwell is defined as the time period during which a fixation or series of temporally contiguous fixations remain within an AOI. That is, an individual dwell is defined as the sum of the durations across all fixations within the current AOI, from entry to exit. To compute fixations, EyeNal ASL was used, which defines a fixation if the observer’ s gaze position remains within a diameter of 0, 5◦of visual angle for at least 120 ms (six consecutive samples, at 50 Hz sampling rate; Eye-Analysis software Manual, v. 1.41, Applied Science Laboratories, 2007). The Dwell Time spent on the Sign and Goal locations was calculated for each trial and then averaged for each participant. The eye-gaze index was calculated as the difference between the Dwell Time on Sign minus the Dwell Time on Goal over the total Dwell Time (Sign −Goal/Sign + Goal), so that a higher value corresponded to a higher Dwell Time toward the Sign (Sign-Tracking behavior) and a lower value corresponded to a higher Dwell Time toward the Goal (Goal-Tracking behavior). Since the interest here was to disentangle two reward-specific CR, only CS+ trials in the second half of the task were considered, when contingency learning was more established. Based on this index, the top and bottom 50% of the total sample were categorized as ST (eye-gaze index between 0.38 and 1.00) and GT (eye-gaze index between −1.00 and 0.27), respectively. FIGURE 2 | Oculomotor response. Eye Tracking E t (Panels A and B) show the eye-gaze index in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) and the two task hemiblocks. (Panel A) represents CS+ trails and (Panel B) represents CS−trials. (Panels C and D) show the eye-gaze index in the two conditions (CS+ = reward-associated cue; CS−= neutral cue) in ST and GT, respectively. (Panel E) shows visual exploratory behavior in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) throughout the task. Bars indicate standard error of the mean. *p < 0.05; **p < 0.01. FIGURE 2 | Oculomotor response. (Panels A and B) show the eye-gaze FIGURE 2 | Oculomotor response. (Panels A and B) show the eye-gaze index in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) and the two FIGURE 2 | Oculomotor response. (Panels A and B) show the eye-gaze index in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) and the two task hemiblocks. (Panel A) represents CS+ trails and (Panel B) represents CS−trials. (Panels C and D) show the eye-gaze index in the two conditions (CS+ = reward-associated cue; CS−= neutral cue) in ST and GT, respectively. (Panel E) shows visual exploratory behavior in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) throughout the task. Bars indicate standard error of the mean. *p < 0.05; **p < 0.01. Frontiers in Behavioral Neuroscience | www.frontiersin.org ST and GT CR In both groups a significant difference Frontiers in Behavioral Neuroscience | www.frontiersin.org Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 5 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino between the two conditions was found. The ST group showed a significantly higher eye-gaze index in the CS+ condition than in the CS−condition (t(21) = 1.69; one-tailed p = 0.03; Cohen’s d = 0.19), indicating a greater tendency to direct contiguous eye-gazes towards the Sign during CS+ trials than during CS− trials (Figure 2C). The GT group showed a significantly lower eye-gaze index in the CS+ condition than in the CS−condition (t(21) = 2.21; one-tailed p = 0.01; Cohen’s d = 0.24), indicating a greater tendency to direct contiguous eye-gazes towards the Goal during CS+ trials than during CS−trials (Figure 2D). FIGURE 3 | Learning rates in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) during Instrumental Conditioning (Panel A) and Pavlovian Conditioning (Panel B). Bars indicate standard error of the mean. ***p < 0.001. g g g Given the specific spatial locations of the Sign and the Goal in the present paradigm, visual exploratory behavior was also considered by analyzing the total dwell time spent on the top and the bottom portions of the screen, in order to exclude the presence of a spatial bias that could account for ST and GT behavior. A mixed-effects model was used, with Group (ST/GT) and AOI (Top/Bottom) as independent variables and Total Dwell Time as dependent variable. Subjects were modeled as a random effect. Assumptions of normal distribution, independence of residuals and sphericity were verified. Results showed a marginal main effect of AOI (F(1,42) = 4.01; two-tailed p = 0.05; part. η2 = 0.09), with more Dwell Time spent on the Top of the screen (mean = 0.76; sd = 0.91) than on the Bottom (mean = 0.41; sd = 0.64) in both groups (Figure 2E). Neither group differences, nor interaction effects emerged (ps > 0.87). These results strengthen the evidence that the behavioral differences observed between ST and GT cannot be ascribed to a mere spatial bias towards the upper or the lower part of the screen. The general difference in time spent looking at the Top and the Bottom of the screen is compatible with the fact that dwell time was calculated during the 5 s of CS presentation. ST and GT CR To ensure that the oculomotor responses used to categorize ST and GT individuals were learned CRs, eye-gaze indices were separately analyzed for CS+ and CS−trials in the first and second halves of the Pavlovian Conditioning task. Two separate mixed-effects models with Group (ST/GT) and Hemiblock (1/2) as independent variables were performed for CS+ and CS−conditions. The eye-gaze index described above was the dependent variable. Subjects were modeled as a random effect. Assumptions of normal distribution, independence of residuals and sphericity were verified. Results from CS+ trials showed a significant interaction effect (F(1,42) = 14.75; two- tailed p = 0.0004; part. η2 = 0.26). Bonferroni-corrected post hoc tests revealed a significant difference (p = 0.003) between ST (mean = 0.35; sd = 0.77) and GT (mean = −0.06; sd = 0.79) in the second Hemiblock (Figure 2A). No other post hoc comparisons were significant (ps > 0.15). Results from CS−trials did not show any significant effects (ps > 0.05; Figure 2B). Overall, these results indicate two important points: first, a bias toward either the Sign or the Goal is a learned CR, since it is not present at the beginning of the task but emerges later in time, when contingencies have been learned (Figure 2A); moreover, this looking bias is specific to the reward-paired cue (CS+), as no differences were observed for the unpaired cue (CS−; Figure 2B). In Figure 2A it is evident how, at the beginning of the Pavlovian task, during CS+ presentation, no tendency seems evident, while, towards the end ST show higher Dwell Time towards the Sign (eye-gaze index increases) while GT show higher Dwell Time towards the Goal (eye-gaze index decreases). Figure 2B, on the other hand, shows that the same pattern is not observable during the presentation of the neutral stimulus (CS−). To further test that this behavior is a reward-specific CR, the eye gaze index was also directly compared between CS+ and CS−trials from the second hemiblock (when contingencies had been learned) within each group. Two separate paired t-tests were performed for the ST and GT groups, using Condition (CS+/CS−) as the independent variable and the eye-gaze index as the dependent variable. ST and GT CR These results thus indicate that both groups spent more time visually exploring the region of the screen where a stimulus was being presented (Top), rather than where there was no stimulus (Bottom). No difference in this spatial bias was found between the two groups (Figure 2E). FIGURE 3 | Learning rates in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) during Instrumental Conditioning (Panel A) and Pavlovian Conditioning (Panel B). Bars indicate standard error of the mean. ***p < 0.001. (mean = 22.09; sd = 9.10; Figure 3A). Neither group differences, nor interaction effects emerged (ps > 0.55). These results indicate that the ST and GT groups learned to discriminate between the rewarding and non-rewarding choices equally well. Pavlovian Conditioning To ensure that Pavlovian learning occurred in both ST and GT groups, reaction times to patch presentation were analyzed. If participants correctly learned to discriminate between the two Pavlovian cues, faster reaction times should be observed for CS+ trials relative to CS−trials. A mixed-effects model was used, with Condition (CS+/CS−) and Group (ST/GT) as independent variables, and reaction times as the dependent variable. Subjects were modeled as a random effect. Assumptions of normal distribution, independence of residuals and sphericity were verified. Results showed a significant main effect of Condition (F(1,842) = 110.24; two-tailed p = 0.0001; part. η2 = 0.72), with faster reaction times for CS+ trials (mean = 306.33; sd = 44.41) relative to CS−trials (mean = 351.21; sd = 50.05; Figure 3B). Neither group differences, nor interaction effects emerged (ps > 0.29). These results indicate that participants generally reacted more quickly to the patch on trials with the reward-paired cue (CS+) than on trials with the unpaired cue (CS−). This reward-specific response facilitation indicates successful Pavlovian conditioning in both ST and GT. Taken together, the last two analyses demonstrated that group differences in the tendency to direct contiguous eye-gazes to the location of the Sign or the Goal cannot be ascribed to a mere spatial bias, but rather reflect a learned reward-related CR. Instrumental Conditioning To ensure that instrumental conditioning was successful in both the ST and the GT groups, so that all participants learned which response leads to a reward, the number of choices (mouse clicks) made on the two white squares were compared. Choosing the square associated with reward was considered a Rewarded Choice, and choosing the square associated with no-reward was considered an Unrewarded Choice. A mixed- effects model was used, with Choice (Rewarded/Unrewarded) and Group (ST/GT) as independent variables and the number of choices as the dependent variable. Subjects were modeled as a random effect. Assumptions of normal distribution, independence of residuals and sphericity were verified. Results showed a main effect of Choice (F(1,42) = 20.88; two-tailed p < 0.0001; part η2 = 0.33), with Rewarded Choices (mean = 32.80; sd = 9.38) occurring more frequently than Unrewarded Choices Pavlovian-to Instrumental Transfer Pavlovian-to Instrumental Transfer To test for PIT, the numbers of Congruent choices (associated with the reward during Instrumental Conditioning) and Incongruent choices (associated with no-reward during Instrumental Conditioning) during CS+ and CS−presentation were compared. A response index was calculated as the probability of selecting the Congruent choice minus the probability of selecting the Incongruent choice (number of congruent—incongruent choices/total number of choices). Higher values correspond to a higher probability of making the Congruent choice, while lower values correspond to a higher probability of making the Incongruent choice. A mixed-effects model was used, with Condition (CS+/CS−) and June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 6 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino FIGURE 4 | Pavlovian-to-Instrumental Transfer (PIT). (Panel A) shows the response index (Congruent-Incongruent/Total) in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) during CS−and CS+ trials. (Panels B and C) show the response index over time by dividing the task into three blocks of two trials. (Panels D and E) show the number of responses. Bars indicate standard error of the mean. *p < 0.05; ***p < 0.001. Subjects were modeled as a random effect. Assumptions of normal distribution, independence of residuals and sphericity were verified. Results showed a significant main effect of Condition (F(1,42) = 6.39; two-tailed p = 0.02; part. η2 = 0.13), a significant Condition × Group interaction (F(1,42) = 7.69; two- tailed p = 0.008; part. η2 = 0.15), and a significant Block × Group interaction (F(1.27,53.32) = 50.61; two-tailed p < 0.001; part. η2 = 0.5; Figures 4B,C). Bonferroni-corrected post hoc tests on the Condition × Group interaction revealed a significant difference (p = 0.003) between CS+ and CS−in ST group but not the GT group, and a significant difference (p = 0.02) between ST and GT groups in CS+ trials (Figures 4B,C). Bonferroni- corrected post hoc tests on the Block × Group interaction revealed a significant difference (p < 0.0001) between ST and GT groups in the third block, but not in the first and second blocks (Figures 4B,C). Figures 4D,E show the number of responses. In line with the results of the first analysis, these results showed that, unlike GT, ST group was more likely to choose the congruent option when they saw the task-irrelevant CS+ than when they saw the CS−, throughout the entire PIT task. Pavlovian-to Instrumental Transfer The only effect of time revealed by this analysis was in the last block, where a group difference in responses emerged. Since this difference was unrelated to the displayed stimulus (CS+/CS−), it does not constitute a difference in PIT. This result instead indicates that the ST and GT groups differed in the proportion of congruent choice made towards the end of the task. Frontiers in Behavioral Neuroscience | www.frontiersin.org Impulsiveness To further investigate differences between ST and GT individuals, self-reported impulsiveness, as rated by the BIS-11 questionnaire (Patton et al., 1995), was compared between the two groups. A two-sample t-test was performed using Group (ST/GT) as the independent variable and BIS-11 scores as the dependent variable. Results revealed a significant difference between the two groups (t(28.75) = 2.06; two-sided p = 0.04, with the ST group (mean = 61.0; sd = 9.91) showing higher impulsiveness than the GT group (mean = 54.09; sd = 8.86; Figure 5). This finding is consistent with previous studies showing significantly higher levels of impulsiveness as compared to GT (Tomie et al., 2000; Flagel et al., 2009). FIGURE 4 | Pavlovian-to-Instrumental Transfer (PIT). (Panel A) shows the response index (Congruent-Incongruent/Total) in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) during CS−and CS+ trials. (Panels B and C) show the response index over time by dividing the task into three blocks of two trials. (Panels D and E) show the number of responses. Bars indicate standard error of the mean. *p < 0.05; ***p < 0.001. FIGURE 4 | Pavlovian-to-Instrumental Transfer (PIT). (Panel A) shows Group (ST/GT) as independent variables and the response index, described above, as the dependent variable. Subjects were modeled as a random effect. Assumptions of normal distribution, independence of residuals and sphericity were verified. Results showed a significant Condition × Group interaction (F(1,42) = 8.22; two-tailed p = 0.006; part. η2 = 0.16). Bonferroni-corrected post hoc comparisons revealed a significant difference (p = 0.001) between CS+ (mean = 0.18; sd = 0.12) and CS−(mean = 0.04; sd = 0.13) only in ST group, and a significant difference (p = 0.04) between ST (mean = 0.18; sd = 0.12) and GT (mean = 0.08; sd = 0.12) during CS+ (Figure 4A). No other comparisons were significant (ps > 0.13). These results indicate that the ST group was more likely to choose the congruent option when they saw the task-irrelevant CS+ than when they saw the CS−. thus revealing a PIT effect. Critically, this bias was stronger in ST than in GT individuals. Group (ST/GT) as independent variables and the response index, described above, as the dependent variable. Subjects were modeled as a random effect. Assumptions of normal distribution, independence of residuals and sphericity were verified. Results showed a significant Condition × Group interaction (F(1,42) = 8.22; two-tailed p = 0.006; part. Discussion Importantly, the present results demonstrate that this oculomotor CR was: (i) acquired over time (i.e., learned), since a specific CR towards the Sign or the Goal only emerged towards the end of the task, when stimulus- reward associations had been acquired selectively during the presentation of reward-paired cues (CS+; Figures 2A,B); and (ii) reward specific, since the CR was only evident when participants saw the reward-related cue (CS+) and not when they saw the neutral cue (CS−; Figures 2C,D). Coherently with what expected, the task-irrelevant CS had a much stronger influence on the ST group than on the GT group during the PIT task. Since the ‘‘Sign’’ and the ‘‘Goal’’ had specific spatial locations (the top and bottom portions of the screen, respectively), it is important to rule out the possibility that spatial biases in gaze direction might account for the difference in the PIT effect between groups. A bias towards looking at the top of the screen might cause result in a stronger influence of the Sign on the ST group just because they spent more time looking at it. Analysis of visual exploratory behavior during Pavlovian Conditioning, however, revealed that the ST and GT groups did not differ in the total amount of time spent looking at the top and bottom of the screen (Figure 2E). Critically, behavioral differences only emerged during CS+ trials towards the end of the task, once the association between the cue and the reward had been learned. Consequently, it is concluded that there was no a priori bias in gaze direction; rather, such a bias emerged during the Pavlovian Conditioning task as a learned reward-specific CR. Moreover, a recent study (Trick et al., 2011) directly investigated the relation between fixation times during Pavlovian learning and the PIT effect. The authors found that fixation times during Pavlovian learning increased with uncertainty (that is, more attention was paid to stimuli with uncertain outcome probabilities, e.g., 50%, than to stimuli with more certain outcome probabilities, e.g., 90%). In contrast, the PIT effect increased with the probability of reward (that is, it was stronger for stimuli associated with a high probability of reward, e.g., 90%, than for stimuli associated with uncertain outcomes, e.g., 50%, or a low probability of reward, e.g., 10%). Thus, Trick et al. Discussion contingencies. In line with the animal literature (Robinson and Flagel, 2009), the Pavlovian cue (CS+) was clearly predictive of reward, since it elicited faster reaction times during Pavlovian conditioning than the neutral stimulus (CS−) did in both groups, along with a CR corresponding to the behavioral profile of each group (ST/GT). Motivated behavior is characterized by a wide span of inter- individual differences in both human and non-human animals. In the present study, the PIT paradigm was used to examine individual differences in the excitatory influence that signals associated with reward can exert on human choices. PIT is a well-known procedure for testing the ability of a Pavlovian reward-paired cue to acquire incentive motivational properties and influence instrumental performance (Estes, 1943, 1948; Rescorla and Solomon, 1967; de Wit and Dickinson, 2009; Holmes et al., 2010). Here, participants performed a standard PIT paradigm composed of three tasks: an Instrumental Conditioning task, during which response-outcome associations were learned; a Pavlovian Conditioning task, during which stimulus-outcome associations were leaned; and a PIT task, in which the ability of a Pavlovian cue to drive instrumental responses was tested. Individual differences were characterized by two distinct oculomotor CR exhibited during Pavlovian Conditioning, corresponding to two different learning styles previously identified and described in animal literature: Sign- Tracking (ST) and Goal-Tracking (GT; Estes, 1943, 1948; Boakes, 1977; Flagel et al., 2011). In the present study, ST behavior consisted of a tendency to direct contiguous eye-gazes towards the cue (CS) that indicated impending reward delivery (Sign); in contrast, GT behavior was characterized by a tendency to direct contiguous eye-gazes towards the location of reward (US) delivery (Goal), even if not available until CS termination. An eye-gaze index was based on the emergence of these two behavioral patterns during presentation of the reward-paired stimulus (CS+) in the second half of the task (when contingencies had been learned), and a median split was used to categorize participants as ST or GT. Impulsiveness η2 = 0.16). Bonferroni-corrected post hoc comparisons revealed a significant difference (p = 0.001) between CS+ (mean = 0.18; sd = 0.12) and CS−(mean = 0.04; sd = 0.13) only in ST group, and a significant difference (p = 0.04) between ST (mean = 0.18; sd = 0.12) and GT (mean = 0.08; sd = 0.12) during CS+ (Figure 4A). No other comparisons were significant (ps > 0.13). These results indicate that the ST group was more likely to choose the congruent option when they saw the task-irrelevant CS+ than when they saw the CS−. thus revealing a PIT effect. Critically, this bias was stronger in ST than in GT individuals. FIGURE 5 | Impulsiveness levels in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) as measured by the Barratt Impulsiveness Scale (BIS-11). Bars indicate standard error of the mean. *p < 0.05. While the first analysis on PIT focused on the overall effect, a second analysis divided the task into three equal blocks of 2 min (four trials) to check for differences in task performance over time. A mixed-effects model was used, with Condition (CS+/CS−), Group (ST/GT) and Block (1/2/3) as independent variables, and the response index as the dependent variable. FIGURE 5 | Impulsiveness levels in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) as measured by the Barratt Impulsiveness Scale (BIS-11). Bars indicate standard error of the mean. *p < 0.05. June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 7 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino Frontiers in Behavioral Neuroscience | www.frontiersin.org Discussion suggesting that the group difference in the PIT effect most likely reflects greater incentive salience to reward cues in ST than in GT individuals. A group difference in the overall amount of congruent responses (during both CS+ and CS−presentation, thus not reflecting PIT) emerged towards the end of the task (Figures 4B,C). for differences in dopamine responsiveness, it has been argued that higher reward-related dopamine release before conditioning might increase attribution of incentive salience to reward-related cues (Wyvell and Berridge, 2000, 2001). Additionally, Flagel et al. (2011) directly demonstrated that dopaminergic transmission is not involved in all forms of learning, but it is necessary for the acquisition of a sign-tracking CR, playing a crucial role in the assignment of incentive salience to reward-related cues. The same study also showed that dopaminergic prediction-error signals, coded by activity in the nucleus accumbens, are present in ST individuals, but not in GT individuals. In the present study, a similar mechanism might occur: high levels of dopamine release might boost attribution of incentive salience to reward-related cues, increasing their ability to motivate and drive behavior. Previous studies have found an association between ST behavior and other traits, such as higher levels of behavioral impulsivity and a greater propensity to develop addiction (Tomie et al., 1998; Flagel et al., 2008; Robinson and Flagel, 2009). In line with these studies, the present study found reduced self- reported impulse control in the ST group than in the GT group (Figure 5). These findings seem to corroborate the idea that ST and GT behaviors are just one expression of a broader profile of individual differences, which might be clinically relevant. Many studies have reported that ST individuals are more impulsive and prone to develop potentially maladaptive behaviors, such as addiction (Tomie et al., 1998; Robinson and Flagel, 2009; Flagel et al., 2011). For example, the propensity to sign-track is associated with a stronger effect of psychomotor sensitization, a higher susceptibility to a form of cocaine-induced plasticity that may contribute to the development of addiction (Flagel et al., 2008). Furthermore, ST behavior in relation to a specific Pavlovian cue (i.e., a cue predicting monetary reward) is also predictive of the propensity to attribute incentive salience to other reward-paired cues, such as food-related or drug-related cues (e.g., cocaine and alcohol; Uslaner et al., 2006; Cunningham and Patel, 2007; Flagel et al., 2008; Clark et al., 2013). Discussion The extent to which such individual differences might play a role in the development of addiction and in the propensity to relapse is not yet clear, but their implications for developing individually targeted treatment programs are promising. g y Future studies might further investigate individual differences in the influence of Pavlovian cues on behavior by taking additional measures into account, such as phasic dopamine levels, psychophysiological indices (e.g., galvanic skin response and heart rate) and as neuroimaging measurements. These methods would allow better comparisons between human and non-human animal research on individual differences in ST/GT behavior and learning styles. A general limitation in the standard PIT paradigm is that the ‘‘Sign’’ and the ‘‘Goal’’ are presented in distinct spatial locations. Thus, unrelated spatial biases in gaze direction might obscure the effect of interest. Although the analysis conducted in this study already confirmed that the present findings cannot be accounted for by any a priori difference in spatial bias between groups, another way to control for this possibility would be to replicate the experiment with the spatial positions of the ‘‘Sign’’ and the ‘‘Goal’’ inverted in the three tasks. In conclusion, the individual differences demonstrated here offer a promising direction for further investigating the degree to which incentive salience is attributed to environmental stimuli associated with rewards, as well as the link between this process and maladaptive behaviors, ranging from over- eating to pathological gambling and addiction (Saunders and Robinson, 2013). Further, the present findings have important implications for the treatment of impulse-control disorders. Overall, these individual differences in PIT offer new insights into the mechanisms underlying the transition from intentional to habitual/compulsive behavior. It should be noted that some recent studies highlighted a more complex scenario relating ST and GT behaviors to addiction. While ST individuals are more susceptible to the influence of discrete cues, GT individuals are more influenced by contextual cues, which can motivate drug-seeking behavior (Robinson et al., 2014). Consequently, these learning styles seem to reflect differences in the kinds of triggers to which the individual is susceptible (e.g., discrete/contextual), rather than a propensity to addiction per se. This finding emphasizes that there are diverse pathways to addiction, and has remarkable implications for the development of personalized treatments in the future. Author Contributions But what exactly is the mechanism underlying the attribution of incentive salience to discrete stimuli, such as Pavlovian cues? A large amount of evidence points to the role of dopaminergic transmission within circuits known to be involved in addiction. The core of the nucleus accumbens, for example, was reported to be involved in ST behavior, and mediates the reinstatement of drug-seeking and drug-taking behavior (Flagel et al., 2007, 2008, 2011; Clark et al., 2013). Furthermore, various studies have supported the involvement of the mesolimbic dopamine system in the emergence of ST behavior. ST individuals are characterized by stronger dopaminergic gene expression and increased levels of dopamine in the nucleus accumbens (correlated with the vigor with which the CR is performed; Flagel et al., 2007, 2008). Even if differences in basic dopaminergic levels cannot fully account All authors conceived of and designed the experiment; S. G. programmed the task, ran the experiment, analyzed the data, wrote the main manuscript text and prepared the figures; all authors read, corrected and approved the final manuscript. Discussion (2011) concluded that the behavioral influence exerted by CS (i.e., the PIT effect) is dissociated from attention to Pavlovian stimuli in humans, (see Kaye and Pearce, 1984, for similar findings in animals). Instead, PIT is linked to the predictive value acquired by stimuli during learning. ST behavior has been explained as a consequence of attributing incentive salience to reward-paired cues (Pavlovian CS), arising from the interaction between previous experience (reinforcement learning processes) and individual propensities (Berridge, 2001; Berridge and Robinson, 2003; Flagel et al., 2011). This incentive salience motivates reward-related action (Tomie et al., 2000; Flagel et al., 2008; Robinson and Flagel, 2009). In the present study, ST and GT groups differed in the extent to which Pavlovian reward-paired cues biased their behavior: relative to the GT group, the ST group showed an increased likelihood of performing the instrumental response independently paired with the same reward when presented with the task-irrelevant reward- paired cue, even if the reward itself was no longer available (i.e., a stronger PIT effect; Figure 4A). Therefore, reward-paired cues exerted a stronger source of influence on the behavior of ST individuals, as predicted. Importantly, time course analysis revealed that this effect occurred early and remained stable throughout the entire PIT test session (Figures 4B,C), thereby Group differences in the PIT effect are not attributable to differences in the strength of Instrumental or Pavlovian learning between the groups, which could have potentially induced a bias towards the rewarded choice in the Instrumental Conditioning task, or a stronger influence of the reward-paired cue in the second Pavlovian Conditioning task. Analyses of both the number of rewarded choices during Instrumental Conditioning, and reaction times during Pavlovian Conditioning, exclude such a possibility by revealing that both the ST and GT groups learned the response-outcome and stimulus-outcome contingencies equally well (Figure 3). Consequently, differences in the PIT effect cannot be explained by group differences in the ability to learn either the instrumental or the Pavlovian June 2015 | Volume 9 | Article 163 8 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino suggesting that the group difference in the PIT effect most likely reflects greater incentive salience to reward cues in ST than in GT individuals. A group difference in the overall amount of congruent responses (during both CS+ and CS−presentation, thus not reflecting PIT) emerged towards the end of the task (Figures 4B,C). Frontiers in Behavioral Neuroscience | www.frontiersin.org References Everitt, B. J., and Robbins, T. W. (2005). Neural systems of reinforcement for drug addiction: from actions to habits to compulsion. Nat. Neurosci. 8, 1481–1489. doi: 10.1038/nn1579 Allman, M. J., DeLeon, I. G., Cataldo, M. F., Holland, P. C., and Johnson, A. W. (2010). Learning processes affecting human decision making: an assessment of reinforcer-selective Pavlovian-to-instrumental transfer following reinforcer devaluation. J. Exp. Psychol. Anim. Behav. Process. 36, 402–408. doi: 10. 1037/a0017876 Flagel, S. B., Akil, H., and Robinson, T. E. (2009). Individual differences in the attribution of incentive salience to reward-related cues: implications for addiction. Neuropharmacology 56, 139–148. doi: 10.1016/j.neuropharm.2008. 06.027 devaluation. J. Exp. Psychol. Anim. Behav. Process. 36, 402–408. doi: 10. 1037/a0017876 Flagel, S. B., Clark, J. J., Robinson, T. E., Mayo, L., Czuj, A., Willuhn, I., et al. (2011). A selective role for dopamine in stimulus—reward learning. Nature 469, 53–57. doi: 10.1038/nature09588 Balleine, B. (1994). Asymmetrical interactions between thirst and hunger in Pavlovian-instrumental transfer. Q. J. Exp. Psychol. B 47, 211–231. Berridge, K. C. (2001). Reward learning: reinforcement, incentives and expectations. Psychol. Learn. Motiv. 40, 223–278. doi: 10.1016/s0079- 7421(00)80022-5 Flagel, S. B., Watson, S. J., Akil, H., and Robinson, T. E. (2008). Individual differences in the attribution of incentive salience to a reward-related cue: influence on cocaine sensitization. Behav. Brain Res. 186, 48–56. doi: 10.1016/j. bbr.2007.07.022 Berridge, K. C., and Robinson, T. E. (2003). Parsing reward. Trends Neurosci. 26, 507–513. doi: 10.1016/s0166-2236(03)00233-9 Flagel, S. B., Watson, S. J., Robinson, T. E., and Akil, H. (2007). Individual differences in the propensity to approach signals vs. goals promote different adaptations in the dopamine system of rats. Psychopharmacology (Berl) 191, 599–607. doi: 10.1007/s00213-006-0535-8 Boakes, R. A. (1977). ‘‘Performance on learning to associate a stimulus with positive reinforcement,’’ in Operant-Pavlovian Interactions, eds H. Davis and H. M. B. Hurwitz (Hillsdale, NJ: Erlbaum), 67–97. Bray, S., Rangel, A., Shimojo, S., Balleine, B., and O’Doherty, J. P. (2008). The neural mechanisms underlying the influence of pavlovian cues on human decision making. J. Neurosci. 28, 5861–5866. doi: 10.1523/jneurosci.0897-08. 2008 Hogarth, L., Balleine, B. W., Corbit, L. H., and Killcross, S. (2013a). Associative learning mechanisms underpinning the transition from recreational drug use to addiction. Ann. N Y Acad. Sci. 1282, 12–24. doi: 10.1111/j.1749-6632.2012. 06768.x Clark, J. J., Collins, A. L., Sanford, C. A., and Phillips, P. E. (2013). Dopamine encoding of Pavlovian incentive stimuli diminishes with extended training. J. Neurosci. 33, 3526–3532. References doi: 10.1523/JNEUROSCI.5119-12.2013 Hogarth, L., Dickinson, A., and Duka, T. (2010). The associative basis of cue- elicited drug taking in humans. Psychopharmacology (Berl) 208, 337–351. doi: 10.1007/s00213-009-1735-9 Colwill, R. M., and Rescorla, R. A. (1988). The role of response-reinforcer associations increases throughout extended instrumental training. Anim. Learn. Behav. 16, 105–111. doi: 10.3758/bf03209051 Hogarth, L., Dickinson, A., Wright, A., Kouvaraki, M., and Duka, T. (2007). The role of drug expectancy in the control of human drug seeking. J. Exp. Psychol. Anim. Behav. Process. 33, 484–496. doi: 10.1037/0097-7403.33.4.484 Corbit, L. H., and Balleine, B. W. (2003). Instrumental and Pavlovian incentive processes have dissociable effects on components of a heterogeneous instrumental chain. J. Exp. Psychol. Anim. Behav. Process. 29, 99–106. doi: 10. 1037/0097-7403.29.2.99 Hogarth, L., Field, M., and Rose, A. K. (2013b). Phasic transition from goal- directed to habitual control over drug-seeking produced by conflicting reinforcer expectancy. Addict. Biol. 18, 88–97. doi: 10.1111/adb.12009 Holland, P. C. (2004). Relations between Pavlovian-instrumental transfer and reinforcer devaluation. J. Exp. Psychol. Anim. Behav. Process. 30, 104–117. doi: 10.1037/0097-7403.30.2.104 Corbit, L. H., and Balleine, B. W. (2005). Double dissociation of basolateral and central amygdala lesions on the general and outcome-specific forms of pavlovian-instrumental transfer. J. Neurosci. 25, 962–970. doi: 10. 1523/jneurosci.4507-04.2005 Holland, P. C., and Gallagher, M. (2003). Double dissociation of the effects of lesions of basolateral and central amygdala on conditioned stimulus- potentiated feeding and Pavlovian-instrumental transfer. Eur. J. Neurosci. 17, 1680–1694. doi: 10.1046/j.1460-9568.2003.02585.x Corbit, L. H., Muir, J. L., and Balleine, B. W. (2001). The role of the nucleus accumbens in instrumental conditioning: evidence of a functional dissociation between accumbens core and shell. J. Neurosci. 21, 3251–3260. Holland, P. C., Petrovich, G. D., and Gallagher, M. (2002). The effects of amygdala lesions on conditioned stimulus-potentiated eating in rats. Physiol. Behav. 76, 117–129. doi: 10.1016/s0031-9384(02)00688-1 Cunningham, C. L., and Patel, P. (2007). Rapid induction of Pavlovian approach to an ethanol-paired visual cue in mice. Psychopharmacology (Berl) 192, 231–241. doi: 10.1007/s00213-007-0704-4 Delamater, A. R. (1995). Outcome-selective effects of intertrial reinforcement in a Pavlovian appetitive conditioning paradigm with rats. Anim. Learn. Behav. 23, 31–39. doi: 10.3758/bf03198013 Holmes, N. M., Marchand, A. R., and Coutureau, E. (2010). Pavlovian to instrumental transfer: a neurobehavioural perspective. Neurosci. Biobehav. Rev. 34, 1277–1295. doi: 10.1016/j.neubiorev.2010.03.007 Kaye, H., and Pearce, J. M. (1984). The strength of the orienting response during Pavlovian conditioning. J. Exp. Psychol. Anim. Behav. Process. 10, 90–109. doi: 10.1037/0097-7403.10.1.90 Delamater, A. R. (1996). Acknowledgments The authors thank Sara Moroni, Francesca Casadei and Chiara Lancioni for helping with data collection. This work was supported by grants from the Ministero Istruzione Università Ricerca (PRIN 2010, protocol number: 2010XPMFW4_009) awarded to GdP. Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 9 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino References Effects of several extinction treatments upon the integrity of Pavlovian stimulus-outcome associations. Anim. Learn. Behav. 24, 437–449. doi: 10.3758/bf03199015 Lovibond, P. F. (1981). Appetitive Pavlovian-instrumental interactions: effects of inter-stimulus interval and baseline reinforcement conditions. Q. J. Exp. Psychol. B 33(Pt. 4), 257–269. doi: 10.1080/14640748108400811 Delamater, A. R., and Holland, P. C. (2008). The influence of CS-US interval on several different indices of learning in appetitive conditioning. J. Exp. Psychol. Anim. Behav. Process. 34, 202–222. doi: 10.1037/0097-7403.34.2.202 Lovibond, P. F., and Colagiuri, B. (2013). Facilitation of voluntary goal- directed action by reward cues. Psychol. Sci. 24, 2030–2037. doi: 10. 1177/0956797613484043 de Wit, S., and Dickinson, A. (2009). Associative theories of goal-directed behaviour: a case for animal-human translational models. Psychol. Res. 73, 463–476. doi: 10.1007/s00426-009-0230-6 Nadler, N., Delgado, M. R., and Delamater, A. R. (2011). Pavlovian to instrumental transfer of control in a human learning task. Emotion 11, 1112–1123. doi: 10. 1037/a0022760 Dickinson, A. D., and Balleine, B. W. (1994). Motivational control of goal-directed action. Anim. Learn. Behav. 22, 1–18. doi: 10.3758/bf03199951 Dickinson, A., and Balleine, B. (2002). ‘‘The role of learning in motivation,’’ in Stevens’ Handbook of Experimental Psychology, ed C. Gallistel (New York, NY: Wiley). Paredes-Olay, C., Abad, M. J. F., Gámez, M., and Rosas, J. M. (2002). Transfer of control between causal predictive judgments and instrumental responding. Anim. Learn. Behav. 30, 239–248. doi: 10.3758/bf03192833 Dickinson, A., Smith, J., and Mirenowicz, J. (2000). Dissociation of Pavlovian and instrumental incentive learning under dopamine antagonists. Behav. Neurosci. 114, 468–483. doi: 10.1037/0735-7044.114.3.468 Patton, J. H., Stanford, M. S., and Barratt, E. S. (1995). Factor structure of the Barratt impulsiveness scale. J. Clin. Psychol. 51, 768–774. doi: 10.1002/1097- 4679(199511)51:6<768::aid-jclp2270510607>3.0.co;2-1 Doya, K. (2008). Modulators of decision making. Nat. Neurosci. 11, 410–416. doi: 10.1038/nn2077 Perks, S. M., and Clifton, P. G. (1997). Reinforcer revaluation and conditioned place preference. Physiol. Behav. 61, 1–5. doi: 10.1016/s0031-9384(96)00243-0 Estes, W. K. (1943). Discriminative conditioning. I. A discriminative property of conditioned anticipation. J. Exp. Psychol. 32, 150–155. doi: 10.1037/h0058316 Prévost, C., Liljeholm, M., Tyszka, J. M., and O’Doherty, J. P. (2012). Neural correlates of specific and general Pavlovian-to-instrumental transfer within human amygdalar subregions: a high-resolution fMRI study. J. Neurosci. 32, 8383–8390. doi: 10.1523/jneurosci.6237-11.2012 Estes, W. K. (1948). Discriminative conditioning; effects of a Pavlovian conditioned stimulus upon a subsequently established operant response. J. Exp. Psychol. 38, 173–177. References doi: 10.1037/h0057525 June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org Frontiers in Behavioral Neuroscience | www.frontiersin.org 10 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino stress-induced corticosterone release and mesolimbic levels of monoamines. Pharmacol. Biochem. Behav. 65, 509–517. doi: 10.1016/s0091-3057(99) 00241-5 stress-induced corticosterone release and mesolimbic levels of monoamines. Pharmacol. Biochem. Behav. 65, 509–517. doi: 10.1016/s0091-3057(99) 00241-5 Rescorla, R. A. (1994a). Control of instrumental performance by Pavlovian and instrumental stimuli. J. Exp. Psychol. Anim. Behav. Process. 20, 44–50. doi: 10. 1037/0097-7403.20.1.44 Trick, L., Hogarth, L., and Duka, T. (2011). Prediction and uncertainty in human Pavlovian to instrumental transfer. J. Exp. Psychol. Learn. Mem. Cogn. 37, 757–765. doi: 10.1037/a0022310 Rescorla, R. A. (1994b). Transfer of instrumental control mediated by a devalued outcome. Anim. Learn. Behav. 22, 27–33. doi: 10.3758/bf03199953 Rescorla, R. A. (1997). Response inhibition in extinction. Q. J. Exp. Psychol. B 50, 238–252. Uslaner, J. M., Acerbo, M. J., Jones, S. A., and Robinson, T. E. (2006). The attribution of incentive salience to a stimulus that signals an intravenous injection of cocaine. Behav. Brain Res. 169, 320–324. doi: 10.1016/j.bbr.2006. 02.001 Rescorla, R. A. (2000). Associative changes with a random CS-US relationship. Q. J. Exp. Psychol. B 53, 325–340. doi: 10.1080/027249900750001338 Rescorla, R. A., and Solomon, R. L. (1967). Two-process learning theory: relationships between Pavlovian conditioning and instrumental learning. Psychol. Rev. 74, 151–182. doi: 10.1037/h0024475 Volkow, N. D., Wang, G.-J., Fowler, J. S., and Telang, F. (2008). Overlapping neuronal circuits in addiction and obesity: evidence of systems pathology. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363, 3191–3200. doi: 10.1098/rstb.2008. 0107 Robinson, T. E., and Flagel, S. B. (2009). Dissociating the predictive and incentive motivational properties of reward-related cues through the study of individual differences. Biol. Psychiatry 65, 869–873. doi: 10.1016/j.biopsych.2008.09.006 Wyvell, C. L., and Berridge, K. C. (2000). Intra-accumbens amphetamine increases the conditioned incentive salience of sucrose reward: enhancement of reward ‘‘wanting’’ without enhanced ‘‘liking’’ or response reinforcement. J. Neurosci. 20, 8122–8130. Robinson, T. E., Yager, L. M., Cogan, E. S., and Saunders, B. T. (2014). On the motivational properties of reward cues: individual differences. Neuropharmacology 76, 450–459. doi: 10.1016/j.neuropharm.2013.05.040 ‘‘wanting’’ without enhanced ‘‘liking’’ or response reinforcement. J. Neurosci. 20, 8122–8130. Wyvell, C. L., and Berridge, K. C. (2001). Incentive sensitization by previous amphetamine exposure: increased cue-controlled ‘‘wanting’’ for sucrose reward. J. Neurosci. 21, 7831–7840. Saunders, B. T., and Robinson, T. E. Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 References (2013). Individual variation in resisting temptation: implications for addiction. Neurosci. Biobehav. Rev. 37(9 Pt. A), 1955–1975. doi: 10.1016/j.neubiorev.2013.02.008 Smeets, P. M., and Barnes-Holmes, D. (2003). Children’s emergent preferences for soft drinks: stimulus-equivalence and transfer. J. Econ. Psychol. 24, 603–618. doi: 10.1016/s0167-4870(03)00004-7 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Talmi, D., Seymour, B., Dayan, P., and Dolan, R. J. (2008). Human pavlovian- instrumental transfer. J. Neurosci. 28, 360–368. doi: 10.1523/JNEUROSCI. 4028-07.2008 Copyright © 2015 Garofalo and di Pellegrino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2015 Garofalo and di Pellegrino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Tomie, A., Aguado, A. S., Pohorecky, L. A., and Benjamin, D. (1998). Ethanol induces impulsive-like responding in a delay-of-reward operant choice procedure: impulsivity predicts autoshaping. Psychopharmacology (Berl) 139, 376–382. doi: 10.1007/s002130050728 Tomie, A., Aguado, A. S., Pohorecky, L. A., and Benjamin, D. (2000). Individual differences in pavlovian autoshaping of lever pressing in rats predict June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 11
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Novel Geometric Series for Application of Computational Science
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MSC Classification codes: 40A05 (65B10) Keywords: computation, theorem, geometric series 1. Introduction Geometric series played a vital role in differential and integral calculus at the earlier stage of development and still continues as an important part of the study in science, engineering, management and its applications. In this article, a new geometric series [1-6] is constructed for application [7] of computational science and engineering. Novel Geometric Series for Application of Computational Science Chinnaraji Annamalai School of Management, Indian Institute of Technology, Kharagpur, India Email: anna@iitkgp.ac.in https://orcid.org/0000-0002-0992-2584 Chinnaraji Annamalai School of Management, Indian Institute of Technology, Kharagpur, India Email: anna@iitkgp.ac.in https://orcid.org/0000-0002-0992-2584 Abstract: Computational science is a rapidly growing multi-and inter-disciplinary area where science, engineering, information technology, management and its collaboration use advance computing capabilities to understand and solve the most complex real life problems. In this article, a new geometric series is constituted for application of computational science and engineering. 3. Conclusion Nowadays, the growing complexity of mathematical and computational modelling demands the simplicity of mathematical and combinatorial equations for solving today’s scientific problems and challenges. This article provides a novel geometric series and its theorem. This idea can enable the scientific researchers for further involvement in research and development. For example, For example, For example, For example, ∑ ∑ ∑ ∑ 2. Novel Geometric Series In today’s world, the growing complexity of mathematical modelling demands the simplicity of mathematical equations for solving today’s scientific problems. For this purpose, the following theorem [1-6] is introduced. ∑ Proof. Let us prove this theorem by the equality given below: If we continue this expansion again and again, we can obtain the following series. p g g g ∑ p g g g ∑ Now, let us prove the theorem shown below: , p https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 ∑ From the Equation (3), we conclude that From the Equation (3), we conclude that From the Equation (3), we conclude that ∑ Hence, theorem is proved. Hence, theorem is proved. Hence, theorem is proved. ∑ Hence, theorem is proved. ∑ F l ∑ ∑ https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 References [1] Annamalai, C. (2018) Annamalai’s Computing Model for Algorithmic Geometric Series and Its Mathematical Structures. Journal of Mathematics and Computer Science, 3(1),1-6 https://doi.org/10.11648/j.mcs.20180301.11. Page | 2 https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 [2] Annamalai, C. (2018) Algorithmic Computation of Annamalai’s Geometric Series and Summability. Journal of Mathematics and Computer Science, 3(5),100-101. https://doi.org/10.11648/j.mcs.20180305.11. [3] Annamalai, C. (2017) Analysis and Modelling of Annamalai Computing Geometric Series and Summability. Mathematical Journal of Interdisciplinary Sciences, 6(1), 11-15. https://doi.org/10.15415/mjis.2017.61002. [4] Annamalai, C. (2018) Novel Computation of Algorithmic Geometric Series and Summability. Journal of Algorithms and Computation, 50(1), 151-153. https://www.doi.org/10.22059/JAC.2018.68866. [5] Annamalai, C. (2017) Computational modelling for the formation of geometric series using Annamalai computing method. Jñānābha, 47(2), 327-330. https://zbmath.org/?q=an%3A1391.65005. [6] Annamalai, C. (2018) Computing for Development of A New Summability on Multiple Geometric Series. International Journal of Mathematics, Game Theory and Algebra, 27(4), 511-513. [7] Annamalai C (2010) “Application of Exponential Decay and Geometric Series in Effective Medicine”, Advances in Bioscience and Biotechnology, Vol. 1(1), pp 51-54. https://doi.org/10.4236/abb.2010.11008. Page | 3 https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 Page | 3
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Phytohormone up-regulates the biochemical constituent, exopolysaccharide and nitrogen metabolism in paddy-field cyanobacteria exposed to chromium stress
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Phytohormone up-regulates the biochemical constituent, exopolysaccharide and nitrogen metabolism in paddy-¦eld cyanobacteria exposed to chromium stress Sanjesh Tiwari  (  tiwarisanjesh@gmail.com ) Ranjan Plant Physiology and Biochemistry Laboratory Anuradha Patel  University of Allahabad Sheo Mohan Prasad  University of Allahabad Anuradha Patel  University of Allahabad Sheo Mohan Prasad  University of Allahabad Research article Version of Record: A version of this preprint was published on July 13th, 2020. See the published version at https://doi.org/10.1186/s12866-020-01799-3. The authors have withdrawn the journal submission associated with this preprint and requested that the preprint also be withdrawn.
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Effects of COVID19 Pandemic on Pediatric Kidney Transplant in the United States
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Effects of COVID19 Pandemic on P Transplant in the United States Olga Charnaya  (  ocharna1@jhmi.edu ) Johns Hopkins University https://orcid.org/0000-0003-1104-2882 Teresa Po-Yu Chiang  Johns Hopkins University https://orcid.org/0000-0003-0601-7420 Richard Wang  Johns Hopkins University Jennifer Motter  Johns Hopkins University https://orcid.org/0000-0003-1750-0119 Brian Boyarsky  Johns Hopkins University https://orcid.org/0000-0001-6902-9854 Elizabeth King  Johns Hopkins University William Werbel  Johns Hopkins University https://orcid.org/0000-0003-2943-5895 Christine M. Durand  Johns Hopkins University https://orcid.org/0000-0003-2605-9257 Robin Avery  Johns Hopkins University https://orcid.org/0000-0001-7692-3619 Dorry Segev  Johns Hopkins University https://orcid.org/0000-0002-1924-4801 Allan Massie  Johns Hopkins University https://orcid.org/0000-0002-5288-5125 Jacqueline Garonzik-Wang  Johns Hopkins University https://orcid.org/0000-0002-2789-7503 Effects of COVID19 Pandemic on Pediatric Kidney Transplant in the United States Olga Charnaya  (  ocharna1@jhmi.edu ) Olga Charnaya  (  ocharna1@jhmi.edu ) Johns Hopkins University https://orcid.org/0000-0003-1104-2882 Elizabeth King  Johns Hopkins University Introduction As the death toll from COVID-19 in the United States surpasses 180,000 fatalities, the pandemic continues to place considerable burden on the health care system. Many transplant centers have been challenged to determine how to proceed with organ transplantation during a time of limited data and resources. These decisions have had profound implications for many patients awaiting transplantation, including the pediatric population, where kidney transplantation (KT) remains the optimal treatment for end-stage renal disease (ESRD) [1]. Adult KT has been significantly impacted by COVID-19, with reduced new wait-list addition and transplantation at many transplant centers across the nation [2,3]. A national survey of adult transplant centers showed that 72% had suspended live donor kidney transplants (LDKT) and 84% had placed significant restrictions on deceased donor kidney transplants (DDKT) by April 2020 [4]. The stringency of these restrictions varied by center, with more restrictive measures in areas with a higher incidence of COVID-19. Several reasons have been cited for these changes. First, there is concern that organ transplant recipients may be at a greater risk for acquiring and experiencing worse clinical outcomes to COVID-19 infection [5-7]. Second, immunosuppressed KT recipients may have prolonged viral shedding and transmissibility, potentially posing a greater risk to public health [8,9]. Third, there is concern that hospitals will not have the resources to safely perform KT and provide the necessary post-operative care [10]. These changes are not without consequences; postponing KT can lead to a higher waitlist mortality, and diverting resources away from transplant recipients may worsen post-operative outcomes [11,12]. It is unclear if the pandemic has resulted in similar challenges and restrictions for the pediatric population. For the most part, children are asymptomatic or have milder infection and fewer complications from COVID-19 compared to adult patients [13,14]. From the limited data available, it appears that disease severity even among immunocompromised children remains less severe than that of the general adult population and remains similar to healthy children [15-17]. Furthermore, while pediatric patients in general have less dialysis-associated morbidity and mortality compared to adults, delaying transplantation and prolonging time on dialysis is associated with increased morbidity and repetitive risk of COVID exposure during in-center dialysis sessions [18,19]. The response of the pediatric transplant community is likely different than adult practices and therefore needs to be studied separately. Research Article Version of Record: A version of this preprint was published at Pediatric Nephrology on September 8th, 2020. See the published version at https://doi.org/10.1007/s00467-020-04764-4. ersion of Record: A version of this preprint was published at Pediatric Nephrology on S ublished version at https://doi.org/10.1007/s00467-020-04764-4. Page 1/14 Abstract In March 2020, COVID-19 infections began to rise exponentially in the United States, placing substantial burden on the healthcare system. As a result, there was a rapid change in transplant practices and policies, with cessation of most procedures. Our goal was to understand changes to pediatric kidney transplantation (KT) at the national level during the COVID-19 epidemic. Using SRTR data, we examined changes in pediatric waitlist registration, waitlist removal or inactivation, and deceased donor and living donor (DDKT/LDKT) events during the start of the disease transmission in the United States compared to the same time the previous year. We saw an initial decrease in DDKT and LDKT by 47% and 82% compared to expected events and then a continual increase, with numbers reaching expected pre-pandemic levels by May 2020. In the early phase of the pandemic, waitlist inactivation and removals due to death or deteriorating condition rose above expected values by 152% and 189%, respectively. There was a statistically significant decrease in new waitlist additions (IRR 0.49 0.65 0.85) and LDKT (IRR 0.17 0.38 0.84) in states with high vs low COVID activity. Transplant recipients during the pandemic were more likely to have received a DDKT, but had similar cPRA, waitlist time and cause of ESRD as before the pandemic. The COVID-19 pandemic initially reduced access to kidney transplantation among pediatric patients in the United States, but has not had a sustained effect. activity. Transplant recipients during the pandemic were more likely to have received a DDKT, but had similar cPRA, waitlist time and cause of ESRD as before the pandemic. The COVID-19 pandemic initially reduced access to kidney transplantation among pediatric patients in the United States, but has not had a sustained effect. Data Source This study used data from the Scientific Registry of Transplant Recipients (SRTR). The SRTR data system includes data on all donor, wait-listed candidates, and transplant recipients in the US, submitted by the members of the Organ Procurement and Transplantation Network (OPTN). The Health Resources and Services Administration (HRSA), U.S. Department of Health and Human Services provides oversight to the activities of the OPTN and SRTR contractors. This dataset has previously been described elsewhere [20]. National cumulative incidence of COVID-19 National COVID-19 incidence data from January – June 2020 were extracted from USA FACTS (https://usafacts.org/issues/coronavirus/) [21]. COVID-19 incidence is reported for all patients and not restricted to children, as many states are not providing data of infections by age groups. Based upon cumulative incidence of COVID-19 positive cases per-million state population (PMP) between March 15 and June 30, states were stratified to be having low (<8000 PMP) or high (>8000 PMP) COVID-19 burden. This cutoff was selected based on an visible difference in distribution of cumulative incidence of COVID- 19 burden in the United States. Introduction To investigate center-specific changes in pediatric transplant practices during the COVID-19 pandemic, we used national registry data to quantify changes to KT waitlist registration, waitlist deaths or removal, rates of DDKT and Page 2/14 LDKT, between February and June 2020. We examined rates of these waitlist events among the overall patient population, and evaluated for differences in patient characteristics during the COVID-19 pandemic. This information will allow us to better understand of the pandemic’s impact on the pediatric kidney transplant population. LDKT, between February and June 2020. We examined rates of these waitlist events among the overall patient population, and evaluated for differences in patient characteristics during the COVID-19 pandemic. This information will allow us to better understand of the pandemic’s impact on the pediatric kidney transplant population. Study Population For waitlist analysis, we included DDKT waitlist registrants aged 0-17 at time of listing (new listing; changed to inactive status; removed due to DDKT, LDKT, death or deteriorating condition, and other causes) were included. For analysis of transplant volume (DDKT, LDKT, DCD, national or regional import) recipients aged 0-17 at time of transplant were included. Weekly counts of waitlist and transplant changes For each week (Sunday-Saturday) starting February 2, 2020 until June 27, 2020, we plotted cumulative counts of new waitlist additions, newly inactive patients, waitlist removal due to death or deteriorating condition, and waitlist removal due to transplant or other causes, using a Lowess smoothing function. We made similar plots for weekly counts of DDKT, LDKT, DCD donor, and regional and national imports. Weekly instead of daily counts were used due to the low number of daily events in pediatric patients to enable statistical analysis. On each plot we also included the average counts during the same period in 2017-2019 as a visual reference of national pre-pandemic pediatric KT volume. Characteristics of pediatric transplant patients during COVID-19 Patient characteristics in three time periods (“Early” Jan 1 – Mar 15 2020, “Middle” Mar 16 – Apr 30 2020, and “Late” May 1 – Jun 30 2020) were examined (Table 1). Patients who received a kidney transplant during the first COVID-19 peak in the United States, Middle period, had similar waitlist time, cPRA and blood type compared to Early and Late periods (p > 0.1). Higher proportion of Black patients received a transplant in the Middle (30.6%) compared to Early (13.1%) and Late (20.7%), (p=0.28). Living donor transplants made up a smaller proportion of total transplants during the Middle period 13.9%, compared to 29.5% and 36.4% in Early and Late, respectively (p=0.035). Median cold ischemia time (CIT) was longer in the Middle period 10.2 hours (IQR 6.5-17.4), compared to the Early 9.0 hours (IQR 4.0-13.2) and Late 7.6 hours (IQR 2.4-10.7) periods, (p=0.02). Weekly count of waitlist changes National weekly pediatric KT waitlist additions ranged from 7 - 41 cases per week between February 2 and June 30, 2020. There was a trend of decreasing new pediatric DDKT registrations, following the national rise of COVID-19 cases mid-March. Since April, none of the weekly pediatric KTs exceeded 21, the 2017- 2019 average counts for the same period (Figure 1A). The numbers of registrants who changed to inactive status also increased in March, with 77.2% of registrants who changed to inactive status in the third and fourth week of March indicating COVID-19 as reason of inactivation (Figure 1C). COVID-19 was added as a refusal code or cause for change in status in UNET on March 25, 2020; however, this classification does not differentiate new COVID-19 infection in the patient vs precaution secondary to the pandemic. Percentage of inactive waitlist registrants rose from 72% to 77% between March 1 and April 15, and remained elevated above previous baseline thereafter (Figure 1D). We observed an increasing trend in waitlist removal due to death or deteriorating condition since March, followed by a trend that returned to previous benchmarks by late-April (Figure 1B). Statistical analysis We presented characteristics of pediatric kidney transplant recipients separately in three time periods: January 1 - March 15, 2020 (“Early”); March 16 - April 30, 2020 (“Middle”); and May 1 – June 30, 2020 (“Late”). Continuous variables were presented as median and interquartile range, and categorical variables were presented as counts and proportion. Comparison between groups were tested using Kuskal-Wallis or Mann Whitney-U, as appropriate, for continuous variables and Fisher's exact test for categorical variables. We used 2015 as reference year to calculate KDPI [22]. We obtained pediatric kidney waitlist changes or transplant volume by center, month and year from January 1, 2016 to February 28, 2020, and constructed a mixed-effects Poisson regression with a center-level random intercept to obtain expected daily counts by center (monthly counts divided by 31), using methods previously described [23]. The expected counts of each time period were the sum of expected center-level counts during the corresponding length of time (March 1 to April 30: 47 days; May 1 – June 30: 61 days). We then compared the observed and expected counts of each time period using Chi-square testing. We used an α of 0.05 to define statistical significance. All analyses were performed using Stata 16.0/MP for Linux (College Station, Texas). Weekly count of transplant events Page 4/14 The national weekly pediatric DDKT volume ranged from 0-16 cases per week between February 1 and June 30, 2020. On average, the weekly DDKT volume in 2017-2019 was 9.6 cases. Between mid-March and the end of June 2020, DDKT volume remained lower than 9.6 except for 4 weeks out of the 15 during observation. There was a trend of decreasing DDKT and LDKT volume seen since March, followed by increase from mid-April to end of June (Figure 2A, 2B). For LDKT, the weekly volumes were never above the 2017-2019 average between mid-March and May 31, but consistently surpassed this volume in June 2020 (Figure 2B). Comparing the observed and predicted waitlist changes The overall observed national volume of waitlist registration was lower (-13.3%, p=0.021) and change to inactive waitlist status was higher (57.2%, p<0.001) compared to the expected volume during March 15 – June 30, 2020 (Table 2). When stratified into the earlier (March 15 – April 30, 2020) and the latter (May 1– June 30, 2020) periods, 6 candidates were removed from the waitlist during the earlier period due to death or deteriorated condition, which was 189% more than the expected 2.1 cases (p=0.005). Similarly, 83 candidates had changed to inactive status during the earlier period, which was 152% more than the expected 32.9 cases (p<0.001). In both cases, the significance was not achieved in the latter period (11.3%, p=0.3 and -11.1%, p=0.5, respectively). Contrarily, the observed counts of new waitlist registration was 23.8% lower compared to the expected during the latter period (p=0.002), though not significantly different in the earlier period (0.4%, p=1.0). Comparing the observed and predicted transplant events There were 157 pediatric KTs performed during March 15-June 30, 2020 (108 DDKT, 49 LDKT), which was 22.8% fewer than the expected 203.3 cases (p=0.001). The 108 DDKT performed during the same period was 29.2% fewer than the expected 103.1 cases (p=0.03), whereas the 49 LDKTs was not significantly different from the expected 64.2 cases (p=0.058). When stratified to the earlier and the latter COVID-19 eras, the observed DDKT, LDKT, and combined total transplant were all significantly less than expected in the earlier era (total: 36 vs. 88.5, -59.3%, p<0.001; LDKT: 5 vs. 27.9, -82.1%, p<0.001; DDKT: 31 vs. 59.1%, -47.6%, p<0.001) but not during the latter period. Regional and National Imports Overall numbers of regional and national imports were extremely low (0-4 per week), with average <1 import per week in 2017-2019 (Figure 2C). During the early period of COVID-19 disease activity in the United States, imports were more common than in previous years. As the pandemic progressed, there was a decline in imports, however the average number of imports continue to remain higher than previous years. Discussion In this national registry study of pediatric KT trends during the COVID-19 pandemic, we found an increase in patients being changed to inactive status by 152%, increase in mortality on the waitlist by 189%, decrease in DDKT by 48% and LDKT by 82% compared to expected in the early COVID-19 time period without a significant impact on new waitlist additions. The COVID-19 pandemic has substantially limited access to KT and increased waitlist mortality in pediatric patients. Many transplant programs significantly altered their routine protocols and stopped performing kidney transplants thereby restricting access to KT during the COVID-19 pandemic [4]. A recent registry of adult SOT recipients infected with COIVD-19 did not show any significant difference in mortality or morbidity compared to non-SOT patients [24]. While extensive data in pediatric solid organ transplant (SOT) recipients is not available, several case reports show that pediatric SOT patients infected with COVID-19 showed only mild disease, even while on immunosuppressive therapy [25,26]. As children have different etiologies of ESRD, different comorbidities and seem to be affected differently by COVID-19, uniform policies affecting access to transplantation for both children and adults are not appropriate nor in the best interest of pediatric patients [16]. The increased mortality while on the waitlist that we showed in pediatric patients is a striking metric that supports the need for an individualized approach for pediatric KT patients. As pediatric KT events are relatively rare compared to adult KT, this affords an opportunity for patient- level versus center-level decisions about risk and benefit of KT in a pandemic setting. The reduction of transplant events in children seen during the Middle (March 15 – April 30, 2020) COVID- 19 era was likely secondary to a combination of factors. As hospitals shifted resources to treat COVID-19 patients there was a decrease in available ICU beds for post-operative management and restrictions on operating room availability. Pediatric KT patients require intensive post-operative care, which may strain healthcare systems already overburdened by COVID- 19 patients [27]. In addition, there was a notable decline in deceased donor organs recovered during March and April [28]. Shortages of COVID-19 testing or delayed results for deceased donors may have impacted center willingness to accept an organ from what would otherwise be an acceptable donor. As testing capacity increased across the country, this limitation was ameliorated. Regional differences in transplant practice and waitlist death by COVID-19 burden Centers situated in states with high COVID-19 burden (NY, NJ, RI, MA, DC, CT,LA, DE, IL, MD, AZ, NE, IA, NS) between March 15 and June 30 had significantly fewer new waitlist registrations (incidence rate ratio (IRR): 0.49 0.65 0.85) and LDKT (IRR: 0.17 0.38 0.84) compared to centers in states with low burden (IRR: 0.82 0.94 1.08) (Table 3). There were no differences in the proportion of expected DDKT and waitlist death between centers in states with high and low COVID- 19 burden. Page 5/14 Page 5/14 Discussion Finally, many centers stopped or significantly reduced elective and non-emergent surgical procedures, which likely had a significant impact on LDKT. For pediatric patients scheduled to receive an LDKT, it should be argued that transplant is not truly an elective procedure whether pre-emptive or not. Dialysis initiation would require at least one surgical procedure to establish dialysis access, and initiation or continuation of hemodialysis would result in a much higher COVID-19 exposure risk [29]. While peritoneal dialysis patients do not require frequent in-center visits and thereby can have minimal healthcare exposure, especially with the advancement of telehealth capabilities across the country, the benefit of transplant over dialysis has been well established in the pediatric ESRD population [30,31]. As healthcare centers lifted restrictions on elective cases and practice patterns changed, there was a large increase in LDKT in June 2020 suggesting cases had been postponed due to the pandemic. Our hypothesis that children who received a transplant during the peak of the pandemic would differ in some characteristics compared to pre-pandemic patients was not supported. There was not a statistically significant difference in donor KDPI, recipient cPRA, or etiology of ESRD. While cold ischemia time in the Middle time period was longer, this is unlikely to be a clinically significant difference. This is the first study to describe characteristics of donors and recipients receiving a KT during the pandemic. As this is a registry study, we are limited in the information that is available for analysis and are not able to delve into granular details of waitlist removal or patient death. While transplant centers have mandatory reporting requirements to Page 6/14 Page 6/14 mission may be delayed due to center practices and pandemic effect on workflow. UNOS/OPTN, data transmission may be delayed due to center practices and pandemic effect on workflow. Nevertheless, we are able to make generalized conclusions about the effects of COVID-19 on access to KT in pediatric patients. We were unable to differentiate regional variability in transplant rates and waitlist changes due to the overall low number of events in pediatric patients. In summary, we found that the COVID-19 pandemic has had a significant impact on pediatric KT waitlist mortality, waitlist registration, DDKT and LDKT. DCD: donation after circulatory death IRR: incidence rate ratio ICU: intensive care unit OR: operating room Discussion Further studies to assess outcomes of pediatric patients who received a KT during this time are necessary to inform changes in policies and practices to optimize pediatric transplant outcomes and ensure access to this life-saving treatment. DISCLAIMER The analyses described here are the responsibility of the authors alone and do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. Government. The data reported here have been supplied by the Hennepin Healthcare Research Institute (HHRI) as the contractor for the Scientific Registry of Transplant Recipients (SRTR). The interpretation and reporting of these data are the responsibility of the author(s) and in no way should be seen as an official policy of or interpretation by the SRTR or the U.S. Government. Abbreviations COVID-19: coronavirus disease 2020 KT: kidney transplant LDKT: live donor kidney transplantation DDKT: deceased donor transplantation ESR y p p g SRTR: Scientific Registry of Transplant Recipients OPTN: Organ Procurement and Transplantation Network PMP: per million population DCD: donation after circulatory death IRR: incidence rate ratio DCD: donation after circulatory death IRR: incidence rate ratio Declarations Funding: This work was supported by grant number KL2TR003099 (Charnaya) from Johns Hopkins Institute for Clinical and Translational Research (ICTR) as well as F32DK124941 (Boyarsky), K23DK115908-01 (Garonzik-Wang), and K01DK101677 (Massie) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and K24AI144954 (Segev) from National Institute of Allergy and Infectious Diseases (NIAID). Conflicts of interest/Competing interests: The authors of this manuscript have no conflicts of interest to disclose. onflicts of interest/Competing interests: The authors of this manuscript have no conf Ethics approval: Approved by the institutional review board at Johns Hopkins University School of Medicine. Consent to participate: Not applicable Consent for publication: Not applicable Availability of data and material: Data is available for review upon request to the corresponding author Code availability: Not applicable Availability of data and material: Data is available for review upon request to the corresponding author Page 7/14 Authors' contributions: Charnaya, Chiang, Motter, Wang and Massie participated in study design, analysis and manuscript preparation. Boyarsky participated in study design and manuscript preparation. Wang, Motter, King, Werbel, Durand, and Segev contributed to significant review of the analysis and manuscript preparation. References 1. 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Verghese PS (2017) Pediatric kidney transplantation: a historical review. Pediatric research 81 (1- 2):259-264. doi:10.1038/pr.2016.207 References American Journal of Transplantation n/a (n/a). doi:10.1111/ajt.16280 25. D’Antiga L (2020) Coronaviruses and Immunosuppressed Patients: The Facts During the Third Epidemic. Liver Transplantation 26 (6):832-834. doi:10.1002/lt.25756 25. D’Antiga L (2020) Coronaviruses and Immunosuppressed Patients: The Facts During the Third Epidemic. Liver Transplantation 26 (6):832-834. doi:10.1002/lt.25756 26. Bush R, Johns F, Acharya R, Upadhyay K (2020) Mild COVID-19 in a pediatric renal transplant recipient. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of 26. Bush R, Johns F, Acharya R, Upadhyay K (2020) Mild COVID-19 in a pediatric renal transplant recipient. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Page 9/14 Page 9/14 Transplant Surgeons. doi:10.1111/ajt.16003 Transplant Surgeons. doi:10.1111/ajt.16003 Tables Table 1. Patient and donor characteristics broken down by three time periods of COVID-19 activity in 2020. Page 10/14 Early Jan 1 – Mar 15 Middle Mar 15 – Apr 30 Late May 1 – Jun 30 p- value N 122 36 121   Recipient Factors Wait time (IQR) 202.5 (81, 618) 201.5 (42.5, 547) 227 (92, 495) 0.87 Female Sex 59 (48.4%) 14 (38.9%) 56 (46.3%) 0.61 Race       0.28 White 95 (77.9%) 21 (58.3%) 86 (71.1%)   Black 16 (13.1%) 11 (30.6%) 25 (20.7%)   Asian 7 (5.7%) 3 (8.3%) 8 (6.6%)   Others 4 (3.3%) 1 (2.8%) 2 (1.7%)   ABO Blood Type       0.94 Type O 70 (57.4%) 21 (58.3%) 71 (58.7%)   Type A 30 (24.6%) 10 (27.8%) 34 (28.1%)   Type B 16 (13.1%) 4 (11.1%) 13 (10.7%)   Type AB 6 (4.9%) 1 (2.8%) 3 (2.5%)   Primary diagnosis       0.065 Glomerulonephritis 21 (17.2%) 2 (5.6%) 17 (14.0%)   FSGS 15 (12.3%) 5 (13.9%) 15 (12.4%)   Hypoplasia 32 (26.2%) 7 (19.4%) 14 (11.6%)   Obstructive 19 (15.6%) 8 (22.2%) 19 (15.7%)   Polycystic 9 (7.4%) 2 (5.6%) 18 (14.9%)   Others 26 (21.3%) 12 (33.3%) 38 (31.4%)   cPRA, mean (SD) 12% (24) 9% (26) 7% (20) 0.29 cPRA ≠ 0, median (IQR) 44% (5, 64) (n=36) 61% (5, 99) (n=7) 11% (0, 52) (n=31) 0.087 cPRA ≥ 0.2 23 (18.9%) 4 (11.1%) 15 (12.4%) 0.33 Donor Factors Age (Yr), median (IQR) 28 (20, 34) 25.50 (21.50, 32) 28 (22, 35) 0.68 Female Gender 52 (42.6%) 16 (44.4%) 51 (42.1%) 0.97 Living donor 36 (29.5%) 5 (13.9%) 44 (36.4%) 0.035 Race       0.55 White 103 (84.4%) 29 (80.6%) 102 (84.3%)   Black 13 (10.7%) 3 (8.3%) 15 (12.4%)   Asian 3 (2.5%) 3 (8.3%) 3 (2.5%)   Other race 3 (2.5%) 1 (2.8%) 1 (0.8%)   KDPI n=86 n=31 n=77   median (IQR) 13.73 (7.65, 26.72) 14.57 (5.93, 28.99) 17.56 (9.71, 28.34) 0.46 mean (SD) 17.30 (13.46) 17.38 (12.63) 19.21 (12.18) 0.61 Cold Ischemic Time (Hours), median (IQR)   9.0 (4.0, 13.2) (n=121)   10.2 (6.5, 17.5) (n=34)   7.6 (2.4, 10.7) (n=57)   0.021 Table 2. Tables Observed compared to expected events in early and later COVID-19 eras of (A) waitlist changes, (B) transplant events   Waitlist changes (pediatric age at listing) March 15- April 30, 2020 May 1 – June 30, 2020 Total Observed expected % change p value observed expected % change p value observed expected % change p value New listing 132 131.5 0.4 0.967 130 170.7 -23.8 0.002 262 302.2 -13.3 0.021 Causes of removal Death 6 2.1 189.0 0.006 3 2.7 11.3 0.852 9 4.8 88.7 0.053 DDKT 42 64.5 -34.9 0.005 93 83.7 11.1 0.309 135 148.2 -8.9 0.279 LDKT 5 27.8 -82.0 < 0.01 44 36.1 21.9 0.188 49 63.9 -23.3 0.062 Changed to inactive status   83   32.9   152.0   < 0.01   38   42.7   -11.1   0.468   119   75.7   57.2   < 0.01 rved compared to expected events in early and later COVID-19 eras of (A) waitlist changes, t events Table 2. Observed compared to expected events in early and later COVID-19 eras of (A) waitlist changes, (B) transplant events ompared to expected events in early and later COVID-19 eras of (A) waitlist changes, Page 11/14 Page 11/14 Transplant (pediatric age at transplant) March 15- April 30, 2020 May 1 – June 30, 2020 Total Observed expected % change p value observed expected % change p value observed expected % change p value Total transplant 36 88.5 -59.3 < 0.01 121 114.8 5.4 0.566 157 203.3 -22.8 0.001 LDKT 5 27.9 -82.1 < 0.01 44 36.3 21.3 0.199 49 64.2 -23.7 0.058 DDKT 31 59.1 -47.6 < 0.01 77 76.8 0.3 1.000 108 135.9 -20.5 0.017 DCD 1 3.2 -68.4 0.224 5 4.1 21.6 0.663 6 7.3 -17.6 0.632 Regional or national import   7   3.7   90.7   0.082   4   4.8   -16.1   0.729   11   8.4   30.4   0.377 Table 3. Observed center-level events as a proportion of expected events, March 15 - June 30, 2020. Bold denotes IRRs that are statistically significantly different from the IRR in states with low COVID-19 disease burden (<8000 cases PMP). COVID-19 rates New listings DDKT LDKT Waitlist death Overall 0.77 0.87 0.98 0.66 0.79 0.96 0.58 0.76 1.01 0.98 1.89 3.63 Low 0.82 0.94 1.08 0.66 0.81 1.00 0.66 0.89 1.20 0.84 1.87 4.16 High* 0.49 0.65 0.85 0.48 0.73 1.12 0.17 0.38 0.84 0.62 1.92 5.96 *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS Table 3. Tables Observed center-level events as a proportion of expected events, March 15 - June 30, 2020. Bold denotes IRRs that are statistically significantly different from the IRR in states with low COVID-19 disease burden (<8000 cases PMP). Table 3. Observed center-level events as a proportion of expected events, March 15 - June 30, 2020. Bold denotes IRRs that are statistically significantly different from the IRR in states with low COVID-19 disease burden (<8000 cases PMP). COVID-19 rates New listings DDKT LDKT Waitlist death Overall 0.77 0.87 0.98 0.66 0.79 0.96 0.58 0.76 1.01 0.98 1.89 3.63 Low 0.82 0.94 1.08 0.66 0.81 1.00 0.66 0.89 1.20 0.84 1.87 4.16 High* 0.49 0.65 0.85 0.48 0.73 1.12 0.17 0.38 0.84 0.62 1.92 5.96 *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS Figures *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS Figures Figures Pediatric patient deceased donor KT waitlist status change; (A) new waitlist additions, (B) removal due to death or deteriorating condition, (C) changed to inactive status, (D) percentage inactive. A. Figures Page 12/14 Figure 1 Pediatric patient deceased donor KT waitlist status change; (A) new waitlist additions, (B) removal due to death or deteriorating condition, (C) changed to inactive status, (D) percentage inactive. A. Figure 1 Pediatric patient deceased donor KT wait deteriorating condition, (C) changed to in Figure 1 Pediatric patient deceased donor KT waitlist status change; (A) new waitlist additions, (B) removal due to death or deteriorating condition, (C) changed to inactive status, (D) percentage inactive. A. Page 13/14 Figure 2 Pediatric transplant events cumulatively by week imports A. Figure 2 Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT, (C) Regional and National imports A. Figure 2 Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT, (C) Regional and National imports A. Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT, (C) Regional and National imports A. Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT, (C) Regional and National imports A. Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT, (C) Regional and National imports A Page 14/14
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The Mersin Greenhouse Workers Study. Surveillance of Work-related Skin, Respiratory, and Musculoskeletal Diseases
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* Ministry of Health, Mersin Public Health Directorate, Mersin, TR † Ministry of Health, Mardin Public Health Directorate, Mardin, TR ‡ Ministry of Health, Manisa Public Health Directorate, Manisa, TR § Ministry of Health, Turkish Public Health Institution, Ankara, TR ‖ Hacettepe University, Faculty of Medicine, Department of Internal Medicine, Division of Occupational Medicine, Ankara, TR The Mersin Greenhouse Workers Study. Surveillance of Work-related Skin, Respiratory, and Musculoskeletal Diseases Farmers and farm work­ ers experience high rates of low back, shoulder, and upper extremity disorders [6]. Pesticide-related illnesses refer to a broad group of health outcomes, including dermatitis, eye injuries, respiratory diseases, and cancers [7]. The use to hazardous chemicals, particularly pesticides [3]. Agri­ culture and forestry, as an occupation, consistently ranks as the third or fourth most hazardous occupations in the European Union [4]. Agricultural workers suffer from sev­ eral conditions and diseases [5]. Farmers and farm work­ ers experience high rates of low back, shoulder, and upper extremity disorders [6]. Pesticide-related illnesses refer to a broad group of health outcomes, including dermatitis, eye injuries, respiratory diseases, and cancers [7]. The use Nuraydın A, et al. The Mersin Greenhouse Workers Study. Surveillance of Work- related Skin, Respiratory, and Musculoskeletal Diseases. Annals of Global Health. 2018; 84(3), pp. 504–511. DOI: https://doi.org/10.29024/aogh.2315 Nuraydın A, et al. The Mersin Greenhouse Workers Study. Surveillance of Work- related Skin, Respiratory, and Musculoskeletal Diseases. Annals of Global Health. 2018; 84(3), pp. 504–511. DOI: https://doi.org/10.29024/aogh.2315 ORIGINAL RESEARCH ¶ Department of Health Sciences of the University of Milan, Milan, IT ** Medical Centre of Vrije Universiteit Amsterdam, Amsterdam, NL †† Hacettepe University, Faculty of Medicine, Department of Public Health, Ankara, TR Corresponding author: Ali Naci Yıldız, MD, PhD (alinaciyildiz@gmail.com) The Mersin Greenhouse Workers Study. Surveillance of Work-related Skin, Respiratory, and Musculoskeletal Diseases Aydın Nuraydın*, Özgür Bilek†, Ali Koray Kenziman*, Mehmet Ali Korkusuz‡, Ali İhsan Atagün*, Nezaket Özpolat Çakar*, Naci Özer*, Serdar Deniz*, Mustafa Kemal Başaralı§, Ahmet Özlu§, Abdulsamet Sandal‖, Gert van der Laan¶,** and Ali Naci Yıldız†† Background: Skin, respiratory, and musculoskeletal diseases in greenhouse workers are frequently observed due to exposure to plant products and pesticides in enclosed conditions and ergonomic risks. Current ­studies on occupational health risks of greenhouse workers in Turkey are insufficient. p g yfi Objectives: The aim of the present study was to assess work-related skin, respiratory, and musculoskel­ etal diseases in greenhouse workers in the Erdemli province, Mersin, an area with a workforce predomi­ nantly active as greenhouse workers. y g Methods: The study population consists of adult greenhouse workers, who visited their family physician between June 12–14, 2017 and were diagnosed with dermatological, respiratory, and musculoskeletal diseases. Immediately after this consultation, occupational physicians conducted face-to-face interviews, asking questions about sociodemographic features and occupational factors including current and previous work, current exposures and relation of current symptoms with work. Results: In total, 423 workers with 555 diagnoses were included in the study. Percentages of diagnoses were 30.1%, 21.6% and 48.3%, for skin, respiratory and musculoskeletal diseases respectively. Nearly half of participants had taken an absence from work due to those diseases. Mean age of onset for greenhouse working was 15.5 years. Almost all participants (96.2%) reported contact with chemicals, and usage of respiratory protection was low (17.3%). Pesticides were regarded as a risk factor by nearly two-thirds of workers with skin or respiratory system disorders. Participants’ answers to questions regarding the rela­ tionship between their diseases and their work was positive for more than half of patients and patients with skin and respiratory diseases. Nearly half of the patients with musculoskeletal diseases attributed their complaints to physical overload at work. p p y Conclusions: We found evidence of work-relatedness in almost half of the 555 greenhouse workers with dermatological, respiratory and musculoskeletal diseases. These findings are helpful in creating an aware­ ness program. There is a need for a more detailed assessment of the cases and the related working condi­ tions to start a tailored prevention program. to hazardous chemicals, particularly pesticides [3]. Agri­ culture and forestry, as an occupation, consistently ranks as the third or fourth most hazardous occupations in the European Union [4]. Agricultural workers suffer from sev­ eral conditions and diseases [5]. Introduction The share of the population working in agriculture greatly varies among countries: while the vast majority of the pop­ ulation in low-income countries work in agriculture, this rate drops to less than 5% in high-income countries [1]. In Turkey, agriculture employs nearly one-fifth (18.9%) of the registered workforce [2]. The work is generally heavy, working hours can be very long, workers are exposed to extreme climatic conditions, and many are also exposed Nuraydın et al: The Mersin Greenhouse Workers Study 505 of pesticides causes at least 7 million cases of acute and long-term nonfatal illness [8]. occupational physicians for data collection via face-to-face interview in FHCs. Data collection forms had 23 questions divided into in two parts, including sociodemographic fea­ tures and features related to work including current and previous work, current exposures, and relation of current diagnosis with work. Diagnoses obtained from the fam­ ily physician visit were listed according to 10th edition of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) codes [13], but no additional examinations were performed due to limited resources, nor was any treatment recommended by the researchers. The preliminary study was performed on ten patients from a different FHC. Greenhouse cultivation, a form of agricultural production, is commonly preferred among farmers because it allows farmers to grow vegetables, fruit, flow­ ers, rare and exotic plants outside the normal seasons in areas covered by glass or plastic, regardless of weather con­ ditions. The work has many physical, chemical, biological and ergonomic risks, and those risks result in a variety of occupational accidents and work-related or occupational diseases [4, 9]. Although climate conditions can be con­ trolled in greenhouse cultivation, the level of occupational exposures to pesticides and plant products and related health risks in greenhouse workers are higher than those of outdoor farm workers due to the enclosed working conditions. The main hazards of working in greenhouses include: injury from materials, problems related to work­ ing position, chemicals including pesticides, endotoxins, dusts, and work at height [4, 10]. Dermatitis, musculoskele­ tal complaints, respiratory system diseases, and neurological diseases are the most common diseases among greenhouse workers [11]. However, the number of studies on health and safety issues of greenhouse workers is limited. Researchers obtained informed consent from each of the participants, whose names were not recorded. Results In three FHCs, 487 greenhouse workers over 18 years of age were diagnosed with skin, respiratory or musculo­ skeletal disease in 23 work days; however, 64 workers did not agree to participate in the study. Some demographic characteristics of 423 participants were summarized in Table 1. More than two-thirds (63.3%) of the participants were between 30–49 years of age and 30 participants (7.1%) were 60 years of age or older. A total of 74.9% of participants started to work in greenhouses at 18 years of age or younger, while 37.5% began such work at 15 years of age or younger. Of all participants, 57.4% who worked in the current greenhouses had previously worked in another workplace (Table 1); 169 people worked at one other workplace, 11 people worked at two and two participants worked at three different workplaces. Approximately one-fifth of these workplaces (22.8%) were greenhouses. Other sectors (n = 152) were outdoor agriculture and livestock (75.0%), industry (10.5%), service sector (10.5%) and construc­ tion sector (3.9%). The mean working duration at previ­ ous workplace was 12.1 ± 7.4 years (min = 1, max = 41, median = 10). There were 60 participants (14.2%) working in additional jobs (40.0% service sector, 31.6% outdoor agriculture and livestock, 13.3% greenhouse manufactur­ ing, 8.3% industry, 6.6% construction). Introduction After the inter­ views, all participants were informed about occupational diseases and advised to refer to the related department of the nearest authorized hospital (Mersin University Hospital) to prepare reference files for final diagnosis of occupational diseases by SSI. Permissions were obtained from Ministry of Health Turkish Public Health Institution, Mersin Public Health Directorate and Erdemli District Governorship, and the results of the research were presented to those units. Ethical approval was obtained from Hacettepe University Ethics Committee for Non-Interventional Clinical Studies (30 May 2017, Report No. 2017/14-846). In Turkey, there are approximately 5 million agricultural workers, but there was only one recognized case of occu­ pational disease in the agricultural work sector between 2012 and 2015, according to official statistics of the Social Security Institution (SSI) [2, 12]. There are no official sta­ tistics of occupational diseases in greenhouse workers. Greenhouse agriculture is common in the southern Turkey where Mersin is located. The Erdemli province of Mersin is one of the leading regions of Turkey’s greenhouse pro­ duction. The main economic activity in the district is agri­ culture, for which greenhouse cultivation is an important form of production. Enterprises are mostly small-scale family enterprises. The main products are tomato, cucum­ ber, pepper, and banana. Despite the extent of this eco­ nomic activity in this region, there is no data about the frequency occupational diseases of greenhouse workers. This gap in the literature was recently noticed by the occu­ pational physicians in the region who are also part of the current research team. Thus, in this study, we aimed to assess the greenhouse workers’ perception and evaluation of occupational risk factors for dermatological, respira­ tory, and musculoskeletal diseases. Statistical analyses were performed using the SPSS software version 24.0. Chi-square test was performed for categorical comparisons and logistic regression analyses were used for multiple comparisons. A 5% type-1 error level was used to infer statistical significance. Materials and Methods The research was conducted between June 12–July 14, 2017 (during 23 work days) in three family health centers (FHCs), which offer health service to a region with numer­ ous greenhouses in the Erdemli province. Nearly 21,000 people are served by nine family physicians working in those three FHCs. Greenhouse workers over 18 years of age who were diagnosed with dermatological, respira­ tory, and musculoskeletal diseases by family physicians within the study period and who agreed to participate to the study were included. After the family physician visit, patients meeting specified criteria were asked whether they wanted to participate in the ongoing research. Patients who accepted were directed to one of the six In terms of duties listed in Table 1, three of four partici­ pants (75.5%) stated that they performed all of the jobs 506 Nuraydın et al: The Mersin Greenhouse Workers Study Table 1: Demographic and work characteristics of participants (n = 423). Table 1: Demographic and work characteristics of participants (n = 423). Characteristic Statistics Age (years), mean ± SD (min-median-max) 41.2 ± 11.2 (18–41–75) Sex, n (%) Male 247 (58.4) Female 176 (41.6) Business type, n (%) Family business 355 (83.9) Business owner 28 (6.6) Salaried seasonal worker 26 (6.1) Paid permanent worker 13 (3.1) Age started to work (years), mean ± SD (min-median-max) 15.5 ± 3.6 (5–15–35) Total working duration in greenhoouses (months), mean ± SD (min-median-max) 215.5 ± 116.1 (12–216–732) Working duration in current greenhouse (months), mean ± SD (min-median-max) 187.5 ± 111.5 (1-180–480) Prior working history, n (%) Absent 243 (57.4) Present* 180 (42.6) Duties performed in greenhouses, n (%) Dibbling 413 (97.6) Weeding 379 (89.6) Spraying 364 (86.1) Spinning 411 (97.2) Fertilization 353 (83.5) Harvesting 415 (98.1) Sorting 403 (95.3) Roofing 323 (76.4) Occupational health practice†, n (%) Contact with chemical substances 405 (96.2) Using work clothes 293 (69.6) Using gloves 266 (62.9) Using work clothes in daily life/at home 192 (45.6) Using a mask 73 (17.3) Consuming food and beverage in the greenhouse 282 (67) min, minimum; max, maximum; SD, standard deviation. *Duration of prior working history (years), mean ± SD (min-median-max) was 12.1 ± 7.4 (1–10–41). †Two cases were missing (n = 421). With 113 people having more than one diagnosis (94 people with two, and 19 people with three systemic dis­ eases), a total of 555 diseases were diagnosed in 423 indi­ viduals (Table 2). Materials and Methods *10th revision of the International Statistical Classification of Diseases and Related Health Proble Of the cases referred by family physicians to the Mersin University Hospital, three were related to musculoskeletal diseases, one for respiratory system diseases, and one for skin diseases. The frequency of any workday loss due to illness in past year was 19.7% (n = 33) for skin diseases, 55.8% (n = 67) for respiratory diseases, 57.5% (n = 153) for musculoskel­ etal diseases, and 45.7% (n = 253) overall. The frequency of workday loss from respiratory and musculoskeletal disorders was significantly higher than that from skin dis­ ease (p < 0.001). Materials and Methods Durations of the complaints in months [n (mean ± standard deviation-median)] were 167 (80.05 ± 66.19–60) for skin diseases, 120 (112.32 ± 85.0–16) for respiratory diseases, and 266 (92.39 ± 68.90–72) for mus­ culoskeletal diseases. listed. In the analysis of duties according to sex, statisti­ cally significant differences (p < 0.0001) were observed for weeding (63.1%–36.9%), spraying (66.5%–33.5%), fertilization (68.8%–31.2%), roofing (72.1%–27.9%) and workers who did all the jobs listed (72.3%–27.7%) (order of percentages follows as men to women). Some features of participants related to occupational health practices are given in Table 1. Logistic regression analysis did not reveal any statistically significant rela­ tionship between presence of the dermatological or res­ piratory disease and contact with chemical substances, features related to personal protectors (e.g. usage of spe­ cial clothes, gloves, and masks at work or changing clothes after work), and consuming food or beverages in green­ houses (p > 0.05). More than half of the patients with skin or respiratory diseases answered open-ended questions about their own beliefs regarding possible occupational factors causing the disease as pesticides (61.1% and 66.7% respectively). People with musculoskeletal disease most frequently indi­ cated heavy lifting (46.6%) and bending (45.1%) as occu­ pational risk factors (Table 3). 507 Nuraydın et al: The Mersin Greenhouse Workers Study Table 2: Distribution of the diseases according to ICD-10* codes. Disease n % Skin (n = 167) L23.9 Allergic contact dermatitis, unspecified cause 125 74.9 L30. 9 Dermatitis, unspecified 11 6.6 L50 Urticaria 9 5.4 L29.9 Pruritus, unspecified 8 4.8 L20.9 Atopic dermatitis, unspecified 6 3.6 R23.8 Other skin changes 5 3.0 R21 Rash and other nonspecific skin eruption 3 1.8 Respiratory System (n = 120) R06.0 Dyspnea 68 56.7 J45 Asthma 32 26.7 J30.4 Allergic rhinitis, unspecified 8 6.7 R05 Cough 7 5.8 J40 Bronchitis, not specified as acute or chronic 2 1.7 R07.0 Pain in throat 2 1.7 J44 Other chronic obstructive pulmonary disease 1 0.8 Musculoskeletal System (n=268) M54.5 Low back pain 195 72.8 M25.5 Pain in joint 25 9.3 M51.9 Unspecified thoracic, thoracolumbar and lumbosacral intervertebral disc disorder 22 8.2 M79.1 Myalgia 20 7.5 M79.6 Pain in limb, hand, foot, fingers and toes 4 1.5 M50 Cervical disc disorders 2 0.7 *10th revision of the International Statistical Classification of Diseases and Related Health Problems. Table 2: Distribution of the diseases according to ICD-10* codes. Discussion In the present study, we evaluated 423 greenhouse work­ ers with total number of 555 diagnoses of skin, respira­ tory, and musculoskeletal diseases. Approximately half of the diagnoses (48.3%) were musculoskeletal diseases, 30.1% were skin diseases, and 21.6% were respiratory diseases. Mean age of onset for working in greenhouses was 15.5 years. Percentages of participants with a previ­ ous history of working in a different workplace and with a current additional job were 42.6% and 14.2% respec­ tively. Nearly half of participants had workday loss due to those diseases. Almost all participants in the survey were exposed to hazardous chemicals, especially pesticides via routes of inhalation and skin contact as well as ingestion with food and beverages consumed in the workplace. Pes­ ticides were claimed as an occupational risk factor trig­ gering the disease by nearly two-thirds of patients with skin or respiratory system disorders. More than half of all patients and patients with skin diseases or patients with musculoskeletal diseases answered positively to all five questions about work-relatedness of their disease, although the number of the cases referred to authorized university hospital by family physicians was quite few. Analysis of answers of participants to questions asking the relationship between their complaints or diseases and their work revealed that more than half of the all patients and patients with skin diseases or patients with muscu­ loskeletal diseases answered positive to all five questions (50.1%, 50.9%, and 57.1% respectively). Only five people (1.2%) answered negatively to all questions (Table 4). In the logistic regression analysis, a positive response to the occupational relationship for all five problems involving the work performed by the patient was not associated with sex, age, previous job, previous occupation, other job, current disease duration, the number of days in which the worker did not work in the last year and the working position (p > 0.05). Compared with the lesser duration of greenhouse work history, those who worked more than 20 years in greenhouses and compared to those diagnosed with a respiratory system disease, those diagnosed with a musculoskeletal system disease were 3.05 times higher (95% CI: 1.55–6.00, p = 0.001) and 2.43 times (95% CI: 1.30–4.50, p = 0.005) answered as yes to all questions respectively. Nuraydın et al: The Mersin Greenhouse Workers Study 508 Table 3: Distribution of factors participants’ answers about their own beliefs regarding possible occupational factors causing the disease. Nuraydın et al: The Mersin Greenhouse Workers Study 509 Nuraydın et al: The Mersin Greenhouse Workers Study A person with an undefined occupational or work- related disease is expected to seek care from a physician in the primary health care services [14]. The nature of work-related or occupational diseases is often not recog­ nized when a patient is consulting a general physician. Lack of awareness and lack of taking a proper occupa­ tional history generally are the causes of underreporting. Several studies address this problem. In general, one of the main problems for underdiagnosis of occupational diseases is the lack of proper occupational history and other related information [15]. Although some basic ques­ tions on whether symptoms arise at work or whether other colleagues have similar symptoms could be impor­ tant clues, occupational history is not usually obtained at the expected level, due to intensive work conditions. Several studies address this problem. A study performed in the UK established that 25% of family physicians did not ask about occupation during their examination [16]. In Turkey, a study showed that 43.9% of the physicians who provided outpatient care didn’t ask patients’ occupa­ tions [17]. Other problems for underdiagnosis of occupa­ tional diseases may include unawareness of notification procedures, need for additional time and effort to com­ plete notification procedures, and unavailability of sys­ tems promoting notification [18]. Although notification of occupational diseases is defined by law and reporting is mandatory in Turkey, notification system has some bar­ riers. The occupational or family physicians should direct the patients with occupational disease to the authorized hospitals for the preparation of the files. The file, which is the basis for the recognition of the occupational disease, is sent to SSI which makes the final decision on suspected occupational disease. an early age and spend time with their parents in an indoor greenhouse environment where they are faced with many unfavorable conditions for every age group. Family mem­ bers can spend the day and even sleep in the greenhouse, especially during the winter months. Moreover, small farmers live where they work, so workplace exposures all too easily migrate into the home.Future studies may be planned to evaluate women’s and children’s health issues related to greenhouse working. The rates of working in another workplace or having additional work and the frequency of workplace change were high in our study. Nuraydın et al: The Mersin Greenhouse Workers Study This phenomenon, which is wide­ spread in the agricultural sector, poses a problem for the implementation and sustainability of occupational health and safety practices and monitoring efforts. Although the average daily working time was within the legal limits on the dates of the research, the workload and the duration of daily working can change according to the seasonal and production stage and can exceed the legal limits. Three-quarters of the participants indicated that they did all kind of work in the greenhouse, although it can be inferred that most of the participants performed all kinds of duties and were exposed to all kinds of effects in the same indoor environment. Lack of statistical significance in the distribution of diseases according to the work may be related to the aforementioned issues. Participants reported higher exposure frequencies than protective device use. Lack of any statistically sig­ nificant difference between groups using and not using personal protective equipment in regards to the presence of skin or respiratory disease may directly be related to the characteristics of the equipment used. Two-thirds of the respondents said they used work clothes, but not all were work-specific clothes. Similarly, although two-thirds of participants reported the usage of gloves, the gloves were mostly inappropriate for the task and of poor quality. Less than one-fifth of participants stated that they used masks. It is common practice for farmers in developing countries to apply hazardous pesticides while working barefoot. The level of skin exposure varies with the fre­ quency of pesticide application, the pesticide-active ingre­ dient concentration and whether personal protectors or other protector equipment is being used correctly [3]. In order to prevent the spread of pesticide-related health conditions, precautions such as providing workers with less toxic substances (such as biological control meth­ ods [23, 24]) adequate protective equipment, training on prevention of pesticide exposures, and implementation of administrative restrictions of working in fields where exposure may occur, ould be taken [3]. In the present study, greenhouse workers with skin, respiratory, and musculoskeletal diseases were evaluated through questions about their work. Although some of the diseases required additional diagnostic procedures (e.g. patch tests for allergic contact dermatitis, serial Peak Expiratory Flow measurements or bronchial challenge tests for asthma), others like low back pain did not, and the algorithm for the diagnosis of occupational diseases began with compatible occupational history [19, 20]. Discussion g Disease n % Skin (n = 167) Pesticides 102 61.1 Dust 58 34.7 Hot working environment 27 16.2 Contact with the produced plant 18 10.8 Humidity 9 5.4 Bending work 2 1.2 Chemical fertilizer 2 1.2 Working upwards 2 1.2 Heavy lifting 1 0.6 Respiratory System (n = 120) Pesticides 80 66.7 Dust 54 45 Humidity 19 15.8 Hot working environment 18 15 Chemical fertilizer 3 2.5 Heavy lifting 1 0.8 Musculoskeletal System (n = 268) Heavy lifting 125 46.6 Bending work 121 45.1 Hot working environment 15 5.6 Working upwards 8 3 Dust 2 0.7 Humidity 2 0.7 Pesticides 2 0.7 Table 4: Distribution of positive answers for questions asking work-relatedness and for all questions about the relation­ ship of diseases to work. Table 4: Distribution of positive answers for questions asking work-relatedness and for all questions about the relation­ ship of diseases to work. tion of positive answers for questions asking work-relatedness and for all questions about the relation­ s to work Table 4: Distribution of positive answers for questions asking work-relatedness and for all questions about the relation­ ship of diseases to work. Skin (n* = 167) Respiratory System (n* = 120) Musculoskeletal System (n* = 268) Total (n† = 423) Question n % n % n % n % Did disease start after started to work in the greenhouse? 141 84.4 97 80.8 230 85.8 353 83.5 Does the disease exacerbated as you work in the greenhouse? 155 92.8 117 97.5 258 96.3 402 95 Are there any others working in the same workplace have similar disease? 102 61.1 56 46.7 186 69.4 262 61.9 Do your complaints decline when you do not work in the greenhouse? 152 91 112 93.3 249 92.9 387 91.5 Is there at least one factor in your workplace causative this disease? 148 88.6 110 91.7 248 92.5 380 89.8 Total number of positive answers n* % n* % n* % n† % 5 85 50.9 46 38.3 153 57.1 212 50.1 4 54 32.3 53 44.2 84 31.3 144 34,1 3 14 8.4 12 10 17 6.3 36 8.5 2 4 2.4 6 5 7 2.6 14 3.3 1 7 4.2 2 1.7 5 1.9 12 2.8 – 3 1.8 1 0.8 2 0.7 5 1.2 *Number of diagnoses. †Number of workers. Nuraydın et al: The Mersin Greenhouse Workers Study By analyzing occupational histories of participants, we obtained clues to diagnose occupational or work-related diseases. Our results also showed that those diseases cause workday loss; thus future studies are needed to evaluate and enhance working conditions of greenhouse workers and to implement specific diagnostic guidelines. Within the scope of the research, we also obtained data on working life, working environment and working con­ ditions that could affect the health level of greenhouse employees. Although all participants were adults, the mean age of onset for working in greenhouses was 15.5 years. Some participants reported much younger starting ages for greenhouse working, which was part of family business, despite strict prohibitions of current legislation [21]. Agricultural work is one of the most common forms and the most dangerous form of child labor [22]. Most of the participants were business owners or unpaid family workers. In the family business, children begin to work at Our study’s strengths include the original study design, the large study population size, its impact in raising awareness among stakeholders, and encouragement of further studies. After the research was completed, basic occupational health and safety trainings were held, bro­ chures and banners about occupational diseases were delivered. Thanks to the efforts of the Mersin Public Health Directorate, the frequency of obtaining occupa­ tion and work records of family physicians in the region has increased. A workshop demonstrating good practice Nuraydın et al: The Mersin Greenhouse Workers Study 510 examples was shared with all stakeholders was also held. Additionally, results of current study will be reinforced by several future intervention studies. 5. Hansen E and Donohoe M. Health issues of migrant and seasonal farmworkers. Journal of Health Care for the Poor and Underserved. 2003; 14(2): 153–164. DOI: https://doi.org/10.1353/hpu.2010.0790 A limitation of the study is that the period of collection of the data, corresponded with both summer months with 38°C in the temperature and the Ramadan month of fast­ ing, so the time period reduced the number of referrals to FHCs compared to other months, despite the absence of any factors causing a selection bias. Additionally, there were no records of occupational health and safety and a lack of information on risk assessments and hazard identi­ fication studies and precautionary measures of entry and periodic examinations, as well as family physicians’ work­ load, duration of loss of capacity due to illness, which may have limited number of referred patients. Competing Interest 13. WHO. International Statistical Classification of Dis­ eases and Related Health Problems 10th Revision Version; 2016. http://apps.who.int/classifications/ icd10/browse/2016/en. Accessed September 12, 2017. The authors have no competing interests to declare. Author Contribution All authors had access to the data and a role in writing the manuscript. 14. Lax MB, Grant WD, Manetti FA and Klein R. Recognizing occupational disease--taking an effec­ tive occupational history. Am Fam Physician. 1998; 58(4): 935–944. Nuraydın et al: The Mersin Greenhouse Workers Study Other limita­ tions include usage of diagnoses made by family physicians without any further standardized verification, inclusion of participants who were currently at a health care facility far from workplace, lack of standardized evaluation of work­ places, possible increased awareness of participants about the relationship between work and their diseases due to family physicians’ awareness about the study. 6. Davis KG and Kotowski SE. Understanding the ergonomic risk for musculoskeletal disorders in the United States agricultural sector. American Journal of Industrial Medicine. 2007; 50(7): 501–511. DOI: https://doi.org/10.1002/ajim.20479 7. Kim K-H, Kabir E and Jahan SA. Exposure to pes­ ticides and the associated human health effects. Science of The Total Environment. 2017; 575(Supple­ ment C): 525–535. DOI: https://doi.org/10.1016/j. scitotenv.2016.09.009 8. ILO, World Health Organization (WHO). Joint Press Release ILO/WHO Number of Work-related Accidents and Illnesses Continues to Increase ILO and WHO Join in Call for Prevention Strategies, 2005. http://www.ilo.org/global/about-the-ilo/ newsroom/news/WCMS_005161/lang--en/index. htm. Accessed September 10, 2017. 9. Coumbis JJ and Anderson RG. Assessment of the Occupational Health of Greenhouse Workers. Uni­ versity of Kentucky, College of Agriculture, Agricul­ tural Experiment Station; 1988. y p y y To conclude, factors such as younger starting ages of working due to family businesses, frequent job changes due to insecurity, and increased physical risk factors such as heavy lifting, bending, or extreme temperature or chemical risk factors like pesticides and chemical fertiliz­ ers, demonstrate that greenhouse work is related to a vari­ ety of diseases. Results of our study emphasize the need to objectively evaluate both health conditions and risk fac­ tors in greenhouse workplaces and to develop solutions. Our findings are important for awareness raising and ini­ tiation of a program for improvement of health and safety in greenhouse workers in the Mersin area, which may be implemented later in the entire greenhouse sector in Turkey. 10. Nordgren TM and Bailey KL. Pulmonary health effects of agriculture. Current Opinion in Pulmonary Medicine. 2016; 22(2): 144–149. DOI: https://doi. org/10.1097/MCP.0000000000000247 11. Jurewicz J, Kouimintzis D, Burdorf A, Hanke W, Chatzis C and Linos A. Occupational risk factors for work-related disorders in greenhouse workers. Journal of Public Health. 2007; 15(4): 265–277. DOI: https://doi.org/10.1007/s10389-007-0129-x 12. Republic of Turkey Social Security Institution. SGK İstatistik Yıllığı 2016. http://www.sgk.gov.tr/ wps/portal/sgk/tr/kurumsal/istatistik/sgk_istatis­ tik_yilliklari. Accessed September 10, 2017. Competing Interest How to cite this article: Nuraydın A, Bilek Ö, Kenziman AK, Ali Korkusuz M, Atagün Aİ, Çakar NÖ, Özer N, Deniz S, Başaralı MK, Özlü A, Sandal A, van der Laan G and Yıldız AN. The Mersin Greenhouse Workers Study. Surveillance of Work-related Skin, Respiratory, and Musculoskeletal Diseases. Annals of Global Health. 2018; 84(3), pp. 504–511. DOI: https://doi.org/10.29024/ aogh.2315 References 1. Roser M. Employment in Agriculture; 2017. https://ourworldindata.org/employment-in- agriculture#long-run-perspective-1300-to-today. Accessed November 27, 2017. 15. Bilir N and Yıldız AN. İş Sağlığı ve Güvenliği. Ankara: Hacettepe Üniversitesi Yayınları; 2014. 16. Elms J, O’Hara R, Pickvance S, et al. The percep­ tions of occupational health in primary care. Occup Med (Lond). 2005; 55(7): 523–527. DOI: https://doi. org/10.1093/occmed/kqi123 2. Turkish Statistical Institute. İşgücü İstatistikleri 2015. http://www.tuik.gov.tr/PreHaberBultenleri. do?id=21567. Accessed September 10, 2017.i 3. International Labour Office (ILO). Safety and health in agriculture. ILO code of practice. Geneva: International Labour Office; 2010. 17. Cimrin AH, Sevinc C, Kundak I, Ellidokuz H and Itil O. Attitudes of medical faculty physi­ cians about taking occupational history. Med Educ. 1999; 33(6): 466–467. DOI: https://doi. org/10.1046/j.1365-2923.1999.00366.x 4. European Commission Directorate-General for Employment Social Affairs and Inclusion. Protecting health and safety of workers in agricul­ ture. Publications Office of the European Union; 2012. 18. Curti S, Sauni R, Spreeuwers D, et al. Interven­ tions to increase the reporting of occupational dis­ eases by physicians: A Cochrane systematic review. 511 Nuraydın et al: The Mersin Greenhouse Workers Study Occup Environ Med. 2016; 73(5): 353–354. DOI: https://doi.org/10.1136/oemed-2015-103209 Workers. Republic of Turkey Ministry of Labor and Social Security, Ankara; 2004. 19. Bepko J and Mansalis K. Common occupational disorders: Asthma, COPD, dermatitis, and musculo­ skeletal disorders. Am Fam Physician. 2016; 93(12): 1000–1006. 22. Öngel S. Çocuk İşçiliği Gerçeği Raporu. 2015. http://disk.org.tr/2015/04/disk-ar-turkiyede- cocuk-isciligi-gercegi-raporu-2015/. Accessed October 30, 2017. 20. Punnett L, Pruss-Utun A, Nelson DI, et al. Esti­ mating the global burden of low back pain attrib­ utable to combined occupational exposures. American Journal of Industrial Medicine. 2005; 48(6): 459–469. DOI: https://doi.org/10.1002/ ajim.20232 23. Birişik N, Kütük H, Karacaoğlu M, Yarpuzlu F, İslamoğlu M and Öztemiz S. Teoriden Pratiğe Biyolojik Mücadele. In: Birişik N (ed.), Örtü Altı Sebze Yetiştiriciliğinde Biyolojik Mücadele. 2012; 13–25. Ankara: T.C. Gıda Tarım ve Hayvancılık Bakanlığı. 24. Tuncer C, Mennan S, Akça İ, Saruhan İ and Akyazı F. Seralarda Zararlılar ile Biyolojik Mücadele. The Journal of Agricultural Facullty of Ondokuz Mayıs University. 2004; 19(2): 83–89. 21. Republic of Turkey Ministry of Labor and Social Security. Regulations on the Fundamentals and Principles of the Employment of Children and Young How to cite this article: Nuraydın A, Bilek Ö, Kenziman AK, Ali Korkusuz M, Atagün Aİ, Çakar NÖ, Özer N, Deniz S, Başaralı MK, Özlü A, Sandal A, van der Laan G and Yıldız AN. The Mersin Greenhouse Workers Study. References Surveillance of Work-related Skin, Respiratory, and Musculoskeletal Diseases. Annals of Global Health. 2018; 84(3), pp. 504–511. DOI: https://doi.org/10.29024/ aogh.2315 Published: 31 August 2018 Published: 31 August 2018 Published: 31 August 2018 Copyright: © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. Annals of Global Health is a peer-reviewed open access journal published by Levy Library Press. OPEN ACCESS
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The use of heat from the CO<sub>2</sub>compression system for production of system heat
E3S web of conferences
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© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). The use of heat from the CO2 compression system for production of system heat Robert Zarzycki1,* 1Energy Engineering Department, Faculty of Infrastructure and Environment, Czestochowa University of Technology, Brzeznicka Street 60A, 42-200 Czestochowa, Poland Robert Zarzycki1,* 1Energy Engineering Department, Faculty of Infrastructure and Environment, Czestochowa University of Technology, Brzeznicka Street 60A, 42-200 Czestochowa, Poland Robert Zarzycki1,* 1Energy Engineering Department, Faculty of Infrastructure and Environment, Czestochowa University of Technology, Brzeznicka Street 60A, 42-200 Czestochowa, Poland Abstract. The study presents the concept and computations of the CO2 compression for the purposes of transport and underground storage of the gas. Cooling between individual stages is needed to reduce the power needed for CO2 compression. Heat obtained from the cooling process can be used to provide heat to the municipal heat power systems. A design of a heat accumulator for storage of excess heat was proposed in order to improve heat supply safety. The solution proposed in the study allows for heating condensing power units using waste heat from the CO2 cooling process. * Corresponding author: zarzycki@is.pcz.pl E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 2 Conventional energy generation As mentioned in the Introduction section, processes of combustion of fossil fuels for the purposes of electricity and heat generation contribute to emissions of various pollutants, including CO2. All the newly built power plants have to be prepared for construction of installations of CO2 capture and separation. With the already existing energy installations, one should take into consideration the increase in unit costs of electricity and heat generation connected with buying permits for CO2 emissions to the atmosphere or the necessity to install CO2 capture and separation systems. y p p y The conventional energy sector can limit CO2 emissions through [8]: • CO2 capture from flue gas. Nowadays, it is possible to use absorption or adsorption technologies; • CO2 capture from flue gas. Nowadays, it is possible to use absorption or adsorption technologies; • Oxygen combustion. In this case, flue gas contains 90–95% CO2; • CO2 capture before combustion. Regardless of the method of limitation of CO2 emissions, it is necessary to compress the gas in order to transport it and store underground. In order to demonstrate the scale of the problem, one can use the following example of newly built power units. Depending on the fuel burnt, a power unit with a capacity of 900 MWe emits around 175 kg CO2/s (hard coal), 225 kg CO2/s (brown coal). The major task of power units in industrial power plants is to generate electricity. Depending on the location (power plant near a big city) and local infrastructure (presence of heat power systems), power units may, besides their main function of electricity generation, be a source of heat needed for municipal heat power system. In the case of big power objects (with condensing turbines), the opportunities for generating additional heat is connected with collecting the steam from the turbine outlet and transfer of the steam to heat exchanger where the water for the network is heated while steam collected from the turbine outlet is condensed. This solution is popular in steam power units and allows for generation of the required amount of heat. This system allows for improving the efficiency of electricity and heat generation. A drawback of this solution is a decline in electricity generation with the increase in heat generation, related to the increased stream of steam transferred to the heat exchanger. 1 Introduction In many countries, including Poland, combustion of fossil fuels have been used to generate electricity and heat. Fossil fuel combustion processes contribute to degradation of the natural environment and climate changes through emissions of combustion products to the atmosphere, including: CO2, SO2, NOx, dusts, and heavy metals, including Hg. Modern technologies of flue gas treatment allow for a substantial limitation of emissions of SO2, NOx and dusts. Technologies of Hg emissions limitation and other heavy metals have been developed [1–3]. The problem of CO2 emissions to the atmosphere has not been solved yet. The regulations known as climate package require limitations of CO2 emissions by 20%, the increase in the use of renewable energy sources to 20% and the increase in energy efficiency to 20% compared to prognoses for 2020. In the development of renewable energy sources, it is mainly the energy sector that uses wind force which helps meet the requirement of 20% energy production from renewable sources. The percentage of electricity generated from renewable sources in Germany increased from 6.3% in 2000 to ca. 41% in 2018, which is double the value of the assumed level of 20% [4]. One should expect further dynamic increase in renewable energy in other countries of the European Union. The studies have been conducted in recent years to improve efficiency of energy generation in power plants and combined heat and power plants [5–7]. Ultra-supercritical power unit technologies are being developed in order to exceed the level of efficiency of 50%. The increase in the use of fossil fuel energy can be achieved by the combined generation of electricity and heat and construction of gas and steam systems. Limitation of CO2 emissions to the atmosphere can be achieved by its capture from flue gas, followed by storage [8]. The process of CO2 capture from flue gas can be performed using various technologies: E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 adsorption, absorption, membrane, and cryogenic. Carbon dioxide isolated from flue gas should be then transported to places where it can be permanently stored. The study presents a proposal for the use of heat from the CO2 combustion process for the purposes of supplying heat to the steam power unit. Furthermore, the opportunities for storing the heat using a heat accumulator were also discussed. E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 Another method to provide heat to the steam power unit is to install an absorption heat pump which is fuelled by steam from the turbine outlet and allows for using a low- temperature heat contained in the water that cools the condenser [12]. Compared to a conventional steam-fuelled heat exchanger, this solution allows electrical power of the steam power unit to be reduced at similar heat generation. Providing heat to a steam power unit using an absorption heat pump helps prepare water with maximal temperature of up to 95°C but it requires installation of a peak heat exchanger for ensuring the required parameters of the network water and heat supply safety. The presented methods to provide heat to condensing steam power units allow first and foremost to improve the efficiency of the combined electricity and heat generation. However, they require additional investment expenditure for building installations of heat exchangers or installations of absorption heat pumps. In order to limit costs of these installations (reduction of installed capacity) and their negative effect on operation of a steam power unit (reduction in electrical power), it is necessary to store heat generated. Installation of the system of heat storage helps make electricity and heat generation process independent in the steam power unit. Therefore, it is possible to ensure optimal electricity and heat generation at higher thermodynamic and economic efficiency. Heat supplies to heat networks and next to receivers require ensuring adequate parameters (temperature, mass flux) of the heat carrier (water). Parameters of heat network operation are contained in the regulation tables which are prepared individually for each heat network and heat consumer. An example of a regulation table is presented in Table 1. From the standpoint of work and installed capacity of the heat source, in addition to the required temperature of network water, important factors also include durations of individual values of external temperatures. Fig. 1 presents an example diagram of external temperatures and the number of hours these temperatures are observed [13]. Table 1. Regulation table [10]. No Air temp. [oC] Feed water temp. 2 Conventional energy generation Another method to provide heat for power unit is to use waste heat contained in flue gas or water that cools the turbine condenser. The efficiency of power units depends on several factors, such as power, combustion technology (pulverized coal-fired boiler, fluidized-bed boiler) and parameters of generated steam or water. However, the biggest effect on energy boiler efficiency is from the fuel and its water content. The most advanced brown coal-fired power boilers reach the efficiency of ca. 88%, whereas those hard coal-fired have an efficiency of even 95% [9]. Despite such high efficiency, the amount of heat contained in flue gas, especially in boilers fired by brown coal or biomass with high water content is sufficient for heat production [10] or using it for other purposes, e.g. preparation of flue gas for CO2 separation using adsorption methods [11]. In this case, the amount of heat generated is proportional to boiler power and, therefore, the capacity of steam power unit. Using this solution, there is a necessity to install a peak heat exchanger that allows for reaching required parameters (temperature, power) of the water in the network. 2 E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 [oC] Return water temp [oC] 1 -16 125 66 2 -15 123 65 3 -14 121 64 4 -13 118 63 5 -12 115 62 6 -11 113 61 7 -10 111 60 8 -9 108 59 9 -8 105 58 10 -7 102 57 11 -6 99 56 12 -5 97 55 13 -4 95 54 14 -3 93 53 15 -2 91 52 16 -1 88 51 17 0 86 50 18 1 84 49 19 2 81 48 20 3 79 47 21 4 76 46 22 5 72 45 23 6 69 44 24 7 66 43 25 8 63 42 26 9 59 41 27 10 56 40 28 11 53 39 29 -12 115 62 Table 1. Regulation table [10]. 3 3 E3S Web of Conferences 49, 00135 (2018) E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 SOLINA 2018 a) 0 1000 2000 3000 4000 5000 6000 7000 8000 35 30 25 20 15 10 5 0 -5 -10 -15 -20 Air temperature [ oC] Time [h] b) -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 0 50 100 150 200 250 300 350 400 450 Hours of air temperature [h] Air temperature [ oC] Fig. 1. The structured graph of outer air temperature for location of a dedicated heat recipient a), hours of presence of external air temperature during the central heating season b) [13]. b) Fig. 1. The structured graph of outer air temperature for location of a dedicated heat recipient a), hours of presence of external air temperature during the central heating season b) [13]. 3 Storing heat For economic and constructional reasons, heat accumulators are unpressurised tanks, and, consequently, maximal temperature of the water stored cannot exceed 100°C. The upper part of the heat accumulator contains hot water with the temperature of the heat network feed, whereas the lower part of the tank is filled with water with the return temperature. There is also a mixture of both types of water between each other, termed thermocline. The difference between temperature of hot and cold water in unpressurised tanks ranges from 30–40°C. The phenomenon of thermal stratification of water is demanded in heat accumulators. In properly designed containers, this phenomenon occurs independently through gravitational forces (differences in hot water and cold water densities). For functional reasons, the heat accumulator should be smooth. The optimal height-to-diameter ratio of heat accumulator ranges from 3 to 4 [15]. The Polish energy system uses heat accumulators with capacities from 12,000 to over 30,000 m3. The heat accumulator installed in Elektrociepłownia Siekierki has capacity of 30,400 m3, with tank diameter of 30 m and height of 47 m. Heat capacity of the accumulator is 1,600 MWh whereas its heat power is 300 MWt. 3 Storing heat Due to substantial capacities of heat power systems, storage of heat requires the equipment and installations which allow for storing huge amounts of heat. There is a number of heat storing methods [14]. However, for technical reasons, the best solution is to store heat in hot water in heat accumulators. Heat accumulators are mostly built near combined heat and power units, which allows for separation of electricity and heat generation. A heat accumulator is a tank which accumulates heat contained in hot water. The accumulator ensures smooth operation of the unit between night and day, allowing for switching the generation to more efficient units and increasing production in co-generation. The heat is collected when the demand for heat is lower and returned when its consumption increases (in the mornings and evenings). Heat accumulator capacities have to be chosen based on the analyses of heat demand and opportunities for operation with the generating unit and heat network and economic analysis connected with investment costs. A heat accumulator is usually an unpressurised water tank included in the heat power system. During "charging" the accumulator (Fig. 2a), hot water is supplied to the upper part of the tank, whereas cold water is received from its bottom part. Therefore, the intermediate layer between the hot and cold water is moved to the lower part of the tank, while the amount of heat collected in the accumulator is increased (level of water in the tank is virtually unchanged). The accumulator is discharged (Fig. 2b) by reception of the hot water Fig. 2. Processes of (a) charging and (b) discharging of a heat accumulator. hot water cold water thermocline hot water cold water thermocline Fig. 2. Processes of (a) charging and (b) discharging of a heat accumulator. 4 4 E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 from the upper part of the tank and supply of the cooled water to its lower part. A direct layer is moved towards the upper part of the container. For economic and constructional reasons, heat accumulators are unpressurised tanks, and, consequently, maximal temperature of the water stored cannot exceed 100°C. from the upper part of the tank and supply of the cooled water to its lower part. A direct layer is moved towards the upper part of the container. n – number of compression stages. The most popular compression systems are those with 3 to 6 compression degrees with cooling between stages, whereas the use of a higher number of compression stages does not produce substantial thermodynamic effects [16] and significantly improves the costs of installation and reduces its efficiency. In the case of multi-stage systems, adequate division of pressures between individual compression stages is critical to minimize compression. It is necessary to conduct multi-dimensional optimization, with optimization criterion being minimization of power necessary for CO2 stream compression. The CO2 obtained from flue gas during separation has to be compressed to reach the parameters that allow for its transport in the liquid phase. With the necessity of supplying the compressed gas to more remote destinations (200 to 300 km), it is critical to obtain high initial pressure which allows for overcoming CO2 flow resistance in the piping and ensures minimal pressure in the pipeline which is not lower than 8 MPa. Therefore, for the purposes of computations presented in the study, the initial CO2 pressure of 15 MPa was adopted, with temperature of 20°C. Computation of the multi-stage compression with cooling between stages was performed for the 900 MWe power unit fired with hard coal (emits CO2 around 175 kg/s) using the dedicated computation software where necessary physical parameters of CO2 were implemented based on the studies [17, 18]. The process of multi-stage CO2 compression is performed near the steam power unit that generates power necessary for driving the compressors (6 to 8% of the power unit capacity) [8]. Depending on the parameters, the heat obtained during cooling in inter-stage coolers can be used in the system for regeneration of water that fuels the steam water, in ORC cycle, and system heat generation or for the purposes of cold generation. The process of multi-stage compression with cooling between stages was performed for the systems containing from 4 to 7 compression stages in the 900 MWe. power unit. A diagram of an example structure of a 7-stage compression system with cooling between steps is presented in Fig. 3. The system is composed of seven compressors denoted with symbols of C1 to C7, with heat exchangers between each other, marked HE1 to HE7. The HE0 heat exchanger is installed before the first compression stage. The temperature of the compressed CO2 after each heat exchanger and before the next compression stage is 60°C. 4 Compression of CO2 The CO2 separated from flue gas, with pressure and temperature similar to conditions of the environment has to be compressed to the pressure of 12–15 MPa and cooled to the temperature of 10–25°C that ensures its transport in the liquid phase. The process of CO2 compression can be performed by means of positive displacement compressors and flow compressors. However, due to substantial fluxes of the compressed CO2, the flow compressors are more often used. The thermodynamic fundamentals of the multi-step CO2 compression were presented in a study [16]. The work [J/(kg CO2)] of the polytropic compression which is actually performed is given by (1): (1)           −       ⋅ − ⋅ ⋅ = − 1 1 1 0 0 m m t p p m m T R L (1) R – individual gas constant; T0 – initial suction temperature; p0 – initial pressure (suction); p – final pressure (pumping); m – polytropic exponent, has value from 1.4 to 1. R – individual gas constant; R – individual gas constant; T0 – initial suction temperature; p0 – initial pressure (suction); p – final pressure (pumping); m – polytropic exponent, has value from 1.4 to 1. m – polytropic exponent, has value from 1.4 to 1. A multi-stage compression process with cooling between stages is used to limit the demand for power for compression of CO2 stream. This solution substantially limits the power needed to drive compressors and allows for the reduction of their size and helps use the heat stream obtained in inter-stage coolers in e.g. the system of boiler water regeneration for heating purposes or supplying heat to a cooler or absorption coolers for cold generation. The increase in the number of compression stages with cooling between stages allows for a reduction in the demand for power while reducing gas temperature following the compression process, which directly impacts maximal temperature of the cooling medium at the outlet from the inter-stage cooler. 4 Compression of CO2 g g The demand for work [J/(kg CO2)] at multi-stage adiabatic compression with n degrees is described by the relationship (2): 5 E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 (2)           −       ⋅ ⋅ − ⋅ ⋅ = − 1 1 1 0 0 κ κ κ κ n t p p n T R L (2) κ – adiabatic exponent; n – number of compression stages. n – number of compression stages. n – number of compression stages. Adoption of this value results from the assumption of the network water temperature of 55°C for the ambient temperature of -5°C (Table 1). It was assumed that polytropic efficiency for the first compressor is 85% and it is gradually reduced to 70% in the last compression stage. This assumption was adopted for all the analysed multi-stage compression systems. 6 https://doi.org/10.1051/e3sconf/20184900135 E3S Web of Conferences 49, 00135 (2018) E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 SOLINA 2018 Fig. 3. The system structure for seven-stage compression process with cooling between stages. C1 C2 C3 C4 C5 C6 C7 HE0 HE7 HE1 HE2 HE3 HE4 HE5 HE6 Fig. 3. The system structure for seven-stage compression process with cooling between stages. For this complex system, a comprehensive optimization analysis of pressures of inter- stage compression pi was performed, with its criterion being a minimal demand for compression power P. The result of the optimization process (minimum power necessary to carry out the compression of CO2 process, mass flow equal to 175 kg/s) are optimal inter- stage pressure values pi opt. Details of the optimization procedure can be found in [16]. opt i i p p f P )} ( min{  = (3) (3) The results of computations for four computational cases for complete optimization, from seven-stage compression to four-stage compression are presented in Figs. 4 to 8. The results of computations for four computational cases for complete optimization, from seven-stage compression to four-stage compression are presented in Figs. 4 to 8. Fig. 4 presents the values of inter-stage pressures for four analysed cases. Figure 5 presents values of CO2 temperature following the compression stages for the four analysed cases. It can be observed that for the system composed of four compression degrees, maximal gas temperature exceeds the level of 190°C, and reduces following individual stages, eventually reaching 150°C following the fourth compression stage. Analysis of the results for greater number of compression stages (5, 6, 7) reveals a reduction in CO2 temperatures after compression. In the case of seven compression degrees, CO2 temperature following the last stage is ca. 95°C. Fig. 6 presents the values of heat flux which should be received in coolers between stages from the compressed CO2 in order to reduce its temperature to 60°C. Figure 7 presents the values of compression power at individual stages depending on the number of compression stages. n – number of compression stages. It can be observed that the increase in the number of compression stages reduces the power of individual stages. Fig. 8 illustrates the demand for power for CO2 compression and total heat flux which should be received in the process of CO2 cooling. It is noticeable that the increase in the number of compression stages leads to the reduction in the demand for power and heat flux from the process of CO2 cooling. Assuming the temperature in the heat accumulator at the level of 97°C, it is possible to use heat from systems composed of 4, 5 or 6 compression stages due to the temperature of CO2 after the compression process following individual stages (Fig. 5). From the standpoint of the demand for power for driving compressors, the best solution is the system composed of 6 compression stages. The demand for power for compression is ca. 65.6 MW, whereas heat flux from CO2 cooling is ca. 98.8 MW. The heat obtained from the compression process can be directly used in the heat network or be stored in the heat accumulator. Assuming the operation of the power unit at maximal power output for 16 hours and minimal power output (40%) for 8 hours over the day, it is necessary to collect 6,829 GJ (1897 MWh) from the compressed CO2 for a system with 6 compression stages. In order to store this amount of heat in the heat accumulator, the required capacity of 41,000 m3 of water is needed assuming the difference between water temperatures (of hot and cold water) of 40°C. Due to the variable demand for heat in the heating season and opportunity 7 E3S Web of Conferences 49, 00135 (2018) E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 of storing heat from the CO2 cooling process, it is advisable to increase the accumulator capacity to ca. 50,000 m3. 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Pressure [MPa] Compression stage number [-] number of compression stages 4 5 6 7 Fig. 4. Distributions of pressure between stages for the analysed four structures of the compression system. Pressure [MPa] Compression stage number [-] . 4. Distributions of pressure between stages for the analysed four structures of the compression system. The research was financed by grants No.BS/PB-404-301/11. n – number of compression stages. 0 1 2 3 4 5 6 7 90 100 110 120 130 140 150 160 170 180 190 CO2 temperature after compression [ oC] Compression stage number [-] number of compression stages 4 5 6 7 Fig. 5. Distributions of temperatures between stages for the analysed four structures of the compression system. Fig. 5. Distributions of temperatures between stages for the analysed four structures of the compression system. 0 1 2 3 4 5 6 7 12 14 16 18 20 22 24 26 28 30 32 34 36 Heat [MW] Compression stage number [-] number of compression stages 4 5 6 7 Fig. 6. Values of CO2 cooling at individual stages depending on the number of compression stages. Fig. 6. Values of CO2 cooling at individual stages depending on the number of compression stages 8 8 E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 0 1 2 3 4 5 6 7 2 4 6 8 10 12 14 16 18 20 22 Power [MW] Compression stage number [-] number of compression stages 4 5 6 7 Fig. 7. Values of compression power at individual stages depending on the number of compression stages. Fig. 7. Values of compression power at individual stages depending on the number of compression stages. 4 5 6 7 65 70 75 80 85 90 95 100 105 Heat, power [MW] Number of compression stages [-] Heat Power Fig. 8. Demand for power for compression, total heat flux from CO2 cooling between steps. Fig. 8. Demand for power for compression, total heat flux from CO2 cooling between steps. 5 Conclusion The computations of the CO2 compression process for the purposes of transport and underground storage presented in this study demonstrated the opportunities for the use of heat from the CO2 cooling system for the municipal heat network. The choice of optimal compression system should take into consideration the parameters of heat network operation, temperatures and opportunities for storing heat and minimal power needed for the CO2 compression system. The system composed of 6 compression stages seems to be an optimal solution for the analysed case. It allows for reaching the heat power at the level of 98.8 MWt. Assuming variability of the load to the steam power unit, it is possible to reach daily heat generation from the compression system of 6829 GJ. In order to store this amount of heat it is necessary to use a heat accumulator with a water capacity of at least 41,000 m3. If power unit had to be performed, the process of CO2 capture and compression for the purposes of transport and storage, this solution would allow for the use of waste heat from the compression process for heating the condensing steam power units. 9 9 https://doi.org/10.1051/e3sconf/20184900135 E3S Web of Conferences 49, 00135 (2018) SOLINA 2018 References 1. R. Zarzycki, M. Wichliński, Energ. Policy Journal, 19(4), 75–86 (2016) 2. R. Zarzycki, M. Wichliński, Energ. Policy Journal, 17(4), 303–316 (2014) 3. R. Zarzycki, M. Wichliński, (Cyclone furnance as a way for mercury removal from lignite, ICCHMT 2018, Cracow, Poland, 21-24 May 2018) 4. https://www.energy-charts.de/energy_pie.htm?year=2018 (access 04. 2018) 5. W. Nowak, T. Czakiert, (Spalanie tlenowe dla kotłów pyłowych i fluidalnych zintegrowanych z wychwytywaniem CO2. Wydawnictwo Politechniki Częstochowskiej, 2012) 6. W. Nowak, W. Rybak, T. Czakiert, (Spalanie tlenowe dla kotłów pyłowych i fluidalnych zintegrowanych z wychwytywaniem CO2. Kinetyka i mechanizm spalania tlenowego oraz wychwytu CO2. Wydawnictwo Politechniki 2013) 7. W. Nowak, M. Chorowski, T. Czakiert, (Spalanie tlenowe dla kotłów pyłowych i fluidalnych zintegrowanych z wychwytywaniem CO2. Produkcja tlenu na potrzeby spalania tlenowego. Wydawnictwo Politechniki Częstochowskiej, 2014) 8. T. Chmielniak, H. Łukowicz, (Modelowanie i optymalizacja węglowych bloków energetycznych z wychwytem CO2. Wydawnictwo Politechniki Śląskiej, 2015) 9. www.rafako.com.pl (access 04.2018) 10. R. Zarzycki, M. Panowski, (The use of waste heat in the process of combined generation of heat and electricity, ICCHMT 2018, Cracow, Poland, 21-24 May 2018) R. Zarzycki, M. Panowski, Energy Res. Technol. 140 (3), 032008-1–032008-7, (2018) 11. R. Zarzycki, M. Panowski, Energy Res. Technol. 140 (3), 032008-1–032008-7, (2018) 12 R Zarzycki M Panowski (Waste heat utilisation for cogeneration of energy 11. R. Zarzycki, M. Panowski, Energy Res. Technol. 140 (3), 032008-1–032008-7, (2018) 12. R. Zarzycki, M. Panowski, (Waste heat utilisation for cogeneration of energy, MECHANIK 3 (2017) 12. R. Zarzycki, M. Panowski, (Waste heat utilisation for cogeneration of energy, MECHANIK, 3, (2017) 13. R. Zarzycki, M. Panowski, P. Komur, Energ. Policy Journal, 17, 375–389 (2014) 14. I. Sarbu, C. Sebarchievici, (A Comprehensive Review of Thermal Energy Storage, Sustainability, 10, 191, 2018) 15. R. Zwierzchowski, M. Kwestarz, (Rola centralnych zasobników ciepła w miejskich systemach ciepłowniczych VIII Forum Ciepłowników Polskich, 216–224. Międzyzdroje 13-15 września 2004) 16. M. Panowski, R. Zarzycki, Energ. Policy Journal, 16(4), 243–256 (2013) 17. R. Span, W. Wagner, J. Phys. Chem. Ref. Data, 25(6), (1996) 18. V. Vesovic, W. Wakeham, G. Olchowy, J. Sengers, J. Watson, J. 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Enhancing the corrosion resistance of reinforcing steel under aggressive operational conditions using behentrimonium chloride
Scientific reports
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Enhancing the corrosion resistance of reinforcing steel under aggressive operational conditions using behentrimonium chloride A. Bahgat Radwan1, Mostafa H. Sliem1, Noor S. Yusuf1, Nasser A. Alnuaimi1,2 & Aboubakr M. Abdullah1* A. Bahgat Radwan1, Mostafa H. Sliem1, Noor S. Yusuf1, Nasser A. Alnuaimi1,2 & Aboubakr M Abdullah1* Aggressive operational conditions e.g. saline media and acidic gases, e.g., CO2 can increase the corrosion rate of reinforcing steel. Accordingly, the necessity to protect the steel under the above conditions without affecting the mechanical properties of the concrete is growing. Herein, the inhibition efficiency of a new corrosion inhibitor, behentrimonium chloride (BTC, C25H54ClN), is explored in a simulated-concrete pore solution (SCP) with 3.5 wt.% NaCl at different pH using electrochemical impedance spectroscopy (EIS) and polarization methods. Using only a 50 μmol L−1 of BTC, we are able to measure an inhibition efficiency of 91, 79, and 71% in SCP solution with 3.5% NaCl at pH of 12.5, 10 and 7, respectively without showing any effect on the mechanical properties on the cured mortars. Temkin isotherm is used to describe the physisorption of BTC inhibitor on the steel surface. Also, the adsorption and influence of the inhibitor on the metal surface are characterized using the scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. In conclusion, this new inhibitor shows high corrosion inhibition efficiencies under different aggressive conditions and can be used in concrete to reduce the corrosion rate of reinforcing steel without decreasing the mechanical properties of the concrete. Corrosion mitigation has attained a vast interest due to the high economic impact of replacing the damaged parts with new ones especially in reinforced concrete structures1. It is well-known that once corrosion starts in reinforc- ing steel, the rust (corrosion product) occupies two to three times more volume than the un-corroded steel. This higher volume induces pressures around the reinforcing bar and causes cracking of the surrounding concrete. In general, a concrete solution has an alkaline nature (pH ~ 13) owing to the existence of sodium oxide (Na2O), and potassium oxide (K2O) in addition to calcium hydroxide as a result of the hydration reaction of cal- cium silicate hydrate in cement (CSH) with water from the surrounding environment2–7. Accordingly, an oxide layer is existing on the reinforcing steel surface within concrete8–11. However, penetrations of aggressive anions like chloride (Cl−) and sulfate (SO4 2−) ions can lead to localized damage of the passive film which increases the corrosion rate of steel1,8,10. www.nature.com/scientificreports www.nature.com/scientificreports 1Center for Advanced Materials, Qatar University, Doha, P.O. Box 2713, Qatar. 2Department of Civil and Architectural Engineering, Qatar University, Doha, P.O. Box 2713, Qatar. *email: bakr@qu.edu.qa Experimentalh p The reinforcing steel samples were abraded by silicon carbide grit papers using a grinding machine (Jean Wirts TG 200, Germany), sonicated with acetone, rinsed by deionized water and after that desiccate in air. The mild steel rebar contains (wt.%) C = 0.128, Si = 0.25, Mn = 0.7, Cu = 0.15, P = 0.04, S = 0.03, and rest was Fe. Saturated calcium hydroxide (Ca(OH)2), were used as an electrolyte to mimic SCP in 3.5 wt.% NaCl. NaCl was purchased from Sigma Aldrich and Ca(OH)2 from Riedel-de Haën. The pH of the SCP solution under investigation was 12.5 for the saturated Ca(OH)2, 10 or 7. The pH was reduced by addition of NaHCO3 powder to the SCP28. The electro- chemical measurements were done at ambient temperatures using a GAMRY 3000 potentiostat/galvanostat/ZRA (Warminster, PA, USA). EIS measurements were investigated in a frequency range of 100 kHz to 0.01 Hz with an AC amplitude of 5 mV. In all electrochemical measurements, a saturated calomel electrode (SCE) and a graphite rod were employed as a reference and counter electrodes, respectively. The mild steel samples with surface areas of 0.765 cm2 were subjected to the SCP. All the mild steel coupons were sited under open circuit (OCP), conditions for 30 min before initiating any electrochemical test to attain the steady-state conditions. Polarization curves were attained from −0.25 to + 0.25 V against the open circuit potential (OCP), with a scan rate of 0.167 mV s−1. Various concentrations (0, 2.5, 5, 10, and 20 ppm) of BTC (molecular weight = 404.164 g mol−1), which are equiv- alent to 12, 24, 37 and 50 μmol L−1, respectively, were synthesized in the simulated saline SCP solutions. The BTC inhibitor was attained from Shanghai Dejun Chemical Technology Co., Ltd, Shanghai China, and its chemical formula is displayed in Fig. 1. Each electrochemical measurement was repeated three times to confirm the repro- ducibility, and the average values were reported. Mechanical characterization. The effect of BTC inhibitor on the compressive and flexural strength of the mortars prepared according to the ASTM C109/C109M and ASTM C348, respectively, was evaluated after differ- ent exposure times in the existence and absence of the BTC corrosion inhibitor29,30. The compressive experiments were performed using a 300 KN Tecnotest 3 compression testing machine (Tecnotest, Modena, Italy). The flexural strength experiments were utilized by a Lloyd LR 50 K universal testing machine (Ametec Inc. USA). Enhancing the corrosion resistance of reinforcing steel under aggressive operational conditions using behentrimonium chloride Anitha et al.23, used the extract of rosa damascene leaves as nature-friendly inhibitor in SCP which achieved an IE% of 82. Wang et al.24, found that using 0.0008 mol L−1 of calcium lignosulfonate (CLS) showed a high IE% of 93.7 after immersion of carbon steel in SCP for 7200 h in comparsion to sodium oleate (SO), that exhibited an IE% of 40–60. Cao et al.25 explored the inhibition behaiour of phytic acid in car- bonated concrete pore solution containing 0.6 mol L−1 NaCl on 20SiMn steel, which displayed an IE% of 84.0 after immersion for 72 h. Asaad et al.26, prepared non- poisonous corrosion inhibitor of silver nano-particles doped palm oil leaf extracts for renforcing steel in salin water. It was found that the addition of silver nanoparticles in the green inhibitor lead to increase the IE% to 94.7 after immersion for 365 d, owing to the presence of excess calcium silicate hydrate and the enhancement of the pore construction and therefore decrease the conductivity of the pore solution. p In this work, the effectiveness of a new inhibitor (behentrimonium chloride, C25H54ClN) for the cor- rosion of reinforcing steel in highly saline SPCs at ambient temperature and different pH values is explored. Behentrimonium chloride (BTC) is commonly used in hundreds of personal care products as conditioning and anti-static agents. Interestingly, Cameron et al.27, found that BTC is biologically safe for humans when used in a concentration range up to 5%. However, the European Union recently restricted its use for more than ≥ 1%. Consequently, we, for the first time, report the use of BTC as a corrosion inhibitor for reinforcing steel in saline SPC solutions of different pH values at significantly low concentrations of 2.5, 5, 10, and 20 ppm using electro- chemical and surface analysis techniques. Enhancing the corrosion resistance of reinforcing steel under aggressive operational conditions using behentrimonium chloride On the other hand, the chemi-physisorped polymethacrylic acid co-acrylamide corrosion inhibitor displayed an IE% of 92.35 in SCP containing 1.8 wt.% chlorides21. Shanmugapriya et al.22, achieved an IE% of 98 in SCP using an aqueous extract of turmeric. Anitha et al.23, used the extract of rosa damascene leaves as nature-friendly inhibitor in SCP which achieved an IE% of 82. Wang et al.24, found that using 0.0008 mol L−1 of calcium lignosulfonate (CLS) showed a high IE% of 93.7 after immersion of carbon steel in SCP for 7200 h in comparsion to sodium oleate (SO), that exhibited an IE% of 40–60. Cao et al.25 explored the inhibition behaiour of phytic acid in car- bonated concrete pore solution containing 0.6 mol L−1 NaCl on 20SiMn steel, which displayed an IE% of 84.0 after immersion for 72 h. Asaad et al.26, prepared non- poisonous corrosion inhibitor of silver nano-particles doped palm oil leaf extracts for renforcing steel in salin water. It was found that the addition of silver nanoparticles in the green inhibitor lead to increase the IE% to 94.7 after immersion for 365 d, owing to the presence of excess calcium silicate hydrate and the enhancement of the pore construction and therefore decrease the conductivity of the pore solution. et al.10,15, highlighted the influence of benzotriazole (C6H5N3) and its derivatives, 5-nitrobenzotriazole (C6H4N4O2) and 5-chlorobenzotriazole (C6H4ClN3), on reinforcing steel inhibition in an SCP solution containing 1 M NaCl. It was found that the IE% of the explored inhibitors dwindled accordingly: 5-chlorobenzotriazole ˃ benzotriazole ˃ 5-nitrobenzotriazole. The maximum attained IE% was 69% in the presence of 5×10−4 M 5-chlorobenzotriazole. Interestingly, 0.0025% of deoxyribonucleic acid (DNA), showed IE% of 94% in SCP solution containing 3.5 wt. % NaCl with an increase of 3.61% in the compressive strength (Fc), after 28 days18. Zhang et al.19, achieved an inhibition efficiency of 83.15% using maize gluten meal extract as an ecologically friendly inhibitor for reinforcing steel in SPC containing 3.5 wt.%NaCl. The synthesized inhibitor of 4-(1-(4-methoxyphenyl) cyclohexyl)phenyl 9-oxodecanoate (MPOD) by Unnisa et al.20, exhibited and IE% of 71.81 in SCP solution including 0.5 M NaCl. On the other hand, the chemi-physisorped polymethacrylic acid co-acrylamide corrosion inhibitor displayed an IE% of 92.35 in SCP containing 1.8 wt.% chlorides21. Shanmugapriya et al.22, achieved an IE% of 98 in SCP using an aqueous extract of turmeric. Enhancing the corrosion resistance of reinforcing steel under aggressive operational conditions using behentrimonium chloride A corrosion current density (icorr), of around 0.2 μA cm−2, indicates active corrosion12, 0.1 μA cm−2, is safe for typical design life requirements of reinforced concrete structures13, while icorr, less than 0.01 μA cm−2, is low enough to avoid corrosion-induced cracking indefinitely14. Consequently, the inhibitors to be used in simulated concrete pore solution should satisfy two conditions; (i) a high inhibition efficiency in the existence of destructive ions, e.g. Cl− ions, at different pH values (from 7 to 12.5) and (ii) no influence on the mechanical attributes of the concrete8,15,16. Abd El Haleem et al.9, used different inorganic inhibitors in saturated calcium hydroxide. The outcomes pointed out that the inhibition efficiency (IE%), of the inhibitors, improved in the following order MoO4 −2 > WO4 −2 > HPO4 −2 > CrO4 −2. However, the disadvantages of using inorganic inhibitors in concrete environments are their toxicity to living beings, cost, and inefficiency for localized corro- sion8,17. Ormellese et al.1, has studied the long-term inhibition effectiveness of over 80 organic compounds from three main categories: amines and alkanoamines, amino alcohols, and carboxylates in SCP solution containing 0.01 M NaOH at pHΣ12.6 in the absence of chloride ions. The results showed an increase in the effectiveness of the inhibitors in the following order: carboxylates > amino acids > amines and alkanolamines. Abd El Haleem Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Figure 1. The chemical structure of behentrimonium chloride surfactant used as a corrosion inhibitor. Figure 1. The chemical structure of behentrimonium chloride surfactant used as a corrosion inhibitor. et al.10,15, highlighted the influence of benzotriazole (C6H5N3) and its derivatives, 5-nitrobenzotriazole (C6H4N4O2) and 5-chlorobenzotriazole (C6H4ClN3), on reinforcing steel inhibition in an SCP solution containing 1 M NaCl. It was found that the IE% of the explored inhibitors dwindled accordingly: 5-chlorobenzotriazole ˃ benzotriazole ˃ 5-nitrobenzotriazole. The maximum attained IE% was 69% in the presence of 5×10−4 M 5-chlorobenzotriazole. Interestingly, 0.0025% of deoxyribonucleic acid (DNA), showed IE% of 94% in SCP solution containing 3.5 wt. % NaCl with an increase of 3.61% in the compressive strength (Fc), after 28 days18. Zhang et al.19, achieved an inhibition efficiency of 83.15% using maize gluten meal extract as an ecologically friendly inhibitor for reinforcing steel in SPC containing 3.5 wt.%NaCl. The synthesized inhibitor of 4-(1-(4-methoxyphenyl) cyclohexyl)phenyl 9-oxodecanoate (MPOD) by Unnisa et al.20, exhibited and IE% of 71.81 in SCP solution including 0.5 M NaCl. Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y Results and Discussion EIS. Figures 2 and 3 display the Bode and Nyquist plots, respectively, for the reinforcing steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 within a frequency range from 0.01 Hz to 100 k Hz at an Ac amplitude of 5 mV. Figure 4 exhibits the equivalent circuit (EC) utilized to fit the measured EIS data to obtain the different parameters that explain the metal/solution interface. The parameters are listed in Table 1 in which Rs and Rct, are credited to the electrolyte resistance and the charge transfer resistance, respectively. However, the constant phase element is expressed by (CPE), which is used for a non-ideal double layer. The imperfectness behavior of the double layer is accredited to the following parameters (i) a non-uniform surface coverage, (ii) surface roughness, and (iii) nonuniform current distribution or corrosion rate.h The admittance and impedance of the CPE is given by34,35: The admittance and impedance of the CPE is given by34,35: ω = Z Y 1/ (j ) CPE p n ω = Z Y 1/ (j ) (1) CPE p n (1) where ZCPE is the CPE impedance (Ω cm−2); Yp is the numerical value of the admittance 1/│Z│, at ω = 1 (rad s−1) and j2 = −1. ω is the angular frequency and n is the deviation element which varies from 0 and 1. When n = 1 or 0, ZCPE is corresponding to an ideal capacitor or resistor, respectively.hli The influence of the thickness and dielectric constant of the double layer is defined by the Helmholtz regime iven by the following formula: εε δ = C A (2) dl o εε δ = C A dl o (2) where Ɛo and Ɛ are the dielectric constant of air and electrolyte (mainly water), respectively and A is the surface rea of the working electrode.hfi g The inhibition efficiency (IE%), is calculated using Eq. 3, g The inhibition efficiency (IE%), is calculated using Eq. Experimentalh The results are obtained by averaging three repeated tests. The mortar with 50 µmol L−1 of BTC was prepared by the mechan- ical mixing, in a stainless steel mixer, one part mass of cement and one and a half part mass of standard sand, with a water/cement ratio of 0.48531. Then, the mold was filled with the mixture under vibration to release air bubbles, and thereafter stored in a moist atmosphere for 2 days. After that, the demolding of the prepared specimens was conducted, and the samples were kept under tap water over the test period32. The cured mortar samples were removed from the water and located in a drying oven at 60 °C for 24 h before the strength test in order to shun the impact of the hydration of the concrete and to increase the strength of the measured samples33. Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Figure 2. (a) Bode spectra for reinforcing steel in 3.5 wt.%NaCl at ambient temperature using variable concentrations of BTC inhibitor (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. Figure 2. (a) Bode spectra for reinforcing steel in 3.5 wt.%NaCl at ambient temperature using variable concentrations of BTC inhibitor (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. Results and Discussion 3, =    −   × IE R R R % 100 (3) ct1 ct2 ct1 (3) Table 1 exhibits that the higher the corrosion inhibitor concertation is, the higher the Rct and lower Cdl values are which indicates that the ability of Cl− ions to attack the reinforcing steel surface declines due to the presence of a protective adsorbed layer of BTC inhibitor. It is worth to mention that lowering the pH lead to alleviat- ing the IE% from 91% at pH=12.5 to 79% and 72% at pH 10 and 7, respectively. Three reasons can justify the chloride-induced loss of passivity of the reinforcing steel. First, an induced de-passivation owing to adsorption of Cl− ion on the passive film at potential values higher than a critical value. Second, the penetration of Cl− ions into the oxide layer leading to the formation of chloride-contaminated oxides. Finally, a mechanical film breakdown Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports cientificreports/ Figure 3. (a) Nyquist plots for reinforcing steel in SCP solution with 3.5 wt.%NaCl at ambient temperature using variable concentrations of BTC (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. www.nature.com/scientificreports/ Figure 3. (a) Nyquist plots for reinforcing steel in SCP solution with 3.5 wt.%NaCl at ambient temperature using variable concentrations of BTC (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. Figure 4. Equivalent circuit utilized to fit the EIS data for reinforcing steel in SCP containing 3.5 wt. % NaCl at variable pH. igure 4. Equivalent circuit utilized to fit the EIS data for reinforcing steel in SCP containing 3.5 wt. % NaCl at ariable pH. due to Cl− ions adsorption which can attenuate the surface tension, thus leading to a localized disturbance in the mechanical stability of the passive layer36.h due to Cl− ions adsorption which can attenuate the surface tension, thus leading to a localized disturbance in the mechanical stability of the passive layer36.h y p y The polarization curves for the reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhib concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 are shown in Fig. Results and Discussion 5.h y p y The polarization curves for the reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhibitor oncentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 are shown in Fig. 5.h The electrochemical corrosion factors such as as the corrosion free potential (Ecorr), pitting potential (Epit), cor- rosion current density (icorr), the polarization resistance, (Rp), cathodic and anodic Tafel slopes (bc and ba, respec- tively), the corrosion inhibition efficiency (IE%) and the surface coverage area (θ), are calculated from Fig. 5, and recorded in Table 2. Additionally, the passive potential window is calculated using the following formula: Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Inhibitor concertation (µmol L−1) Rct, Ω cm2 Y × 10−6 sn ohm−1 cm−2 n Cdl, × 10−5 F Inhibition efficiency (IE%) Surface coverage (ϴ) Goodness of fit (χ2) pH = 7 Blank 377 ± 5.3 96 ± 22 0.88 ± 0.02 6.1 — — 2.1 × 10−5 12 461 ± 42 78 ± 10 0.84 ± 0.11 4.1 18 0.18 1.5 × 10−5 24 637 ± 52 70 ± 12 0.81 ± 0.08 3.4 41 0.41 3.4 × 10−6 37 924 ± 28 66 ± 6 0.78 ± 0.12 3.1 59 0.59 5.2 × 10−5 50 1362 ± 100 59 ± 8 0.75 ± 0.21 2.5 72 0.72 1.8 × 10−5 pH = 10 Blank 643 ± 18 89 ± 11 0.85 ± 0.02 5.3 — — 6.5 × 10−6 12 863 ± 24 74 ± 15 0.81 ± 0.09 3.8 25 0.25 4.2 × 10−5 24 1349 ± 31 61 ± 17 0.80 ± 0.03 3.2 52 0.52 1.1 × 10−4 37 1953 ± 52 50 ± 13 0.79 ± 0.05 2.7 67 0.67 2.4 × 10−6 50 3105 ± 120 44 ± 28 0.72 ± 0.12 2.1 79 0.79 1.1 × 10−5 pH = 12.5 Blank 960 ± 66 80 ± 32 0.82 ± 0.06 4.5 — — 8.1 × 10−5 12 2678 ± 133 64 ± 9 0.75 ± 0.13 3.5 64 0.64 7.7 × 10−6 24 5009 ± 149 49 ± 15 0.72 ± 0.09 2.8 80 0.80 3.6 × 10−5 37 7680 ± 352 39 ± 10 0.66 ± 0.17 2.1 87 0.87 8.1 × 10−6 50 10526 ± 420 28 ± 15 0.56 ± 0.27 1.1 91 0.91 1.9 × 10−5 Table 1. Results and Discussion The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7. Figure 6. Temkin adsorption plots for reinforcing steel in SCP solution conatining 3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of 12.5, 10, and 7. Figure 6. Temkin adsorption plots for reinforcing steel in SCP solution conatining 3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of 12.5, 10, and 7. =    −   × IE i i i % 100 (5) 1 2 1 (5) where i1 and i2 are the corrosion current densities of reinforcing steel in the absence and existence of the BTC corrosion inhibitor, respectively.h where i1 and i2 are the corrosion current densities of reinforcing steel in the absence and existence of the BTC corrosion inhibitor, respectively. Th f (θ) i l l t d tili i E 611 y The surface coverage area (θ) is calculated utilizing Eq. 6 θ = IE% 100 (6) (6) and the polarization resistance (Rp), was detected using the Stern–Geary equation37. and the polarization resistance (Rp), was detected using the Stern–Geary equation37. = . + R b b i b b 2 303 ( ) (7) p c a corr c a (7) The polarization curves show the breakdown of the passive film before and after the addition of BTC inhibitor at pH 12.5, see Table 2. However, increasing the inhibitor concentration shifts the pitting potentials (Epit), towards the more noble values indicating that the passive layer formed more stabilized by the presence of BTC inhibitor. Moreover, the passive potential window at pH 12.5 is 0.18, which increased to 0.25, 0.27, 0.32 and 0.38 by the addition of 12, 24, 37, 50 μmol L−1 of the BTC inhibitor, respectively. Results and Discussion Electrochemical elemen ts attained from the EIS spectra of the reinforcing steel in SCP solution with 3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values. Table 1. Electrochemical elemen ts attained from the EIS spectra of the reinforcing steel in SCP solution with 3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values. Table 1. Electrochemical elemen ts attained from the EIS spectra of the reinforcing steel in SCP solution with 3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values. Figure 5. Polarization curves for reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of (a) 12.5, (b) 10 and (c) 7. Figure 5. Polarization curves for reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of (a) 12.5, (b) 10 and (c) 7. Results and Discussion − E E (4) pit corr (4) The corrosion inhibition efficiency (IE%), is calculated using the following formula37, The corrosion inhibition efficiency (IE%), is calculated using the following formula37, Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ pH values Inhibitor concertation (µmol L−1) icorr (µA cm−2) ipit (µA cm−2) Epit (mV) SCE Ecorr (mV) SCE Epit-Ecor (mV) SCE Bc (V decade−1) Ba (V decade−1) Rp Ω cm2 IE% surface coverage (ϴ) 12.5 Blank 13 2.0 × 10−4 −0.41 −0.601 0.187 0.11 0.076 1502 — — 12 5.2 6.7 × 10−5 −0.34 −0.598 0.254 0.081 0.061 2888 56 0.56 24 3.2 5.0 × 10−5 −0.33 −0.601 0.271 0.077 0.059 4590 76 0.76 37 2.5 4.2 × 10−5 −0.28 −0.602 0.322 0.072 0.056 5471 81 0.81 50 1.6 3.9 × 10−5 −0.21 −0.594 0.384 0.068 0.048 7636 88 0.88 10 Blank 20 — — −0.71 — 0.091 0.091 988 — — 12 15 — — −0.639 — 0.087 0.081 1214 22 0.22 24 10 — — −0.665 — 0.075 0.079 1671 52 0.52 37 8 1.23 × 10−5 — −0.635 0.058 0.061 0.066 1721 63 0.63 50 5 9.7 × 10−5 — −0.585 0.376 0.058 0.058 2518 78 0.78 7 Blank 33 — — — — 0.098 0.120 709 — — 12 28 — — — — 0.087 0.110 755 15 0.15 24 21 — — — — 0.080 0.109 958 36 0.36 37 14 — — — — 0.071 0.111 1344 57 0.57 50 9.5 — — — — 0.116 0.091 2332 71 0.71 Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7. Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions l di 3 5 t %N Cl d BTC i hibit t ti f 12 24 37 50 l L−1 t H l f 12 5 10 d Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 Table 2. Results and Discussion The attack of chloride species to the reinforc- ing steel surface in SCP can lead to loss of its passive layer if the concentration of the chloride species is adequately high. For reinforcing steel in concrete, the degree to which Cl¯ ions can damage the passive layer is related to the alkalinity of the environment. In chloride-free alkaline conditions, the passive layer on the mild steel breaks Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ pH values Slope α Intercept Kads × 104, (L mole−1) ∆G°ads, (kJ mol−1) 12.5 −0.33 0.17 −3 0.88 −32 10 −0.36 0.14 −3.5 1.6 −33 7 −0.39 0.13 −3.9 2.2 −34 Table 3. The calculated thermodynamic parameters derived from Temkin plot. pH values Slope α Intercept Kads × 104, (L mole−1) ∆G°ads, (kJ mol−1) 12.5 −0.33 0.17 −3 0.88 −32 10 −0.36 0.14 −3.5 1.6 −33 7 −0.39 0.13 −3.9 2.2 −34 Table 3. The calculated thermodynamic parameters derived from Temkin plot. Table 3. The calculated thermodynamic parameters derived from Temkin plot. Table 3. The calculated thermodynamic parameters derived from Temkin plot. Table 3. The calculated thermodynamic parameters derived from Temkin plot. Figure 7. SEM images for the reinforcing steel (a,c,e) before and (b,d,f) after immersion in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b) 12.5, (c,d) 10, and (e,f) 7 for 24 h. Figure 7. SEM images for the reinforcing steel (a,c,e) before and (b,d,f) after immersion in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b) 12.5, (c,d) 10, and (e,f) 7 for 24 h. Figure 7. SEM images for the reinforcing steel (a,c,e) before and (b,d,f) after immersion in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b) 12.5, (c,d) 10, and (e,f) 7 for 24 h. Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ www.nature.com/scientificreports ure.com/scientificreports/ Figure 8. AFM images for the reinforcing steel surface (a,c,e) before and (b,d,f) after immersing the reinforcing steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b) 12.5, (c,d) 10, and (e,f) 7. Figure 8. Results and Discussion In order to understand and estimate the adsorption route of BTC on steel surface, different adsorption isotherms are checked using the measured data from the poanlarization plots e.g. Langmuir, Frumkin and Temkin isotherms using the following equations: mkin and Temkin isotherms using the following equations: Langmuir Langmuir θ = + C K C 1 (8) inh ads inh (8) Frumkin α ×   θ −θ   = . + θ log C logK { 1 } 2 303 2 (9) (9) Temkin α − θ = K C exp( 2 ) (10) ads (10) where θ is the surface coverage of the reinforcement steel, C is the concentration of the BTC inhibitor species, α is the adsorbate interaction factor and Kads is the adsorption– desorption equilibrium constant. The fitting outcomes showed that BTC inhibitor obeys Temkin isotherm, see Fig. 6.t y g After reorganizing Eq. (10), the following expression is attained: After reorganizing Eq. (10), the following expression is attained: θ α α = − + − lnc lnK 1 2 1 2 (11) ads (11) It can be deduced from Eq. (11) that both of the slope and intercept are calculated from 1/−2α and (1/−2α) lnKads, respectively. It can be deduced from Eq. (11) that both of the slope and intercept are calculated from 1/−2α and (1/−2α) lnK respectively Knowing the Kads values at various pH values, the standard Gibbs free energy change of adsorption (∆G°ads) are calculated using Eq. 12. = . −∆ K e 1 55 5 (12) G RT ads ads o (12) Table 3 summarizes the values of the α, Kads and ∆G°ads. Values of ∆G°ads ≥ −20 kJ mol−1, showing a phy- sisorption adsorption, while ∆G°ads ≤ −40 kJ mol−1 depicts chemisorption adsorption reactions. Consequently, the intermediate values of ∆G°ads shown in Table 3 (−32, −33 and −34 kJ mol−1), usually elucidate that chemi-physisorption of BTC inhibitor occurs on the reinforcing steel at different pH values of 12.5, 10 and 7, respectively. However, since there is no free electron pair existing in the molecular structure of the BTC inhib- itor that can form coordinated covalent bond with the vacant d-orbitals in Fe (chemisorption), therefore it is more favorable that the adsorption mechanism is a strong physisorption ratherthan a chemi-physisorption one. Results and Discussion AFM images for the reinforcing steel surface (a,c,e) before and (b,d,f) after immersing the reinforcing steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b) 12.5, (c,d) 10, and (e,f) 7. pH Ra in absence of BTC (nm) Ra in presence of BTC (nm) 12.5 27 15 10 47 23 7 70 36 Table 4. The surface roughness values (Ra), of the reinforcing steel before and after addition of BTC inhibitor in saline water for 24 h at variable pH values. Table 4. The surface roughness values (Ra), of the reinforcing steel before and after addition of BTC inhibito saline water for 24 h at variable pH values. down at a potential of +560 mV SCE38. The highest attained IE% was 88% at 50 μmol L−1 of the BTC inhibitor at pH = 12.5. It can be seen that the values of ba and bc diminished upon the addition of the inhibitor indicating that BTC is a mixed type inhibitor. it is noteworthy to mention that the tabulated values of the corrosion density (icorr), shifts towards decreases with increasing the concertation of the BTC inhibitor. However, these values are not in accordance with reported articles in references12,39. In fact, the diffusion of chloride species (Cl−), in cementitious materials immersed in saline water is a difficult process, which includes numerous chemical and physical inter- actions. Cl− ions can bound chemically or physically through the cement paste, thus reducing the segment of free Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Figure 9. XPS survey scan composition of the mild steel after immersion for 24 h in SCP solution containing 3.5 wt.%NaCl in the existence of 50 μmol L−1 of BTC corrosion inhibitor at pH 12.5. Figure 9. XPS survey scan composition of the mild steel after immersion for 24 h in SCP solution containing 3.5 wt.%NaCl in the existence of 50 μmol L−1 of BTC corrosion inhibitor at pH 12.5. Cl− species that can diffuse easily in the concrete pore solutions. Furthermore, the internal electric field generated from the anions and cations will accelerate the ions that possess low diffusion coefficients and decelerate the ions that have high diffusion coefficients in order to keep the electro-neutrality status40. Adsorption isotherm. Results and Discussion Physical adsorption takes place rapidly because of weak bondings such as Van der Waal’s or electrostatic attrac- tive forces between inhibitor species and metal surface, and is directly influenced by the electronegativity of the inhibitor compounds. The residence time for a physically adsorbed inhibitor is short, and its interaction with Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y 9 www.nature.com/scientificreports/ Figure 10. High resolution XPS spectrum of (a) Cl 2p, (b) C 1 s, (c) Ca 2p, (d) N 1 s (e) O 1 s and (f) Fe 2p after immersing the reinforcing steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at pH Σ12.5. Figure 10. High resolution XPS spectrum of (a) Cl 2p, (b) C 1 s, (c) Ca 2p, (d) N 1 s (e) O 1 s and (f) Fe 2p after immersing the reinforcing steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at pH Σ12.5. the steel surface is directly associated with the corrosion free potential of the metal corrosion with respect to the potential of zero charge.t Figure 7 exhibits the SEM of the reinforcing steel coupons after immersion in SCP including 3.5 wt.% NaCl at different pH values of 12.5, 10, 7 in the existence and absence of 50 μmol L−1 of the BTC inhibitor for 24 h. It is clear that in case of the absence of the corrosion inhibitor, deep pits were formed and their number is suppressed as the pH of the medium increases. Nonetheless, in the existence of the corrosion inhibitor, the number and pits size are considerably reduced at the same pH values. Moreover, the pH values before and after immersion are measured. It is found that the pH values in the absence of the corrosion inhibitors are lowered from 12.5, 10 and 7 to 10, 8.7 and 5.7, respectively. However, in the existence of the BTC inhibitor the pH values slightly dropped from 12.5, 10 and 7 to 11.5, 9.3 and 6.3, respectively. p y Surface topography and surface roughness of the reinforcing steel are explored after immersion in 3.5 wt.%NaCl of variable pH values for 24 h in the existence and absence of 50 μmol L−1 of the BTC inhibitor using AFM, as depicted in Fig. 8. Results and Discussion It is noted that the surface roughness (Ra), escalates as the pH alleviates in the absence Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y 10 www.nature.com/scientificreports/ Exposure time (days) Flexural strength Maximum Bending Stress at Break, (MPa) Compression strength (N mm−2) Blank BTC Blank BTC 7 5.9 5.6 42.2 39.1 14 6.3 6.3 44. 2 44.2 21 6.5 6.2 47.31 47.1 28 6.6 6.4 47.4 47.3 Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing times. Exposure time (days) Flexural strength Maximum Bending Stress at Break, (MPa) Compression strength (N mm−2) Blank BTC Blank BTC 7 5.9 5.6 42.2 39.1 14 6.3 6.3 44. 2 44.2 21 6.5 6.2 47.31 47.1 28 6.6 6.4 47.4 47.3 Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing times. Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing times. Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing times. of the corrosion inhibitor, see Table 4. However, Ra is decreased significnatly in the existence of the BTC inhibitor in the deleterious medium signifying the construction of an adsorbed protective layer of BTC inhibitor on the metal surfaces, which retards the attack of the Cl− species Th d ( ) d h h h l ( ) b d ft of the corrosion inhibitor, see Table 4. However, Ra is decreased significnatly in the existence of the BTC inhibitor in the deleterious medium signifying the construction of an adsorbed protective layer of BTC inhibitor on the metal surfaces, which retards the attack of the Cl− speciesht The wide scan spectrum (Fig. 9) and the high resolution XPS spectra (Fig. 10) are obtained after immersing the reinforcing steel for 24 h in SCP including 3.5 wt.% NaCl in the existence of 50 μmol L−1 of BTC corrosion inhibitor at pH 12.5.h p The Cl 2p spectrum is deconvoluted into two components at BE of 199.1 and 200.7 eV which are attributed to Cl− and FeClx, respectively41, see Fig. 10a. It is noteworthy that the peak intensity of Cl− species is low; indicating that the adsorbed BTC inhibitor lowers the adsorption affinity of the Cl− ions to the metal surface which subse- quently lessens the corrosion rate. Results and Discussion The adsorption of the BTC inhibitor on the reinforcing steel surface is further confirmed by the analysis of C spectra, which showed the presence of CN+-R3 and (C-C & C-H) at BE of 284.5 and 287.7 eV, respectively42,43, see Fig. 10b. However, Ca 2p spectrum shown in Fig. 10c is decomposed into two bands at 347.8 eV and 351.5 eV that are credited to Ca 2P3/2 and 2P1/2 respectively, of CaO/Ca(OH)2 8. Figure 10d shows the appearance of N 1 s peaks at 399.4 and 402.5 eV, characteristics of N-CH2 and N+ quaternary nitrogen, respectively43-46. This approves the adsorption of the BTC inhibitor on the reinforcing steel surface. There is no peak for C–N–Fe bonding was observed at 397.7–398.6 eV, suggesting that BTC inhibitor was adsorbed on the reinforcing steel through physisorption47. The XPS spectrum of O 1 s in Fig. 10e, exhibits three peaks at 530.3, 531.7 and 534.2 eV, that are credited to O2− of iron oxides, OH− of hydrous iron oxides (FeOOH), and H2O, respectively43,46. On the other hand, Fe 2p spectra in Fig. 10f is deconvoluted into six peaks. In fact, the interpre- tation of Fe 2p spectra is a complex owing to the existence of iron (Fe), in variable oxidation states of Fe°, Fe2+, Fe3+, and satellites of Fe3+ species. The (Fe 2p3/2), XPS spectra at high resolution involves four bands at 707.1 eV that is related to the metallic iron, 710.9 eV for Fe3+ of Fe2O3/ FeOOH and 713.9 eV, which could be attributed to a mixture of (Fe2+ & Fe3+), in different forms of iron (II) oxide (FeO), iron (II) hydroxide Fe(OH)2, iron (III) hydroxide Fe(OH)3, FeOOH, iron (III) oxide (Fe2O3), and magnetite (Fe3O4)48. The shake up phenomenon found at 716.6 and 719.8 eV is ascribed to Fe2+ and Fe3+, respectively. The spectra of the Fe 2p1/2 peaks at BE of 722.7 and 725.3 eV can be ascribed to Fe2O3 and FeO(OH), respectively49. Mechanical properties. Although corrosion inhibitors can protect steel against corrosion, however it can badly affect its mechanical features50. Therefore, the mechanical characteristics of cured mortars are investigated in the presence and absence of a 20 ppm (50 μmol L−1), of the BTC inhibitor. This is achieved through measuring the flexural and compressive strength of cement, see Table 5. Results and Discussion It can be observed that there is almost no change in the mechanical properties of the concrete after the addition of the inhibitor, which is attributed to the low con- centration of the BTC. Conclusions A new BTC corrosion inhibitor for reinforcing steel is investigated in 3.5 wt.% NaCl at different pH values. Taflel plots indicated that BTC is a mixed type inhibitor. The BTC inhibitor showed a corrosion inhibition efficiency (IE%) of 88, 78 and 71% in 3.5 wt.% NaCl using 50 μmol L−1 at pH values of 12.5, 10 and 7, respectively, which is effective if carbonation of the concrete happens and the pH of the concrete is lowered. Based on the adsorption isotherm calculations, BTC inhibitor showed the best fitting with Temkin isotherm. XPS results illustrate that BTC inhibitor is physisorbed on the reinforcing steel surface, which is matching with the ∆G°ads calculations. The surface roughness of the metal surface is significantly decreased upon the addition of BTC inhibitor as shown in the AFM results confirming a high corrosion inhibition efficiency of BTC. Additionally, no change in the mechanical features of concrete is observed upon the addition of BTC inhibitor, which allows using it in concrete without any reservation. Data availability The raw/processed data required to reproduce these findings could not be shared at this time due to time limitations. However, it will be available on request. Received: 31 July 2019; Accepted: 30 October 2019; Published: xx xx xxxx Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ References The effect of functionalized polycarboxylate structures as corrosion inhibitors in a simulated concrete pore solution. Applied Surface Science 441, 895–913, https://doi.org/10.1016/j.apsusc.2018.02.012 (2018). p pp f p g j p 22. Shanmugapriya, S., Prabhakar, P. & Rajendran, S. Corrosion Resistance Property of Mild Steel in Simulated Concrete Pore Solu Prepared in Well Water by Using an Aqueous Extract of Turmeric. Materials Today: Proceedings 5, 8789–8795, https:/ ( ) 2. Shanmugapriya, S., Prabhakar, P. & Rajendran, S. 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Corrosion Science 52, 1472–1481, https://doi.org/10.1016/j.corsci.2010.01.015 (2010).h p g j ( ) 38. Martin, F. & Olek, J. The Nature of Passivity of Reinforcing Steel. ASCE Materials Congress, Washington, D.C (1996). Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y Acknowledgementsh g The publication of this article was funded by the Qatar National Library. Author contributions Ahmed Bahgat Radwan, Mostafa Sliem and Noor Yusuf were responsible for the experimental work including preparation and characterizataion. Dr. Nasser Alnuaimi supervised the concrete preparation and mechanical testing section. Dr. Aboubakr Abdullah was resposnsible for the corrosion section and the overall manuscript gathering, reviewing and submission. Additional information Correspondence and requests for materials should be addressed to A.M.A. Correspondence and requests for materials should be addressed to A.M.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and i tit ti l ffili ti Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y 13
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THE USE OF CONSONANT ELISION BY SRI MULYANI DURING HER INTERVIEW SESSION WITH THE BANKER
Eltin Journal : Journal of English Language Teaching in Indonesia
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Firdhani, Indrayani & Mahdi: The Use of Consonant… Firdhani, Indrayani & Mahdi: The Use of Consonant… ABSTRACT This research is entitled “The Use of Consonant Elision by Sri Mulyani During Her Interview Session with The Banker”. The objects of this research are the consonant elisions that are produced by Sri Mulyani when she was interviewed by The Banker. The objectives of this research are to describe and analyze the frequency of consonant elision used by Sri Mulyani and to find out the phonemes that are often being elision-ed by Sri Mulyani in her interview with The Banker. The analysis of this research was done by using descriptive qualitative analysis method. This study uses Carr’s (2008), Giegerich’s (1992), and Roach’s (2001) theories about phonological rules and elision. This study shows that during her interview, Sri Mulyani uses consonant elision for 26 times and she tends to do the omission to the phoneme /t/ (19 times), /d/ (5 times), /r/ (1 times), and /s/ (1 times). Keywords: Consonant Elision, Elision, Phonology, Phonological Rules, Sri Mulyani Keywords: Consonant Elision, Elision, Phonology, Phonological Rules, Sri Mulyani THE USE OF CONSONANT ELISION BY SRI MULYANI DURING HER INTERVIEW SESSION WITH THE BANKER Anggi Rizky Firdhani1, Lia Maulia Indrayani2 , Sutiono Mahdi3 anggi12001@mail.unpad.ac.id, lia.maulia@unpad.ac.id, sutiono.mahdi@unpad.ac.id Keywords: Consonant Elision, Elision, Phonology, Phonological Rules, Sri Mulyani B. LITERATURE REVIEW 1. Elision In English pronunciation, sometimes a speaker simplifies the pronunciation of some words. According to Carr (2008), this process of simplification is counted as a part of the phonological rules. In his book, Carr mentions that simplification consists of various types, those are reduction, lenition, assimilation, and elision. Sometimes, the simplification occurs in order to ease the native speakers in expressing their feelings. It is common for them to speak English in high speed along with their emotions, and as the result, they make a ‘shortcut’ to ease their pronunciation. Sometimes, the native speakers of English make a complete disappearance of one or more sounds in a word or phrase as an attempt to make a word or phrase easier to pronounce, and that phenomenon is called elision (Crystal, 2008). According to Giegerich (1992) in Febriyanti (2015), one of the most common elisions in English is the omission of phoneme /t/ and /d/. Although the use of elision is often used by native speakers, in fact, native speakers are not the only person who use elision in uttering some words in English. Many non-native speakers often use elision when they are speaking in English, including Indonesians. One example of the native speakers of Indonesian who use elision while speaking in English is the Minister of Finance of the Republic of Indonesia, Sri Mulyani. Sri Mulyani is one of the prominent figures in Indonesia, so it is not surprising that Sri Mulyani is often required to speak English. Sri Mulyani's speaking style in English caught the attention of the researcher, especially when the researcher watched her being interviewed about the tax amnesty in Indonesia by The Banker, a British financial media, for approximately nine minutes. In the interview, the researcher saw that Sri Mulyani had her own pattern when she tried to speak English and she used a lot of elisions, particularly consonant elision when she was pronouncing certain words. The researcher saw that the use of elision by Sri Mulyani while being interviewed by The Banker is interesting to be examined in this research. Based on the explanation above, the researcher is interested to find out how often Sri Mulyani used elision while being interviewed and to find out the phonemes that are often being elision-ed by Sri Mulyani in her interview with The Banker. A. INTRODUCTION In English pronunciation, there are various ways of pronunciation for each individual. This phenomenon occurs because of numerous factors, such as areas of origin, cultural influences, and social environment. English pronunciation involves the production of each sound and pronunciation of words, phrases, and sentences with correct spelling, emphasis and/or intonation. In addition, there is a way to read the word correctly called 'phonetic transcription', which is defined as a kind of alphabetic writing in which each letter represents a sound (Fromkin, Rodman, & Hyams, 2003). The purpose of phonetic transcriptions is to provide clear and unambiguous information to language learners, such as which sounds should be used on a word or phrase, and in what order to use the sound. The branch of linguistics which studies the sound system of languages is called phonology. The pronunciation of speech sound in English is governed by certain rules, and the rule is called the phonological rules. This statement was approved by Fromkin, Rodman, and Hyams (2003) because, in their book, they state that "phonological rules relate to phonemic representations and phonetic representations of the speaker’s knowledge of the language." With the existence of these phonological rules, we cannot pronounce any speech sounds carelessly, especially in pronouncing speech sounds in English. There are several phonological rules that we need to obey in pronouncing English speech sound, such as the use of pressure, intonation, and so forth. The aim of studying the rules of phonology is to give a phonetic information or guideline on how to pronounce a word in a correct way, so 53 ELTIN Journal, Volume 6/II, October 2018 the meaning that conveyed from the spoken word can be conveyed correctly. Although the specific rules of phonology differ from language to language, the kind of rules, what they do, and the natural classes they refer to are the same cross-linguistically (Fromkin, Rodman, & Hyams, 2003). the meaning that conveyed from the spoken word can be conveyed correctly. Although the specific rules of phonology differ from language to language, the kind of rules, what they do, and the natural classes they refer to are the same cross-linguistically (Fromkin, Rodman, & Hyams, 2003). 2. Types of Simplification As the researcher mentioned in the explanation above that in English pronunciation, sometimes, a speaker simplifies the pronunciation of some words. The simplification occurs in order to ease the native speakers in expressing their feelings. It is common for them to speak English in high speed along with their emotions, and as the result, they make a ‘shortcut’ to get the ease of their pronunciation. According to Carr (2008), this process of simplification is counted as a part of the phonological rules. In his book, Carr mentions that simplification consists of various types, those are reduction, lenition, assimilation, and elision. Elision is going to be the focus of this research. 54 Firdhani, Indrayani & Mahdi: The Use of Consonant… Firdhani, Indrayani & Mahdi: The Use of Consonant… Crystal (2008) defines that elision is a complete disappearance of one or more sounds in a word or phrase as an attempt to make a word or phrase easier to pronounce. The process of elision occurs when speakers leave or delete a phoneme out of pronunciation as an attempt to fasten and get rid of difficulties in speech. Elision happens with both consonants and vowels. Carr (2008) states that where the cluster of consonants arise through the combination of words into phrases, they are frequently reduced in this way. For example, the phrase crisp bowl is often pronounced as [khrɪsboʊl], with the final stop in crisp elided. Carr also states that vowel elision may result in the loss of an entire syllable as in university when it is pronounced as [juː.nɪˈvɜː.sti]. According to Giegerich (1992) cited in Febriyanti (2015), elision often happens before sonorant consonants which give the consonants an opportunity to be syllabic so that they will occupy the peak of the syllable. The process is called vowel elision, for examples are button /bʌtən/ or /bʌtn/ and little /lɪtəl/ or /lɪtl/. Giegerich also explains that the most common elision in English words is the omission of sounds /t/ and /d/ at the word boundary: they are positioned at end of a word before going to the next word. For example, the elision of phoneme /t/ in the word postman is missing, so its pronunciation from [poʊstmən] changed to [poʊsmən] because of the speed of the speaker’s speech. In the case of phoneme /t/, the preceding consonant must be voiceless; in the case of /d/, the preceding consonant must be voiced. 2. Types of Simplification In both cases, elision happens with any following consonant, except /h/ and sometimes /j/, e.g. Christmas becomes [’krɪsməs], listen becomes [lɪsən], left foot becomes [lefʊ:t], but it doesn’t happen in left hand [left hænd], left you [lefʧʊ], and old man [oʊld mæn]. Roach (2001) agreed with that explanation: Roach states the first type of elision that can be found in connected speech involves a loss of weak vowels after /p/, /t/, /k/. Roach pointed out that the vowel in the first syllable may be omitted. That is, the aspiration of the initial plosive may take up the whole of the middle portion of the syllable as in “potato”, “tomato”, “canary” “perhaps” and “today” C. RESEARCH METHODOLOGY The method used in this research is descriptive qualitative analysis method. The descriptive qualitative analysis method is a research method that produces descriptive data in the form of written words or oral things that can be observed (Bogdan and Taylor, 1975). Qualitative descriptive analysis research relates to ideas, perceptions, opinions, and all of them cannot be measured by numbers. This qualitative descriptive study aims to obtain information about existing circumstances (Mardalis, 1999) and is designed to collect information about current ongoing realities (Cevilla, 1993). In doing this study, the researcher transcribes the audio via video obtained from Youtube. The video that is chosen by the researcher is an interview video between Sri Mulyani and The Banker entitled View from IMF: Interview with Sri Mulyani Indrawati, Finance Minister, Indonesia. To perform the audio transcription, the researcher listened to the speech sounds produced by Sri Mulyani carefully, then the researcher marked the pronunciation of any elision-ed words that are produced by Sri Mulyani. After that, the pronunciation of some words spoken by Sri Mulyani compared with the General American English (GA) pronunciation. 55 ELTIN Journal, Volume 6/II, October 2018 D. FINDINGS AND DISCUSSION The process of elision involves the omission of a phoneme in its pronunciation. The researcher found several cases which belong to elision in Sri Mulyani’s utterance during her interview, and most of them the elision of phoneme /t/ and /d/. In Sri Mulyani's interview with The Banker, which lasted approximately for 9 minutes, the researcher found out that Sri Mulyani used elision 26 times. More detailed information can be seen in the table below. Table 1. The frequency of elision uttered by Sri Mulyani during her interview with The Banker Frequency of the Elision of Phoneme /t/ Frequency of the Elision of Phoneme /d/ Frequency of the Elision of Phoneme /r/ Frequency of the Elision of Phoneme /s/ 19 5 1 1 TOTAL 26 Table 1. The frequency of elision uttered by Sri Mulyani From the table above, we can see that the phoneme that is often omitted by Sri Mulyani is phoneme /t/, followed by phoneme /d/, /r/, and /s/. Here is the list of pronunciation realizations by Sri Mulyani that are being elision-ed, compared with General American English Pronunciation (GA). Table 2. Li f i i li i b S i M l i h b i li i d Table 2. List of pronunciation realizations by Sri Mulyani that are being elision-ed No Word GA English Pronunciation Realization by Sri Mulyani 1. Months /mʌnθs/ [mʌns] 2. Month / mʌnθ/ [mʌn] 3. Went /went/ [wen] 4. Export /ek.spɔːrt/ [ekspɔr] 5. Import /ɪm.pɔːrt/ [ɪmpor] 6. Left /left/ [lef] 7. Lengthen /leŋ.θən/ [leŋhən] 8. Project /prɑː.dʒekt/ [prɑː.dʒek] 9. Affect /əˈfekt/ [əˈfek] 10. Past /pæst/ [pæs] 11. Percent /pəˈsent/ [pəˈsen] 12. Wealth /welθ/ [welh] 13. Robust /roʊˈbʌst/ [roʊˈbʌs] 14. Government /ɡʌv.ɚn.mənt/ [ɡʌvən.mən] 15. Department /dɪˈpɑːrt.mənt/ [dɪpɑːrmən] 16. Important /ɪmˈpɔːr.tənt/ [ɪmpɔrtən] 17. Conduct /kənˈdʌkt/ [kənˈdʌk] 18. Want /wɑːnt/ [won] Table 2. List of pronunciation realizations by Sri Mulyani that are being elision-ed 56 Firdhani, Indrayani & Mahdi: The Use of Consonant… Firdhani, Indrayani & Mahdi: The Use of Consonant… 19. Trust /trʌst/ [trʌs] 20. Behind /bɪˈhaɪnd/ [bɪˈhaɪn] 21. Land /lænd/ [læn] 22. Spending /spen.dɪŋ/ [spenɪŋ] 23. Abandoned /əˈbæn.dənd/ [əˈbæn.dən] 24. Second /sek.ənd/ [sek.ən] 25. Course /kɔːrs/ [kɔs] 26. Maintenance /meɪn.tən.əns/ [meɪn.tən.ən] From the table above, the researcher sees the phonological pattern that is used by Sri Mulyani when she is talking. E. CONCLUSION Based on the explanations above, the researcher can conclude that in Sri Mulyani's interview with The Banker, which lasted approximately for 9 minutes, Sri Mulyani used elision for 26 times and most of them are the elision of phoneme /t/ (19 times) and /d/ (5 times). Mostly, the omission of the phoneme /t/ and /d/ was done by Sri Mulyani when she had to utter the sonorant consonantal sounds such as the liquid sounds (l, r) and nasal sounds (n, m, ŋ) that appear before the phoneme /t/ and /d/. That phenomena show that plosive alveolar sounds such as /t/ and /d/ is quite difficult to pronounce when those sounds are placed after the liquid sounds or after the nasal sound. Therefore, the elision occurs as the way of Sri Mulyani in maintaining the speed of her speech in order to make the pronunciation easier. D. FINDINGS AND DISCUSSION According to the table, we can see that mostly, the omission of the phoneme /t/ and /d/ was done by Sri Mulyani when she had to utter the sonorant consonantal sounds such as the liquid sounds (l, r) and nasal sounds (n, m, ŋ) that appear before the phoneme /t/ and /d/. The phenomena indicate that plosive alveolar sounds such as /t/ and /d/ is quite difficult to pronounce when those sounds are placed after the liquid sounds or after the nasal sound. Therefore, the elision occurs as the way of Sri Mulyani in maintaining the speed of her speech in order to make the pronunciation easier. Roach, P. (2001). English Phonetics and Phonology: A Practical Course. Cambridge: Cambridge University Press. ELTIN Journal, Volume 6/II, October 2018 F. REFERENCES Bogdan, R., & Taylor, S. (1975). Introduction to Qualitative Research Methods. New York: John Willey and Sons. Carr, P. (2008). English Phonetics and Phonology: An Introduction. West Sussex: Blackwell Publishing. Cevilla, C. G. (1993). Pengantar Metode Penelitian. Jakarta: Universitas Indonesia. Crystal, D. (2008). A Dictionary of Linguistics and Phonetics. Oxford: Blackwell Publishing. Febriyanti, D. N. (2015). Assimilation, Reduction and Elision Reflected in the Selected Song Lyrics of Avenged Sevenfold. Journal of Language and Literature, 14-19. Fromkin, V., Rodman, R., & Hyams, N. (2003). An Introduction to Language. Boston: Thomson/Heinle. Giegerich, H. (1992). English Phonology: An Introduction. Cambridge: Cambridge University Press. Mardalis. (1999). Metode Penelitian Suatu Pendekatan Proposal. Jakarta: Bumi Aksara. Bogdan, R., & Taylor, S. (1975). Introduction to Qualitative Research Methods. New York: John Willey and Sons. Carr, P. (2008). English Phonetics and Phonology: An Introduction. West Sussex: Blackwell Publishing. Cevilla, C. G. (1993). Pengantar Metode Penelitian. Jakarta: Universitas Indonesia. Crystal, D. (2008). A Dictionary of Linguistics and Phonetics. Oxford: Blackwell Publishing. Febriyanti, D. N. (2015). Assimilation, Reduction and Elision Reflected in the Selected Song Lyrics of Avenged Sevenfold. Journal of Language and Literature, 14-19. Fromkin, V., Rodman, R., & Hyams, N. (2003). An Introduction to Language. Boston: Thomson/Heinle. Giegerich, H. (1992). English Phonology: An Introduction. Cambridge: Cambridge University Press. g y Mardalis. (1999). Metode Penelitian Suatu Pendekatan Proposal. Jakarta: Bumi Aksara. 57 57 ELTIN Journal, Volume 6/II, October 2018 Roach, P. (2001). English Phonetics and Phonology: A Practical Course. Cambridge: Cambridge University Press. ELTIN Journal, Volume 6/II, October 2018 Roach, P. (2001). English Phonetics and Phonology: A Practical Course. Cambridge: Cambridge University Press. 58
https://openalex.org/W2469438062
https://www.jstage.jst.go.jp/article/jmj/62/2/62_96/_pdf
English
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Have Host Defense Peptides Been Acting in Innate Immunity Since the Trilobites of the Cambrian Period 540 Million Years Ago?
Juntendo iji zasshi
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1,307
Current Topics: Current Status and Future Perspective on the Application of Host Defense Peptides to Medicine Juntendo Medical Journal 2016. 62(2), 96-97 Current Topics: Current Status and Future Perspective on the Application of Host Defense Peptides to Medicine Juntendo Medical Journal 2016. 62(2), 96-97 ISAO NAGAOKA* *Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan ISAO NAGAOKA* *Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan Antimicrobial peptides (AMPs), molecules evolu- tionarily conserved among various species, are usually positively charged, having both hydropho- bic and hydrophilic domains. They are also called host defense peptides (HDPs), because they partici- pate in the innate immune response by exerting antimicrobial activities against Gram-negative and -positive bacteria, viruses and fungi. Moreover, some HDPs (such as human cathelicidin peptide LL-37 and guinea pig cathelicidin CAP11) directly bind with Gram-negative bacterial endotoxin (lipopolysaccharides, LPS) and neutralize its biolog- ical activity 1)-5). Based on their potent and broad spectrum of activities, they are expected to become novel therapeutic agents. In fact, HDP derivatives are being developed as antibiotics, anti-inflammato- ries and antifungals. In addition to their direct antimicrobial properties, HDPs have the ability to enhance immunity and link innate and adaptive immunity by functioning as immunomodulators. dins are characterized by a highly conserved N- terminal cathelin-domain, and a variable C-terminal antimicrobial peptide domain (12-80 amino acid residues). Cathelicidin peptides have been isolated from many different mammalian species. In this issueʼs Current Topics, findings about the “Current status and future perspective on the appli- cation of host defense peptides to medicine”are reviewed. First, Dr. Hu demonstrated LL-37 to be a promising candidate for sepsis, based on its poten- tial abilities, including the inhibition of pyroptosis (a recently identified caspase-1 dependent macro- phage cell death pathway), and modulation of inflammatory cytokine production and antimicro- bial activity. Second, Dr. Suzuki suggests the possibility that during sepsis (endotoxemia) LL-37 may not only suppress LPS-induced endothelial cell apoptosis through the inhibition of LPS-binding to the cells but also induce the clearance (uptake) of LPS by endothelial cells via complex formation with LPS. Third, Dr. Ishibashi characterizes insect (beetle) defensin-derived AMPs, and demon- strates their therapeutic applications to bacterial infections and their use as novel antimicrobial materials such as AMP-immobilized fibers. Fur- thermore, Dr. Niyonsaba reviews the roles of HDPs in human skin diseases, based on their antimicrobial There are two major families of HDPs, defensins and cathelicidins. Defensins are small cysteine-rich cationic peptides consisting of 18-45 amino acids, and include six to eight conserved cysteine residues with intramolecular disulfide bonds. Defensins are found in both vertebrates and invertebrates (including arthropods), and also in plants. In contrast, cathelici- Copyright © 2016 The Juntendo Medical Society. Isao Nagaoka Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan TEL: +81-3-5802-1032 FAX: +81-3-3813-3157 E-mail: nagaokai@juntendo.ac.jp 〔Received Apr. 27, 2016〕 ISAO NAGAOKA* *Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan This is an open access article distributed under the terms of Creative Commons Attribution Li- cense (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original source is properly credited. doi: 10.14789/jmj. 62.96 96 Juntendo Medical Journal 62(2), 2016 Dermis A B C Figure-1 Horseshoe crab and trilobite A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spine horseshoe crab; B. A decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A trilobite fossil (Elrathia kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). Dermis B C C B A Figure-1 Horseshoe crab and trilobite Figure-1 Horseshoe crab and trilobite A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spine horseshoe crab; B. A decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A trilobite fossil (Elrathia kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). Figure-1 Horseshoe crab and trilobite A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spine horseshoe crab; B. A decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A trilobite fossil (Elrathia kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). Figure 1 Horseshoe crab and trilobite A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spin decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). and immunomodulatory actions in the skin. By understanding the roles of HDPs in the pathophysi- ology of skin diseases, novel therapeutic targets can be identified, and potential treatments can be developed for these skin conditions. Lastly, Dr. Tamura reviews the Limulus amoebocyte lysate (LAL) test, which is the most sensitive and reliable assay for the detection of trace amounts of bacterial endotoxin (LPS); thus, the LAL test can be utilized for the diagnosis of sepsis, or endotoxemia. ISAO NAGAOKA* *Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan How- ever, it is important to note that the LAL test can be affected by the presence of HDPs, since HDPs with potent LPS-neutralizing activities (such as LL-37 and CAP11) can mask the endotoxin active site when they bind. relatives of trilobites (ancient arthropods), which appeared in the Cambrian period, 540 million years ago (https://en.wikipedia.org/wiki/Trilobite, http: //www.uh.edu/engines/epi2496.htm)(Figure-1C). Thus, it is reasonable to speculate that AMPs/ HDPs were present in trilobites and have been acting in innate immunity since the Cambrian period. Now, we are attempting to apply these ancient host defense peptides as novel therapeutic agents to medicine. References 1) Nagaoka I, Hirota S, Niyonsaba F, et al: Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14 + cells. J Immunol, 2001; 167: 3329-3338. Interestingly, LAL is prepared from amoebocytes (hemocytes) of horseshoe crabs (Figure-1A and B). Horseshoe crab hemocytes contain two types of granules; large granules contain the essential components of the LAL test, including serine protease zymogens (factor C, factor G and factor B), proclotting enzyme and the clotting factor coagulogen, whereas small (dense) granules con- tain AMPs 6). Hoemocytes are highly sensitive to LPS, and LPS stimulation induces the degranulation and secretion of granular components and results in clot formation. These responses are important for host defense of horseshoe crabs in engulfing and killing invading microbes as well as preventing hemolymph leakage. Horseshoe crabs are called living fossils, since they have been living on earth since the late Ordovician period, 450 million years ago (https://en.wikipedia.org/wiki/Horseshoe_crab). Notably, horseshoe crabs are the closest living 2) Nagaoka I, Hirota S, Niyonsaba F, et al: Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial pepti- des by replacement with hydrophobic and cationic amino acid residues. Clin Diagn Lab Immunol, 2002; 9: 972-982. 3) Nagaoka I, Kuwahara-Arai K, Tamura H, Hiramatsu K, Hirata M: Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions. Inflamm Res, 2005; 54: 66-73. 4) Okuda D, Yomogida S, Tamura H, Nagaoka I: Determi- nation of the antibacterial and lipopolysaccharide-neu- tralizing regions of guinea pig neutrophil cathelicidin peptide CAP11. Antimicrob Agents Chemother, 2006; 50: 2602-2607. 5) Okuda D, Yomogida S, Kuwahara-Arai K, Hiramatsu K, Tamura H, Nagaoka I: Augmentation of the antimicro- bial activities of guinea pig cathelicidin CAP11-derived peptides by amino acid substitutions. Int J Mol Med, 2009; 23: 501-508. 6) Kawabata S: Immunocomponent molecules and their response network in horseshoe crabs. Adv Exp Med Biol, 2010; 708: 122-136. 97
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Efeito do ácido indolbutírico no enraizamento de estacas de ramos semilenhosos de pessegueiro
Pesquisa Agropecuária Brasileira
2,002
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An experimental analysis of the fluid flow on performance and frost formation in exhaust air energy recovery heat exchanger
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1 Introduction efficiency values are usually evaluated without taking into account a dynamic change of fluid flow velocity or flow rate as well as outdoor and indoor air conditions which significantly affecting the reduction of heat recovery efficiency. In 2010 the Energy Performance of Buildings Directive (EPBD) [1] had been introduced by the European Commission. This document states that from 2019 all public buildings in UE will have to meet the requirements for nearly zero-energy buildings. Additionally during the winter air temperature in cold and moderate climate zones may drop below minus 5℃ which results in danger for HE icing. This phenomenon is caused by water dropping out from the exhaust air and occurs at the contact of the heat exchanger surface with cold air drawn from the outside [15,16]. Under the temperatures below zero Celsius, the condensate deposited in HE change its phase and cause a significant decrease in the heat recovery and finally increase the flow resistance. During low outdoor air temperatures, there is a risk of frost large formation on the heat exchanger surface which can lead to high-pressure drop, flow velocity decrease or even serious unit damage. To prevent heat exchanger from this situation and to keep constant fluid flow velocity, additional energy supply has to be implemented. Such a system is usually energy consuming and significantly reduce system performance. In the literature, various solutions have been proposed to melt ice deposited on HE surface. One of the most popular solutions includes the implementation of an electrical heater inside of HE in the supply air duct. Protection systems warm up the heat exchanger surface and melt the ice layer. The electrical heater significant increase the energy consumption and decrease central unit efficiency. Often a fresh air bypass is also applied. Different manufacturers also apply own solution, in which to warm-up the heat exchanger they turn-off the fresh air fans. However, the fresh air flow prevention Heating, as well as air conditioning systems, are responsible for substantial energy consumption. In many cases, this consumption exceeds 60% of the total energy in operated buildings. Due to new regulations, such systems have to be designed to meet the requirements for the maximum use of non-renewable energy for heating, ventilation, cooling and hot water purpose. On the other hand, heating/cooling and ventilation units are often increasingly applicable in the domestic application as well as in industry [2-5]. An experimental analysis of the fluid flow on performance and frost formation in exhaust air energy recovery heat exchanger Marek Jaszczur1,*, Marek Borowski2, Michał Karch2, Sławosz Kleszcz1,3 1AGH University of Science and Technology, Faculty of Energy and Fuels, Kraków, Poland 2AGH University of Science and Technology, Faculty of Mining and Geoengineering, Kraków, Poland 3Frapol Sp. z o.o., Kraków, Poland Marek Jaszczur1,*, Marek Borowski2, Michał Karch2, Sławosz Kleszcz1,3 1AGH University of Science and Technology, Faculty of Energy and Fuels, Kraków, Poland 2AGH University of Science and Technology, Faculty of Mining and Geoengineering, Kraków, Poland 3Frapol Sp. z o.o., Kraków, Poland 1AGH University of Science and Technology, Faculty of Energy and Fuels, Kraków, Poland 2AGH University of Science and Technology, Faculty of Mining and Geoengineering, Kraków, Poland 3Frapol Sp. z o.o., Kraków, Poland Abstract. In the present paper, results for fluid flow, temperature and air humidity experimental measurements for innovative energy recovery counterflow air-to-air heat exchanger have been shown. For the low outdoor air temperatures case, frost formation is also observed and analysed. Base on the results of analysis the temperature, humidity and total efficiency of the exchanger are presented. The analysis showed that fluid flow rate, humidity and system efficiency significantly drop down under unfavourable conditions in the winter period when the outdoor temperature decrease below -5oC. In the present system, the heat exchanger equipped with a set of opposing air dampers can use intake air to absorb the moisture from the exhaust air. This solution has the advantage to prevent the exchanger frosting as well as melt ice layer if any growth on heat exchanger walls. * Corresponding author: jaszczur@agh.edu.pl © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). EPJ Web of Conferences 26 EFM 2019 , 01023 (2022) 9 EPJ Web of Conferences 26 EFM 2019 , 01023 (2022) 9 https://doi.org/10.1051/epjconf/202226901023 DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) 1 Introduction Public buildings as also quite often with modern ventilation systems [6,7]. With increasing share of ventilation, the efficient energy recovery appears to be an essential issue and the unique solutions for energy saving [7-9]. But such ventilation type is necessary to keep certain indoor air quality [10-13]. With the implementation of energy recovery exchanger, it is a possibly significant decrease building energy consumption. The typically used for this purpose heat exchanger (HE) are characterised by high (often above 90%) recuperation efficiency [14]. Catalogue sheets of heat exchangers typically provide the temperature efficiency under optimum fluid flow conditions. Such high-efficiency also results from the fact that it is usually determined under laboratory conditions and for very low fluid flow velocity through the heat exchanger. However, these relatively high- EPJ Web of Conferences 26 , 01023 (2022) 9 EPJ Web of Conferences 26 EFM 2019 , 01023 (2022) 9 https://doi.org/10.1051/epjconf/202226901023 https://doi.org/10.1051/epjconf/202226901023 The experimental measurements were done under real conditions reproduced in the laboratory to generate low air temperature and the possibility of occurrence of the phenomenon of heat exchanger frosting (Table 1). The experimental measurements were done under real conditions reproduced in the laboratory to generate low air temperature and the possibility of occurrence of the phenomenon of heat exchanger frosting (Table 1). fully disturbs the air flow in the entire building not ensuring acceptable comfort [17]. In many situations, the ice layer can also be melted by different the ratio of air supplied to the exhausted application. But such a solution reduces the amount of fresh air delivery to the building as well as create substantial negative pressure in the ventilated space. p g g ( ) The test section consisted of the central unit with a control system, which activates the anti-icing mode when the fresh air temperature 𝑡21 was below zero Celsius. The air handling unit was also equipped with a fresh air bypass, which was closed and deactivated for the current analysis. The time of dampers switching was set to 300s. The central unit was placed in an insulated chamber, inside which the air temperature was about 20+/-1oC. The air was supplied to the air handling unit by two ducts equipped with glycol heaters/coolers. Figure 1 shows the measurement section of the heat exchanger. 1 Introduction The temperature of the exhaust air from the room was maintained on a constant level of 23oC, while the temperature of the fresh air was equal to -18oC. JUMO EE650-T2L200 airspeed converters were used to record the volume flow rates (𝑉11 ,𝑉12, 𝑉21, 𝑉22). JUMO EE210-HT3xPBFxB hygroscopic humidity and temperature converters were used to measure the air temperature and humidity (𝑡11, 𝑡12, 𝑡21, 𝑡22 and 𝜙12, 𝜙12, 𝜙21, 𝜙22). The data acquisition from sensors was carried out using a Mitsubishi Electric FX5U module and a MAPS HMI 750 panel. The measured values were recorded with 1-second intervals during the time equal to 2000s. The data recording was activated at the time of receiving constant temperature of the fresh air. p In most of the central units, parallel plate counterflow air-to-air energy recovery heat exchanger are used. This type of heat exchanger was analysed in [18] in order to provide a guideline for optimal ventilation unit design. In their study two-dimensional model has was used in order to find analytical expressions. In other work [19] authors examined a new type of geometry for HE used in ventilation systems in cold and moderate climate zones. The methodology for system performance was presented base on the theoretical model and verified with experimental data. Fluid flow in parallel-plate turbulent open channel [20] has been studied successfully using Direct Numerical Simulation methods which provide very accurate results. In [21] the influence of channel geometrical configuration on the counter flow heat exchanger efficiency has been studied. In this work, flow, temperature and humidity for innovative energy recovery counterflow air-to-air heat exchanger equipped with a set of opposing air dampers have been studied for the selected set of temperatures. Base on the results of analysis the temperature, humidity and total efficiency of the exchanger is evaluated. For the low outdoor air temperatures case, frost formation is also analysed. The present study was aimed to implement this solution to large industrial units. Fig. 1. Diagram of the test section. Fig. 1. Diagram of the test section. Moisture content in the air: The absolute humidity is defined as a ratio of water vapour mass in the air to the dry air mass. Using Dalton’s law and the gas equation of state, it is possible to obtain a formula to evaluate the moisture content x in the air based on temperature and relative humidity as follows: Additionally, the central unit is equipped with four counter-rotating aluminium dampers controlled by the actuators. Switching position allows changing the direction of air in different part of the exchanger. The fourth tightness class (according to EN-1751) prevent fresh and used air mixing. The measurement was done to determine the efficiency of the air handling unit with a new generation of the high-performance periodic counter-flow air-to-air heat exchanger with dampers. 𝑥= 0.622 ∙ 𝜑∙𝑝𝑔𝑠 𝑝𝑏−𝜑∙𝑝𝑔𝑠 (1) (1) where 𝑝𝑔𝑠= 6.1121 ∙𝑒 17,502𝑇 𝑇+240,97 (2) 𝑝𝑏= 1013.25 (2) 2 Methodology The periodic-flow heat exchanger unit under consideration consists of a standard counter-flow exchanger equipped with a set of opposing air dampers with a short opening/closing time, used in order to cyclically modify air flow direction. With this solution, the heat exchanger can use intake air to absorb the moisture from the exhaust air. It has also advantage to prevent the exchanger frosting as well as melt ice layer if any appears. The analysed HE used four identical counterflow exchanger arranged in the right order. Fig. 1. Diagram of the test section. where V, t, ϕ are volume flow rate, air temperature and humidity respectively. 3 Results and discussion It is worth notice that despite critical air conditions due to dumpers implementation and flow direction variation, no frost layer was observed on the heat exchanger surfaces (see Fig. 7). Despite of not full water droplets evaporation (condensate), heat exchanger surface was at good condition. The average amount of condensed water from extract air was about 5.9 g/kg, and the evaporated water in supply air increase was about 4.72 g/kg. The deposited water content was observed as the iced surface of drip tray shown in Figure 8. It is worth notice that despite critical air conditions due to dumpers implementation and flow direction variation, no frost layer was observed on the heat exchanger surfaces (see Fig. 7). Despite of not full water droplets evaporation (condensate), heat exchanger surface was at good condition. The average amount of condensed water from extract air was about 5.9 g/kg, and the evaporated water in supply air increase was about 4.72 g/kg. The deposited water content was observed as the iced surface of drip tray shown in Figure 8. Figures 2 to 5 shows the experimental results of the air flow rate of fresh air V21, and exhaust air V11, temperatures t21, t11 and relative humidity ϕ21, ϕ11 of the air on each side involved in the energy exchange process. The volume flow rate of both flows was set-up to be 2000 m3/h. On the basis of laboratory and experimental test measurements, the interval of rapid air flow damper position changes was set-up at the level of 300 s. Different the dumpers position results in different the direction of air flow through the air-to-air heat exchanger, enabling the condensate accumulated evaporation from the walls of the exchanger in both operating modes. The deposited at the wall surface water droplets are evaporated and absorbed by the stream of outside air. The results in Figure 2 shows that by switching the air damper position result in the air flow direction change through the heat exchanger. At the same time the resistance of the channel flows has changed which impact on the fluid flow velocity on both sides of the heat exchanger. The relative humidity is presented in Figure 4. During the measurements, it changes dynamically between 40%–60% on supply air and 80–90% on waste air. Temperature efficiency: Table 1. Flow Conditions during the study. Parameter V21 t21 ϕ 21 V11 t11 ϕ 11 Unit m3/h oC % m3/h oC % Value 2000 -18 70 2000 23 46 The temperature 𝜂𝑡 efficiency is calculated according to the following equations: The temperature 𝜂𝑡 efficiency is calculated according to the following equations: η𝑡= 𝑡22−𝑡21 𝑡11−𝑡21 (3) (3) where V, t, ϕ are volume flow rate, air temperature and humidity respectively. where 𝑡22, 𝑡21, are the temperature of supply and outdoor air, while 𝑡11 is exhaust air temperature. where 𝑡22, 𝑡21, are the temperature of supply and outdoor air, while 𝑡11 is exhaust air temperature. 2 EPJ Web of Conferences 26 , 01023 (2022) 9 EPJ Web of Conferences 26 EFM 2019 , 01023 (2022) 9 https://doi.org/10.1051/epjconf/202226901023 https://doi.org/10.1051/epjconf/202226901023 3 Results and discussion During the study, the supplied air had the average humidity level higher than 50%, which effectively prevented the drying of the ventilated rooms, contributing to maintaining the feeling of thermal comfort for the users. Fig. 4. Fresh 𝜑21, supply 𝜑22, exhaust 𝜑11 and waste 𝜑12 air relative humidity changes. 0 20 40 60 80 100 3000 3500 4000 4500 5000 Relative humidity, % Time, s φ21 φ22 φ11 φ12 Fig. 2. Fresh V21 and exhaust V11 air volume flow rates. 0 1000 2000 3000 3000 3500 4000 4500 5000 Airflow, m3/h Time, s V21 V11 Fig. 4. Fresh 𝜑21, supply 𝜑22, exhaust 𝜑11 and waste 𝜑12 air relative humidity changes. Fig. 2. Fresh V21 and exhaust V11 air volume flow rates. On the basis of the measurements of temperature and relative humidity values of the air flowing through the heat exchanger, the moisture content changes in each of the four air flow streams were evaluated. All these values, which contribute to the heat and moisture exchange processes, are presented in Figure 5. The temperature of the supply air t22 presented in Figure 3 was about 14oC, with the temperature values for the intake t21 and extract air t11 stable all equal to -18oC (+/- 0.5oC), and 23oC (+/-0.5oC), respectively. The above conditions result in the estimated temperature efficiency of the exchanger with dumpers in the range of 76-78% (see Fig. 6). Fig. 5. Fresh 𝑥21, supply 𝑥22, exhaust 𝑥11 and waste 𝑥12 air humidity changes. 0 2 4 6 8 10 3000 3500 4000 4500 5000 Humidity, g/kg Time, s x21 x11 x12 x22 0 2 4 6 8 10 3000 3500 4000 4500 5000 Humidity, g/kg Time, s x21 x11 x12 x22 of the exchanger with dumpers in the range of 76 78% (see Fig. 6). Fig. 3. Fresh t21, supply t22, exhaust t11 and waste t12 air temperature changes. Fig. 5. Fresh 𝑥21, supply 𝑥22, exhaust 𝑥11 and waste 𝑥12 air humidity changes. -30 -20 -10 0 10 20 30 3000 4000 5000 Temperature, oC Time, s T21 T22 T11 T12 0 2 4 6 8 10 3000 3500 4000 4500 5000 Humidity, g/kg Time, s x21 x11 x12 x22 Fig. 3. Fresh t21, supply t22, exhaust t11 and waste t12 air temperature changes. 3 Results and discussion -30 -20 -10 0 10 20 30 3000 4000 5000 Temperature, oC Time, s T21 T22 T11 T12 -30 -20 -10 0 10 20 30 3000 4000 5000 Temperature, oC Time, s T21 T22 T11 T12 Fig. 5. Fresh 𝑥21, supply 𝑥22, exhaust 𝑥11 and waste 𝑥12 air humidity changes. Fig. 3. Fresh t21, supply t22, exhaust t11 and waste t12 air temperature changes. 3 EPJ Web of Conferences 26 , 01023 (2022) 9 EPJ Web of Conferences 26 EFM 2019 , 01023 (2022) 9 https://doi.org/10.1051/epjconf/202226901023 Fig. 9. Air process heating on Mollier chart (x=const). Pressure Temperature Density Humidity Ethalpy Fig. 6. The temperature efficiency over time. 0% 20% 40% 60% 80% 100% 3000 3500 4000 4500 5000 Efficiency, % Time, s Fig. 6. The temperature efficiency over time. Based on the experimental measurement the average humidity efficiency was evaluated to be equal to approx. 64%. According to Table 2 when humidity is not exchanged (as the case in traditional standard ventilation units), the process of heat exchange between fresh and exhaust air can be shown on Mollier chart - Figure 9. On these terms, the relative humidity would be only 10%. Table 2. Average value of temperature, relative humidity and humidity. 𝑡21 −17.8℃ 𝜑21 70.7% 𝑥21 0.66 g/kg 𝑡22 13.8℃ 𝜑22 55.4% 𝑥22 5.38 g/kg 𝑡11 23.3℃ 𝜑21 45.6%, 𝑥11 8.04 g/kg 𝑡12 −5.6℃, 𝜑22 85.7% 𝑥12 2.13 g/kg Table 2. Average value of temperature, relative humidity and humidity. Fig. 9. Air process heating on Mollier chart (x=const). 4 Conclusions Fig. 7. The heat exchanger surface at the end of measurements. In this research, the results of experimental measurements of an air handling unit equipped with an innovative periodic parallel-plate counterflow heat exchanger with dumpers are presented. Based on the analysis it is seen that traditional “anti-freezing” system which utilises different flow rates or even prevents fresh air flow to melt ice layer on heat exchanger surface should not be used at low-temperature conditions and can’t be applied without an additional usually energy consuming protection method. Additionally, such a solution doesn’t fulfil its primary role - proper building ventilation. The deficit of fresh air will result in significant growth of CO2 concentration in the room as well as in the air temperature drop. The implementation of the heat exchanger with a system of air dampers which change air flow direction may solve wholly issue of heat exchanger icing. With dumpers, it is possible to achieve very high-efficiency values - not available by any other commercial unit. The proposed solution, remove anti-freeze systems (usually electrical heaters) completely. For the stable fresh air temperature equal to -18oC the evaluated temperature efficiency of the exchanger with dumpers was in the range of 76-78%. In addition to that, based on the experimental measurement the average humidity efficiency is equal to approx. 64%. The mean amount of condensed water from extract air was 5.9 g/kg, while the evaporated water in supply air increase was 4.72 g/kg. During the analysis, Fig. 7. The heat exchanger surface at the end of measurements. Fig. 8. Iced drip tray at the end of measurements. Fig. 8. Iced drip tray at the end of measurements. 4 EPJ Web of Conferences 26 , 01023 (2022) 9 EPJ Web of Conferences 26 EFM 2019 , 01023 (2022) 9 https://doi.org/10.1051/epjconf/202226901023 19. J. Kragh, J. Rose, T. R. Nielsen, S. Svendsen, Ener. Build. 39, 1151 (2007) 20. M. Jaszczur, Arch. of Mech. 63,77 (2011) 21. M. I. Hasan, A. A. Rageb, M. Yaghoubi, H. Homayoni, Int. J. Therm. Sci. 48, 1607 (2009) the supplied air the average humidity was higher than 50%, which entirely prevents the drying of the ventilated space. It is important to notice that low moisture content in air flow stream may lead to health problems, malaise and worsens the thermal comfort. At autumn and winter seasons there is even recommended to humidify the air in dry rooms. 4 Conclusions However using additional devices people are exposed to unnecessary investment, operational and maintenance costs. The proposed periodic-flow heat exchanger with flow direction switching using dampers may be an excellent solution for all types of buildings (public, private, schools or hospitals), allowing to keep the necessary measure at a comfort level. It has been found that it is important to precisely determine the time of changing the direction of the fluid flow (the positions of the air dampers) with correlation to the outside and indoor conditions thus enabling a controlled recovery of moisture from the extract air. The works on the periodic-flow heat exchanger are supported in the Smart Growth Operational Programme 2014-2020 by the Polish National Centre for Research and Development and presentation by the Polish Ministry of Science Grant AGH No. 11.11.210.312. 19. J. Kragh, J. Rose, T. R. Nielsen, S. Svendsen, Ener. Build. 39, 1151 (2007) 20. M. Jaszczur, Arch. of Mech. 63,77 (2011) 21. M. I. Hasan, A. A. Rageb, M. Yaghoubi, H. Homayoni, Int. J. Therm. Sci. 48, 1607 (2009) References 1. Directive 2012/27/EU of the European Parliament and of the Council of 25 Oct. (2012) 2. M. Mijakowski, J. Sowa, P. Narowski, Civil Engineering 4, 107 (2010) 3. G. P. Vasilyev, A. Tabunshchikov, M. M. Brodach, V. A. Leskov, N. V. Mitrofanova, N. A. Timofeev, V. F. Gornov, G. V. Esaulov, Ener. Build. 112, 96 (2016) 4. V.Yu. Borodulin, M.I. Nizovtsev, App. Therm. Eng. 130, 1246 (2018) 5. S. Koestera, M. Falkenberga, M. Logemanna, M Wessling, J. Memb. Sci. 525, 68 (2017) 5. S. Koestera, M. Falkenberga, M. Logem Wessling, J. Memb. Sci. 525, 68 (2017) 6. B. R Hughes, H. N. Chaudhry, J. K. Calautit, Appl. Energy 113,127 (2014) Energy 113,127 (2014) 7. C. Simonson, Energy Build. 37, 23 (2005) 8. X. P. Liu, J.L. Niu, Appl. Energy 129, 364 (2014) 9. El Fouih, Y. Stabat, P. Rivie`re, P. Hoang, P. Archambault, Ener. Build. 54, 29 (2012) 10. M. Orme, Energy Build. 33, 199 (2001) 11. J. K. Calautit, B. R. Hughes, H. N. Chaudhry, S. A. Ghani, Appl. Energy 112, 576 (2013) 12. M Jaszczur, M Branny, M Karch, M Borowski, J. Phys. Conf. Ser. 745, 032049 (2016) 13. M. Borowski, M. Karch, Chłodnictwo & Klimatyzacja 4, 65 (2015) 14. Mure, Monitoring of energy efficiency trends and policies in the EU, 36 (2015) 15. M. Besler, M. Skrzycki, Rynek Instalacyjny 3, 26 (2013) 16. D. Seker, H. Kartas, N. Egrican, Int. J. Refrig. 27, 367 (2004) 17. L. Huang, Z. Liu, Y. Liu, Exp. Ther. Fluid Sci. 33, 1049 (2009) 18. M. Vera, A. Linan, Int. J. Heat Mass Tran. 53, 4885 (2010) 5
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The Role of Methyl Jasmonate During the Biotic Stress in Matricaria Chamomilla L.
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The Role of Methyl Jasmonate During the Biotic Stress in Matricaria Chamomilla L. Zuzana Dučaiová  (  zuzana.kovalikova@uhk.cz ) Pavol Jozef Stafarik University in Kosice Faculty of Science: Univerzita Pavla Jozefa Safarika v Kosiciach Prirodovedecka fakulta Highlights MeJA pre-treatment ameliorate negative effect of thrips thrips feeding enhanced PAL activity and coumarins content diploid cultivar reacted more significantly compared to the tetraploid one Research Article Keywords: Matricaria chamomilla, Echinothrips americanus, methyl jasmonate, coumarin-related compounds Posted Date: September 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3333626/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: September 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3333626/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract The objective of the present study was to evaluate the effect of methyl jasmonate (MeJA) in plant resistance to polyphagous thrips. Eight-weeks old diploid and tetraploid chamomile plants (Matricaria chamomilla) in stage of leaf-rosettes were pre-treated with 0.22 mM MeJA for 72 h and then co-cultivated with thrips for next 96 h. In both cultivars, there was an increase in the content of hydrogen peroxide and superoxide radical, with a maximum in plants after thrips infestation. The pre-treatment with MJ led to a reduction, but the measured values were still higher than those in the control plants. The accumulation of total phenols and flavonoids corresponded with the activity of phenylalanine ammonia-lyase (PAL). The highest PAL activity and the highest levels of total phenols were observed in plants infected with thrips. MeJA pre-treatment slightly decreased their values. Within the cultivars we noticed differences in the accumulation of monitored coumarins: (Z)- and (E)-GMCA, herniarin and umbelliferone. In diploids MeJA significantly ameliorated their accumulation caused by thrips. On the other hand, in tetraploids, changes were minimal. The amount of umbelliferone sharply increased in both cultivars which points to its role as a stress metabolite. INTRODUCTION Various fungal, bacterial and parasitic diseases degrade the quality of important medical herbs and crops. During the long co-evolution plants have developed various defensive strategies. In addition to always expressed constitutive defence represented by physical (e.g. thorns, trichomes) or chemical (e.g. repellent or toxic chemicals) barriers, plants respond with a more specific induced defence. It is triggered specifically after an herbivore attack and thus allows plants to effectively manage their energy reserves via synthesis defence compounds only when needed. At the same time, volatile substances can act as attractants for herbivore predators (Mouden and Leiss 2021; War et al. 2012). In general, feeding stimuli by chewing herbivores or certain phloem feeder are transduced via a signalling pathway mediated through the synthesis of jasmonate (JA) compounds (Lazebnik et al. 2014). Artificial manipulation of jasmonates induces the expression of JA-responsive genes, resulting in the formation of chemical and physical barriers in a pattern similar to herbivory. For example, triggering of synthesis of primary metabolites such as amino acids or sugars (Chen et al. 2020; Wei et al. 2021), secondary metabolites (Chen et al. 2020), enhancing antioxidant activity (Tianzi et al. 2018) or trichome density (Mouden et al. 2020). This state of preparedness for the attack of herbivores can subsequently manifest itself as a higher percentage of plant survival after the insect attack (Tierranegra-García et al. 2011), reducing oviposition or increasing larval and adult’s mortality (Kawazu et al. 2013; Senthil-Nathan 2019). Page 2/20 Page 2/20 In addition, some author suggests that application of volatile methyl jasmonate (MeJA) in inducing jasmonate response could be more effective than that of JA itself due to its high volatility and easy entry through stomata (Jang et al. 2014; Jiang and Yan 2018). In addition, some author suggests that application of volatile methyl jasmonate (MeJA) in inducing jasmonate response could be more effective than that of JA itself due to its high volatility and easy entry through stomata (Jang et al. 2014; Jiang and Yan 2018). Echinothrips americanus is a polyphagous thrip species that in a very short time occupy large areas of plants. Their original territory was east part of North America and their first European catch was recorded in 1989. Since then, this species infested rapidly at least in 19 European countries. It can be easily recognized due to the white stripes pass across the chest and at the base of the wings. INTRODUCTION It has piercing- sucking mouthparts and feed on plant juices sucking from a wide range of host plants (Varga et al. 2010) German chamomile (Matricaria chamomilla L.) belongs to a major group of cultivated medicinal plants. It owes its therapeutic activity to different groups of effective substances; such as sesquiterpenes, flavonoids, coumarins (herniarin and its glucosidic precursors (Z)- and (E)-2-β-d-glucopyranosyloxy-4- methoxycinnamic acid (GMCA); umbelliferone) and polyacetylenes (Franke and Schilcher 2012). Several papers, dealing with the changes of mentioned metabolites as a result of plant reaction to biotic stimuli, approved their involvement in plant defence processes (Repcak and Suvak 2013; Repcak et al. 2001). The results of previously published works (Repcak et Suvak 2013; Ducaiova et al. 2016) pointed out that the impact of phytophagous insects or/and at the same time MeJA increased the level of chamomile secondary metabolites. Since the used MeJA concentration (0.4 mM) significantly affected the accumulation of the total phenols and coumarins (Ducaiova et al. 2016), here we selected lower concentration, only 0.2 mM, for stimulation the defence processes. Based on these studies, we examined whether the exogenously added low concentration of MeJA before attack of E. americanus could ameliorate its negative impact. The induced stress intensity was evaluated at biochemical level by measuring of reactive oxygen species and total proteins content, as well as phenolic metabolites involved in defence responses, i.e. total phenolic and flavonoid content and coumarin content. Polyploidisation, or genome duplication, is common in higher plants and is considered to be an adaptation mechanism which played significant role in plant speciation. Induces polyploidisation can influence overall plant physiological and biochemical processes and affect the biosynthetic pathways of primary and secondary metabolites (Madani et al. 2021). In several species the production of primary and secondary metabolites was higher in polyploids than in their diploid counterparts, even qualitative differences were displayed (Moetamedipoor et al. 2022; Park et al. 2021). Although overall enlargement of the plant organs was observed in chamomile in tetraploid cultivars, a clear correlation between the increase in the level of active compounds was not recorded (Repčák and Krausová 2009). However, under the impact of environmental stimuli or hormone treatment, the responses of diploid plants were more stimulated (Ducaiova et al. 2020; Petruľová et al. 2014). Therefore, the second aim of the present study was to evaluate defence responses of two chamomile cultivars differing in ploidy level (diploid vs. tetraploid). MATERIAL AND METHODS MATERIAL AND METHODS Page 3/20 Plant cultivation and experimental design Seeds of chamomile (Matricaria chamomilla L.), diploid cv. 'Novbona' and tetraploid cv. 'Lutea', were germinated in sterile sands. Three-week-old plants were transferred into plastic pots filled with garden substrate. Eight-week-old plant in stage of leaf rosettes were used for the experiment. The cultivation was held in a growing chamber (Fitotron®, Weiss Technik) under controlled 12-h photoperiod lighting conditions and temperature 25/20°C (day/night). The soil water-holding capacity was maintained to 60% with daily moistening. Thrips (Echinothrips americanus Morgan) were collected on the leaves of the Alocasia macrorrhiza in the greenhouse of the Botanical Garden using the exhauster. Overall, four different variants were tested: C – control plants sprayed with distil water C – control plants sprayed with distil water MeJA – plant treated with 0.22 mM aq. solution of MeJA T – plant treated with 100 pieces of thrips for 96 hours T – plant treated with 100 pieces of thrips for 96 hours MJT – plant pre-treated with MeJA for 72 hours and subsequently treated with 100 pieces of thrips for the next 96 hours. All plants were placed under the glass bells to prevent the spread of volatile MeJA and insect. A total of three glass bells with 8 plants were used for each variant. Whole leaf rosettes were used for all analyses. Damage caused by thrips feeding (hereinafter referred to as “silver damage”) was evaluated visually and expressed as total damaged leaf area in mm2. Quantification of stress-related parameters Potassium phosphate buffer (50 mM; pH 7.0) homogenates were used for hydrogen peroxide and superoxide radical determination. The accumulation of hydrogen peroxide was estimated according to titanium chloride method (410 nm) and superoxide radical was estimated using hydroxylamine for the nitrite formation (530 nm). The amount of malondialdehyde (MDA) monitored by thiobarbituric acid reactive substance assay (TBARS) was used as an expression of the level of membrane lipid peroxidation (Dučaiová et al. 2016). Quantification of total phenols, flavonoids a Total soluble phenols (TP) and flavonoids (TF) were extracted from fresh leaf rosettes with 80% methanol. Their content was determined spectrophotometrically at 750 nm using the Folin–Ciocalteu method (Ducaiova et al. 2016) with gallic acid as standard (Sigma-Aldrich) for TP, and at 420 nm using aluminium chloride method (Ordoñez et al. 2006) with quercetin (Sigma-Aldrich) as standard for TF. The activity of phenylalanine ammonia-lyase (PAL) was measured as a production of t-cinnamic acid (t- CA) from phenylalanine in sodium borate buffer (Ducaiova et al. 2016). The HPLC conditions: column Kromasil SGX C18 7 µm (150 × 4.6 mm); flow rate 0.8 ml min-1; mobile phase: 55% acetonitrile. Detection Page 4/20 was performed at 275 nm using t-CA standard compound (Sigma-Aldrich). The activity of PAL was expressed as nmol t-CA min-1 mg-1 proteins. Total soluble proteins were quantified in phosphate buffer extracts according to Bradford (1976) with bovine serum albumin (Bio Rad) as standard. Quantification of secondary metabolites Quantification of secondary metabolites The 80% methanol was used for extraction of secondary metabolites. The separation of coumarin-related compounds was performed using HPLC-DAD (Agilent Technologies 1260 Infinity device) on a reversed- phase column LiChroCart 4–4, C-18 (5 µm). The mixture of mobile phases A (water with 1% trifluoroacetic acid) and B (55% acetonitrile) was used in gradient programme with flow rate of 0.7 ml min-1: 0–15 min 40% B; 15–25 min 100% B; 25–35 min 100% B; 35–40 min 0% B. A wavelength of 320 nm was used for quantitative analysis. For the separation of dicycloethers the mobile phase was changed to the mixture of A (acetonitrile:water:trifluoroacetic acid; 19:80:1) and B (90% acetonitrile) in gradient programme: 0–23 min 70% B; 23–25 min 0% B (Ducaiova et al. 2020). Commercially available standards were used to construct calibration curves: herniarin (≥ 98%; Extrasynthese), umbelliferone (≥ 99%; Fluka), skimmin (Apin Chemicals), daphnin (≥ 95%; Extrasynthese), chlorogenic acid (≥ 98%; Fluka) and 1,5-dicaffeoylquinic acid (≥ 95%; Fluka). For commercially unavailable standards (Z) - and (E) -GMCA, 5-feruloylquinnic acid and dicycloethers isolated and purified substances with confirmed identity based on NMR analyses were used. Statistical analysis The comparison of differences in experiment was based on one-way analysis of variance (ANOVA) and Tukey’s test at the significance level p < 0.05 (GraphPad Prism 9 Software, LLC, San Diego, CA, USA). Six individual plants mixed from three glass bells were used for analyses of each parameter. The whole experiment was repeated twice. Effect of thrips on plant oxidative status During the experiment, any significant increment or drop of biomass compared to the control plants was record (0.11 g ± 0.003 for both cultivars). MeJA treated plants exhibited mild leaves curling with prolonged isolation inside a glass bell. Exposure to thrips led to the visible changes in plants. On a closer study, numerous silver or brownish diffuse spots were observed, which in older leaves changed into the small necrotic lesions. This negative effect was in MJ pre-treated plants significantly reduced (Fig. 1). In both cultivars, there was an increase in the content of hydrogen peroxide, with a maximum in plants after thrips infestation, more than 2-fold for diploid and 1.5-fold for tetraploid plants (Fig. 2). The pre- treatment with MeJA led to a reduction, but the measured values were still higher than those in the control plants. In the case of the superoxide radical (Fig. 2), a similar accumulation trend was observed. The highest values were found in T variants, 2-fold for diploid and 1.2-fold for tetraploid plants. In the diploid Page 5/20 Page 5/20 cultivar, the addition of MeJA significantly reduced the level of superoxide radical, while in tetraploid plants the reduction was negligible. Changes in reactive oxygen species (ROS) accumulation led to a membrane damage only in the plants affected by thrips, where an approximately 1.3-fold increase in MDA content in both cultivars was observed (Fig. 2). The addition of MeJA reduced the content of MDA, but significantly only in diploid cultivar. Overall, the MDA content in tetraploid plants was approximately twice as high as in diploid ones. The maximum values of total soluble proteins were recorded in T variants and MeJA pre-treatment reduced their values, but significantly only in case of diploid plants. Overall diploid plants showed higher values and subsequent increment was much more pronounced here, almost 2-fold. Effect of thrips on phenol metabolism The content of TP (Fig. 3) sharply increased and the maximum values were recorded in T variants, 1.6- fold increase for diploid and 2-fold increase for tetraploid plants. A similar accumulation trend was observed for TF (Fig. 3). Overall, the addition of MeJA before the thrips ameliorate their negative impact only in case of TF, where there was a significant decrease, but the values were still higher compared to control values. The accumulation of TP and TF corresponded to the PAL enzyme activity (Fig. 3). The highest enzymatic activity was observed in plants infested with thrips, 1.8-fold for diploids and 1.3-fold for tetraploids. Pre-treatment with MeJA slightly decreased their values. Surprisingly, a significant drop in the activity after application of MeJA alone was monitored in both cultivars. Overall, the values in tetraploid plants were lower compared to diploid ones. A more pronounced difference occurred in the case of flavonoids and PAL activity, where the values in MJT and T variants did not rise as rapidly as in the case of the diploid cultivar. The effect of thips and MeJA pre-treatment was variously reflected in the accumulation of coumarins and their glucosidic precursors (Fig. 4) in both cultivars. In diploid plants, thrips infestation resulted in a significant increment compared to control almost in all monitored coumarins, with exception for (Z)- GMCA where the increase was only modest but still significant. Pre-treatment with MeJA reduce elevated level, but significantly only in case of umbelliferone and its glucosidic precursor skimmin, 3.7-times and 3.2-times, respectively. MeJA alone increased the values of coumarins, but their levels still remained lower in comparison to MJT and T variants. The accumulation trend in tetraploid plants was similar to diploids, with exception for skimmin and (Z)- GMCA. Here, a significant increment over control values was observed, but with moderate differences between tested variants. Like in diploids, sharp, almost 2-fold increment was in case of stress coumarin umbelliferone. Changes of three simple phenylpropanoids, namely chlorogenic acid, 1,5-dicaffeoylquinic acid, and 5- feruloylquinic acid, we monitored. The thrips invasion significantly raised their levels (Fig. 5). Pre- Page 6/20 treatment with MeJA reduced their levels, more pronounced in diploid plants in the case of 1,5- dicaffeoylquinic acid and 5-feruloylquinic acid, more then 1.5-times. Effect of thrips on dicycloethers As shown in Fig. 6, more enhanced accumulation of both (Z)- and (E)- isoforms of dicycloethers after thrips infestation was in the case of diploid plants, 3.5-fold and 1.8-fold, respectively. The protective effect of MeJA showed a significant decrease only in the case of (E)-isoform. In tetraploid plants, the increment was moderate, around 1.4-times for both isoforms. Here, in (Z)-isoform, the MeJA pre- treatment reduced its level to the control values. DISCUSSION (2018) pointed out that MeJa-treated leaves of Populus x euramericana led to increased ROS generation, which was subsequently reflected in enhanced synthesis of antioxidant enzymes in Clostera anchoreta larvae fed on these leaves. et al. 2014). The study of Tianzi et al. (2018) pointed out that MeJa-treated leaves of Populus x euramericana led to increased ROS generation, which was subsequently reflected in enhanced synthesis of antioxidant enzymes in Clostera anchoreta larvae fed on these leaves. Overall, the characteristics of ROS accumulation can vary depending on the plant type and the invading insect. For example, in Arabidopsis, accumulation maximum of hydrogen peroxide after the oviposition of P. brassicae was after 72 hours (Little et al. 2006), while the attack of the phloem feeding aphid Brevicoryne brassicae showed changes after only 48 hours (Kunierczyk et al. 2008). In the case of Bemisia tabaci, no oxidative burst occurred even after 21 days of feeding (Kempema et al. 2006). In the aforementioned works, in parallel with the increasing concentration of ROS, an increased activity of not only antioxidant enzymes, specifically superoxide dismutase, catalase, peroxidase or polyphenoloxidase, but also other effective antioxidants, such as ascorbate and glutathione, was recorded. At the same time a number of corresponding genes associated with oxidative stress were reported to be upregulated (Kempema et al. 2006; Kunierczyk et al. 2008). Malondialdehyde (MDA) represents a decomposition product of peroxidation of polyunsaturated fatty acid, and its concentration is usually correlated with the degree of membrane lipid degradation. Moreover, it has been suggested that elevated level of MDA may assist in the synthesis of more complex defence compounds and activate antioxidative enzymes. In addition, the emissions of leaf volatiles partially involved in indirect plant defence, are induced by lipid peroxidation after herbivore damage. Our results are in line with earlier reports, where MDA level was induced in cucumber plants infested with Bemisia tabaci (Zhang et al. 2008) or in rice plants attacked by Cnaphalocrocic medinalis (Chen et al. 2022). The rate of MDA formation can also depend on the insect type, e.g. scraping-sucking feeding activity is more stressful due to its concertation on large surface of plant tissues than the phloem-sucking insects which affect only vascular bundles (Kuster et al. 2022). In addition to secondary metabolites, which are the main components of chemical defence, proteins also play an important role. DISCUSSION The ability of plants to recognize and adequately respond to herbivores attack is essential for their survival. Important hormones in such induced resistance are JA or salicylic acid, which trigger many defence mechanisms that reduce herbivore survival, reproductive capacity, or preference for a plant. It has been well documented that exogenous applications of JAs can protect plants against herbivores by inhibiting or disturbing their normal development and reproduction processes, e.g. reduction in egg numbers (Shahabinejad et al. 2014), larval and pupal average weight (Jiang and Yan 2018) or fecundity (Tianzi et al. 2018). Even with increasing MeJA concentration, the total weight of larvae and adults and their survival time decreased (Senthil-Nathan 2019). These results suggest that pre-treatment of plants with JAs could provide a greater opportunity for plant survival via the activation of JA-responsive genes (Selig et al. 2016). E. americanus represent phloem feeding insect whose infestation of piercing-sucking mouthparts is manifest as silver dots, which later turn into necrotic lesions. In our work, numerous silver or brownish diffuse spots were observed which were slightly reduced in MeJA pre-treated plants. Similarly, a foliar application of JA caused a marked reduction in silver damage caused by western flower thrips which had dose-dependent trend (Mouden et al. 2020) or locally-dependent trend (Chen et al. 2020). Insect feeding activity provokes a local wound, and consequently triggers various metabolic responses driven by ROS formation. Apoplastic ROS burst acts as a first barrier against the pathogens and herbivores attack. Their elevated levels are not only toxic to the invasive organisms, but also to the plants themselves. Oxidative damage to significant biomolecules can lead to programmed cell death, which is part of a hypersensitive reaction (War et al. 2012). Among ROS, highly stabile and freely diffusible hydrogen peroxide may also act as secondary messenger by triggering the expression of defence genes and synthesis of various secondary metabolites (Fürstenberg-Hägg et al. 2013). Moreover, hydrogen peroxide has been found to have direct toxicity against insects (Maffei et al. 2007). In our experiment, we noted significantly higher ROS level after thrips invasion, which was ameliorated via MeJA pre-treatment. Similar results were also recorded in white cabbage infected with larvae of Pieris brassicae or Phyllotreta nemorum (Kovalikova et al. 2019) or in sorghum plants attacked by the butterfly Chlio partellus (Hussain Page 7/20 Page 7/20 et al. 2014). The study of Tianzi et al. DISCUSSION Proteins are the main nutrient for insect growth and changes in their content can affect the overall physiological state of herbivores by reducing their growth rate, adult size or survival rate. In this study, a sharp increase in protein content was noted. Hussain et al. (2014) reported similar results, where their content increased even more after the addition of JA. Increased levels of proteins may reflect the plants' current need for antioxidant enzymes or other protein-based defensive compounds. For example, MeJA spraying on Larix olgensis seedlings significantly increased the bio-activities of superoxide dismutase, polyphenol oxidase and two proteinase inhibitors, which resulted in strongly reduced larval/pupal weights and survivals, as well as the fecundity of Lamantria dispar (Jiang and Yan 2018). Among the secondary metabolites, plant phenols constitute one of the most common and widespread group of defensive compounds, which play a major role in plant resistance against herbivores, including insects. In plants, polyphenols are synthesized through the phenylpropanoid pathway in which L- phenylalanine is converted to cinnamic acid via PAL enzyme. Further, different enzymes at different levels Page 8/20 Page 8/20 modulate the structure of the compounds formed at different levels of the pathway (Singh et al. 2021). PAL together with polyphenol oxidase have been considered as key enzymes in phenol metabolism, and their activity has been correlated to plant protection against insect feeding. In our study, the accumulation of phenols and flavonoids corresponded with the enzymatic activity of PAL with the maximum in plants attacked by thrips. Pre-treatment with MeJA slightly reduced PAL activity together with phenol levels. Similar results were also found in sorghum plants (Hussain et al. 2014), but on the contrary, an increased level of phenols and flavonoids was recorded in lettuce under the influence of MeJA and pill bugs (Tierranegra-García et al. 2011). Moreover, the level of activity may also depend on the type of plant cultivar. Aphid induced stronger enzyme elevation during the early period after infestation in resistant cultivars of chrysanthemum than to susceptible (He et al. 2011). Changes were also noted at the level of gene expression. For example, in pepper plants, the relative expression of PAL gene increased with prolonged invasion of western flower thrips (Zheng et al. 2019). Surprisingly, Selig et al. DISCUSSION This negative effect is reduced by their sequestration into an inactive form in vacuoles, mostly by conjugation with glucose. With an increased need for active aglycones, the bond is broken by a specific glucosyltransferase (Singh et al. 2021). Occurrence of cellular membrane disruption, monitored here as an elevated level of MDA, should lead to a decrease in glycosidic form, GMCA and skimmin, and an increase in aglycones, herniarin and umbelliferone. Surprisingly, continuous increment in all mentioned coumarins was monitored here. Similar results were found in chamomile plants treated with salicylic acid (Ducaiova et al. 2013) or MeJA (Ducaiova et al. 2016). The involvement of coumarins in defence processes suggests both their elevated concentrations in stressful conditions and also their antioxidant potential linked with the presence of conjugated system, ester bonds and with the number of bound hydroxyl groups. According to Lin et al. (2008), coumarins with higher amount of hydroxyl groups express better scavenging properties than unhydroxylated coumarins. From hydroxylated coumarins, we observed strong increment in the content of umbelliferone (7-hydroxycoumarin), but surprisingly, daphnetin (7,8-dihydroxycoumarin), was found only in trace amounts, but its glucosidic precursor daphnin (daphnetin-7-O-glucoside) significantly rose in MJT and T variants. The work of Petruľová-Poracká et al. (2013), points out that there are significantly higher concentrations of daphnetin in the anthodia than in the leaves. Umbelliferone plays an important role, both as a stress coumarin and as a precursor for the synthesis of other coumarins (herniarin, esculetin or skimmin) (Chu et al. 2017). In this way, low amounts of umbelliferone under normal physiological conditions and elevated levels under stressful conditions were expected. Both in our experiments and in abovementioned works, a significant increase in umbelliferone was noted. Polyacetylenes of spiroketal enol ether type are known secondary metabolites synthesized by Asteraceae plants. Together with other substances such as monoterpenes or sesquiterpenes, they are part of the volatile substances that create the typical aroma. In addition, they play an important role in plant defence thanks to their toxic properties, such as growth and reproductive inhibitors, repellents, and oviposition- deterrent (Minto and Blacklock 2008). Several studies showed that essential oils or substances alone showed strong larvicidal potential against mosquito (Ali et al. 2018; Govindarajan and Benelli 2016). DISCUSSION (2016) pointed out that the expression level can also be influenced by the type of insect itself, when phloem-feeder thrips upregulated and cell-content feeder aphid downregulated soybean PAL2 expression. Among the phenolic acids, chlorogenic acid is the most abundant among the caffeoylquinic acid isomers in nature. It is considered as a defence metabolite in plants that provides protection against different herbivores via its oxidation to chlorogenoquinone which binds to free amino acids and proteins and reduced their digestibility. Higher accumulation of chlorogenic acid has been described in several plant species regardless of the feeding method, i.e. stem boring (Lee et al. 2017), piercing–sucking (Leiss et al. 2009) and chewing (Kundu et al. 2018). In these studies, anti-herbivore effect of chlorogenic acid was also confirmed by dietary tests, when a growth retardation, survival rate or larval weight reduction was detected. In our work, we noted an increase not only in chlorogenic acid, but also in 1,5-dicaffeoylquinic acid and 5-ferruloylquinic acid, both due to MeJA and thrips. Overall, the levels were higher in the case of the diploid cultivar, which, according to the conclusions of the study by Leiss et al. (2009), indicated that cultivars with higher values of the mentioned metabolites showed higher resistance. In addition to chlorogenic acid, other phenolic acids can also play an important role in protection. For example, exogenous application of cinnamic and vanillic acid negatively affected the oviposition and feeding preference of the larvae of Achaea janata and Spodoptera litura on castor plants (Usha Rani and Pratyusha 2014). Among other phenolic metabolites we focused on coumarins, which content was significantly influenced by thrips infestation. Almost in all cases, pre-treatment with MeJA reduced their levels. A significant elevation not only due to the effect of thrips, but also due to the effect of MeJA itself was monitored in the work Repčák and Suvák (2013), so we can conclude that concentration (0.4 mM) used here could already have a stressful rather than acclimatizing effect. Increased levels of coumarins in chamomile plants were also observed under the influence of various abiotic, e.g. heavy metals (Kováčik et al. 2012), nitrogen nutrition (Sajko et al. 2018) or UV radiation (Petruľová et al. 2014), and biotic stimuli, e.g. Erypside cichoracearum infection (Repčák et al. 2001). Page 9/20 Page 9/20 Many phenolic metabolites, including coumarins, are toxic not only to herbivores, but also to the plants themselves. DISCUSSION The literature data about the changes of dicycloethers in chamomile plants during the stress conditions are expressed in two way, as their sum or as free form, so it is difficult to compare to our results. Similar to our results, an increasing trend was observed after biotic stress (Repčák et al. 2001). However, MeJA application alone resulted in different accumulation trends, an increase in the (Z)- isoforms and a decrease in the (E)-isoforms (Dučaiová et al., 2016). In the case of other abiotic stressors, a predominantly declining trend was observed (Repčák et al. 2001, Sajko et al. 2018). An increased ploidy level not only leads to a higher accumulation of pharmaceutically important metabolites, but can also help better adaptation to environmental stimuli. Yang et al 2021 showed that plants with higher ploidy level produced more phenolic metabolites with allelopathic effects, which Page 10/20 Page 10/20 allowed seedlings to survive better and colonize new habitats faster. The effect of ploidy level on the rate of attack by herbivores is not yet completely clear. Authors report both a lower (Nuismer et Thompson 2001) and a higher attack rate (Arvanitisa et al. 2010) in polyploid plants. Furthermore, it is known that polyploidy can cause several changes in physiology, morphology, and gene expression that can impact resistance to herbivores. For example, in Brassicales, genome duplication led to an increased rate of glucosinolate synthesis, which play a key role in defence against invading butterflies (Edger et al. 2015). Qualitative changes of terpenes in Solidago gigantea, namely an increase in sesquiterpenes in tetraploids and, conversely, an increase in monoterpenes and diterpenes in hexaploids, led to changes in the survival and growth of invading herbivores (Hull-Sanders et al. 2009a, b). Due to the fact that in the case of our experiment diploid and tetraploid plants were separated, it is not possible to clearly determine the preference of invading thrips. Macroscopically observable manifestations of feeding in the form of silver and brown spots did not show significant differences in number between cultivars. Activation of defence mechanisms was manifested by increased accumulation of total proteins (possibly increased synthesis of antioxidant enzymes) and increased accumulation of coumarins and phenolic acids with potential antioxidant properties (chlorogenic acid, 5- dicaffeoylquinic acid, and 5-feruloylquinic acid). A higher content of phenolic metabolites in diploid Centaurea phrygia plants compared to tetraploid plants was also noted by Münzbergová et al. DISCUSSION (2015), which led to increased resistance to insects. However, the total content of hydrogen peroxide and the rate of its increase was significantly higher in the case of diploid plants, which indicates a higher level of stress. Overall, we can say that diploid plants responded to stress to a higher extent, but the use of MeJA as a protective agent alleviated the subsequent insect-induced stress more significantly than tetraploid plants. ACKNOWLEDGEMENTS The authors are grateful to Excellence project PřF UHK 2216/2022-2023 for the financial support. We the authors declare that this manuscript is original, has not been published before, and is not currently being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. The authors have no competing interests to declare that are relevant to the The authors have no competing interests to declare that are relevant to the content of this article. competing interests to declare that are relevant to the content of this article. editing: Zuzana Kovalikova. All authors revised the manuscript and approved its final version. editing: Zuzana Kovalikova. All authors revised the manuscript and approved its final version. AUTHOR CONTRIBUTION Page 11/20 Conceptualization: Zuzana Kovalikova, Veronika Petrulova; methodology: Zuzana Kovalikova, Veronika Petrulova; data curation: Zuzana Kovalikova; writing—original draft: Zuzana Kovalikova; writing—review & editing: Zuzana Kovalikova. 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Petruľová-Poracká V, Repčák M, Vilková M, Imrich J (2013) Coumarins of Matricaria chamomilla L.: aglycones and glycosides. Food Chem 141:54–59 44. Repčák M, Imrich J, Franeková M (2001) Umbelliferone, a stress metabolite of Chamomilla recutita (L.) Rauschert. J Plant Physiol 158:1085–1087. 44. Repčák M, Imrich J, Franeková M (2001) Umbelliferone, a stress metabolite of Chamomilla recutita (L.) Rauschert. J Plant Physiol 158:1085–1087. Page 14/20 45. Figures Figures Figures Figure 1 Feeding damage by thrips on chamomile leaf with or without MeJA pre-treatment. Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p< 0.05) References Wei X, Vrieling K, Kim HK, Mulder PPJ, Klinkhamer PGL (2021) Application of methyl jasmonate and salicylic acid lead to contrasting effects on the plant’s metabolome and herbivory. Plant Sci 303:110784 58. 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Arthropod-Plant Interact 13:289–297 Aleyrodidae) infestation. Arthropod-Plant Interact 2:209–213. Aleyrodidae) infestation. Arthropod-Plant Interact 2:209–213. Aleyrodidae) infestation. Arthropod-Plant Interact 2:209–213. 60. Zheng X, Chen Y, Zhao L et al. (2019) Tripartite interactions between jasmonic/salicylic acid pathways, western flower thrips, and thrips-transmitted tomato zonate spot virus infection in Capsicuum annuum. Arthropod-Plant Interact 13:289–297 60. Zheng X, Chen Y, Zhao L et al. (2019) Tripartite interactions between jasmonic/salicylic acid pathways, western flower thrips, and thrips-transmitted tomato zonate spot virus infection in Capsicuum annuum. Arthropod-Plant Interact 13:289–297 Figure 1 Feeding damage by thrips on chamomile leaf with or without MeJA pre-treatment. Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p< 0.05) Page 16/20 Figure 2 Figure 2 Effect of MeJA pre-treatment and thrips infestation on the content of hydrogen peroxide (µmol g-1 DW), superoxide radical (µg g-1 DW), malondialdehyde (µmol g-1 DW), and total soluble proteins (mg g-1 DW) in chamomile leaf rosettes. Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Effect of MeJA pre-treatment and thrips infestation on the content of hydrogen peroxide (µmol g-1 DW), superoxide radical (µg g-1 DW), malondialdehyde (µmol g-1 DW), and total soluble proteins (mg g-1 DW) in chamomile leaf rosettes. Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Page 17/20 Page 17/20 Figure 3 Effect of MeJA pre-treatment and thrips infestation on the content of total soluble phenols (mg g-1 DW), flavonoids (mg g-1 DW) and PAL activity (nmol t-CA min-1 mg-1 prot.) in chamomile leaf rosettes. Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 3 Effect of MeJA pre-treatment and thrips infestation on the content of total soluble phenols (mg g-1 DW), flavonoids (mg g-1 DW) and PAL activity (nmol t-CA min-1 mg-1 prot.) in chamomile leaf rosettes. Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Effect of MeJA pre-treatment and thrips infestation on the content of total soluble phenols (mg g-1 DW), flavonoids (mg g-1 DW) and PAL activity (nmol t-CA min-1 mg-1 prot.) in chamomile leaf rosettes. Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Page 18/20 Figure 4 Effect of MeJA pre-treatment and thrips infestation on the content of coumarin-related compounds (m 1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 4 Effect of MeJA pre-treatment and thrips infestation on the content of coumarin-related compounds (mg g- 1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Page 19/20 Page 19/20 Page 19/20 pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1 means ± SDs (n = 12). Values within column, followed by the same regular or italic significantly different according to Tukey’s test (p < 0.05) pre-treatment and thrips infestation on the content of dicycloethers (mg g-1 DW). Data are = 12). Values within column, followed by the same regular or italic letter(s), are not ferent according to Tukey’s test (p < 0 05) Figure 5 Effect of MeJA pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 5 Figure 5 Effect of MeJA pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Effect of MeJA pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 6 Effect of MeJA pre-treatment and thrips infestation on the content of dicycloethers (mg g-1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 6 Effect of MeJA pre-treatment and thrips infestation on the content of dicycloethers (mg g-1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Page 20/20
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Malaria Epidemics and Surveillance Systems in Canada
Emerging infectious diseases
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J. Dick MacLean,* Anne-Marie Demers,* Momar Ndao,* Evelyne Kokoskin,* Brian J. Ward,* and Theresa W. Gyorkos† J. Dick MacLean,* Anne-Marie Demers,* Momar Ndao,* Evelyne Kokoskin,* Brian J. Ward,* and Theresa W. Gyorkos† tem should be strengthened and used more proactively to help identify appropriate preventive measures. In the past decade, fluctuations in numbers of import- ed malaria cases have been seen in Canada. In 1997 to 1998, malaria case numbers more than doubled before returning to normal. This increase was not seen in any other industrialized country. The Canadian federal malaria surveillance system collects insufficient data to interpret these fluctuations. Using local (sentinel), provincial, feder- al, and international malaria surveillance data, we evaluate and interpret these fluctuations. Several epidemics are described. With an ever-increasing immigrant and refugee population of tropical origin, improved surveillance will be necessary to guide public health prevention policy and practice. The Canadian experience is likely to be general- izable to other industrialized countries where malaria is a reportable disease within a passive surveillance system. All 10 provincial and 3 territorial health authorities in Canada are required by law to report diagnoses of malaria and other selected diseases to federal authorities at Health Canada (2). Summary reports of these diseases are pub- lished by both levels of reporting in provincial and territo- rial news bulletins and by Health Canada in the Canada Communicable Diseases Report. In several instances over the past decade, malaria inci- dence in Canada as a whole, or in individual provinces, reached epidemic levels (7). Why some were not immedi- ately identified and why no comprehensive analysis has been published as part of government surveillance systems are questions that will be addressed. Failing to recognize these epidemics has limited the ability of public health officials to assess and intervene appropriately to control the illness and death associated with imported malaria in Canada. M alaria has been a reportable communicable disease in Canada since 1929, when a surveillance system for communicable diseases was first developed. Although no longer endemic in Canada, malaria has remained an impor- tant imported disease, principally in immigrants and travel- ers (1–3). Rarely, it has been transmitted in blood products (4). Published reports document delays in clinical and lab- oratory diagnoses of malaria and lack of understanding of malaria prophylaxis and fever management in travelers (3). J. Dick MacLean,* Anne-Marie Demers,* Momar Ndao,* Evelyne Kokoskin,* Brian J. Ward,* and Theresa W. Gyorkos† The Canadian infectious disease surveillance system has reported an average of 538 malaria cases per year since 1990, and Statistics Canada reported an average of one death per year (5,6, Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). The present federal surveillance system reports the age and sex of a patient and does not document malaria death rate, malaria species, nor the likely country of acquisition. While malar- ia-related deaths may be few, that any exist is a matter of concern. The continued incidence of malaria cases and deaths in Canada suggests that the malaria surveillance sys- M This study evaluated and summarized data collected over the past 22 years by local, provincial, and federal malaria surveillance systems, from Canadian federal immigration and refugee data resources and from interna- tional tourist resources, to identify and explore the causes of malaria epidemics. In addition, geographic patterns and Plasmodium spp. profiles of malaria are examined. This analysis led us to conclude that changes are needed in both the surveillance reporting instruments and how these sur- veillance results are analyzed and used. *Montreal General Hospital, McGill University Centre for Tropical Diseases, Montreal, Québec, Canada; and †McGill University, Montreal, Québec, Canada TDC Database Overall, 553 clinical cases of malaria were seen at TDC from 1981 to 2002, with some fluctuation over time but an overall gradual increase (Figure 1). In these 553 cases, 562 microscopy diagnoses were made; Plasmodium falci- parum 295 (52%), P. vivax 218 (39%), P. ovale 26 (5%), P. malariae 16 (3%), and unknown species 7 (1%). Nine (2%) of the clinical cases were mixed infections, involving P. falciparum with either P. malariae or P. vivax. Seven patients were seen two or three times with relapses of P. vivax (recurrence >2 months later). The relative frequency of species changed over time, with a gradual increase in the proportion of P. falciparum cases from 20% to 30% in the early 1980s to 60% to 70% in the 1990s and to 70% to 80% in the present decade (Figure 2). Over this 22-year period, only one fatality occurred (3). Sixty-one countries were identified as the most likely sources of the malaria exposure. Sub-Saharan Africa was the region where most patients contracted malaria, 353 case-patients (65%), followed by south Asia (23%), Southeast Asia (6%), Central America (5%), and South America (1%). However, India, with 110 cases (20%), was the single most frequent source country. Tourists (29%), immigrants or refugees (29%), and foreign workers (24%) represented the categories most frequently reported. A shift over time occurred in the importance of sub-Saharan Africa as a source of malaria cases. In the 1980s, 50% of malaria infections were acquired in Africa; in the 90s, 70%; and, since 2000, 85%. Patients of all categories were more like- ly to contract P. falciparum in Africa (74.3%) while it has been an uncommon species in south Asia (5.8%). The increase in P. falciparum cases over time correlated with Provincial reportable disease databases have included, in the past 10 years, patient characteristics such as age, sex, and malaria species, but not the likely country of acquisition. Because 90% of all malaria cases in Canada were reported by the Provinces of British Columbia (Monica Naus [British Columbia Centre for Disease Control], pers. comm.), Ontario (Lorraine Schiedel [Ontario Ministry of Health and Long-Term Care], pers. comm.), and Québec (Colette Colin [Ministère de la santé et des services sociaux, Québec], pers. comm.), the present analysis focuses on their data, primarily for 1990–2002 (11). Methods The databases used for the present analysis include 22 years of records from a local malaria reference center in Montreal, Canada (the McGill University Centre for Tropical Diseases [TDC]) and up to 13 years of quality assurance and notifiable disease surveillance databases of the provincial and federal governments of Canada, France, India, Switzerland, the United States, and the United Kingdom. TDC is a clinical and laboratory facility that *Montreal General Hospital, McGill University Centre for Tropical Diseases, Montreal, Québec, Canada; and †McGill University, Montreal, Québec, Canada 1195 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 PERSPECTIVES terns of Canadians to the tropics by the World Tourism Organization, Madrid (27). provides care to 800 to 1,100 new patients per year (approximately 60% primary cases and 40% consult cases), drawn mainly from the Montreal region. The TDC database from 1981 to 2002 has allowed previous detailed reviews of changing patterns of malaria in its patient pop- ulations (8–10). Malaria-relevant data captured include category of traveler (tourist, immigrant, refugee, expatri- ate, missionary, and volunteer), countries visited, and malaria species. A diagnosis of malaria is made if para- sites are noted on a blood smear (thin, thick, or buffy coat) or if, in the last 5 years, the patient had a positive result on a malaria antigen-capture test (e.g., Macromed [Nova Century Scientific, Inc., Burlington, Ontario, Canada], ICT Malaria P.f. [ICT Diagnostics, Brookvale, New South Wales, Australia], or OptiMAL [Flow Inc., Portland, OR]). While active surveillance studies during this period included polymerase chain reaction (PCR) as a screening tool, PCR-positive cases were not included in any of the passive surveillance statistics unless they were also independently confirmed by either malaria antigen capture or smear. TDC Database Quality assurance data for the province of Québec (1994–2002) were provided by TDC and the Laboratoire de Santé Publique du Québec. The federal government’s notifiable disease database from 1990 to 2002 is a compi- lation of selected information from individual provincial databases and includes patient age and sex for each report but no malaria species or country of acquisition (Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). Figure 1. McGill University Centre for Tropical Diseases malaria cases by year and origin (N = 553). International malaria surveillance data (1990–2002) were acquired from the World Health Organization (WHO) Regional Office for South East Asia (Rakish Mani Rastogi, pers. comm.), the WHO Regional Office for Europe (12), and the United States (13–24). Malaria rates for all coun- tries were based on population data of the U.S. Census Bureau (25). Trends in Canadian immigration and refugee data for the years 1990–2002 were provided by Citizen and Immigration, Canada (Karen Tremblett [Medical Services Branch, Citizen and Immigration Canada], pers. comm.), data on language by Statistics Canada (26), and travel pat- Figure 1. McGill University Centre for Tropical Diseases malaria cases by year and origin (N = 553). 1196 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Malaria Epidemics and Surveillance Systems in Canada Figure 2. Relative rates of Plasmodium species (N = 553). malaria incidence was observed in males and females. British Columbia had the highest rate per 100,000 (3.6 ± 2.8) over this period, followed by Ontario (2.2 ± 0.98), and Québec (1.3 ± 0.67) (Figure 3). However, the highest cumulative numbers for the 12-year period were reported from Ontario (N = 3,222), followed by British Columbia (N = 1,763), and Québec (N = 1,246). The Canadian data suggest that an epidemic occurred from 1995 to 1997, reflecting higher than average numbers of malaria cases in these years from British Columbia, Ontario, and to a less- er extent, from Québec (Figure 3). This epidemic was almost entirely due to increased P. vivax being reported in these provinces (Figure 4). From 1990 to 1999, two events occurred in Québec that did not occur in other Canadian provinces. In 1994, a quality assurance program for the province was initiated by TDC, in collaboration with the Laboratoire de Santé Publique du Québec. TDC Database Provincial malaria rates for Québec, Ontario, and British Columbia (6,11, Colette Colin [Ministère de la santé et des servic- es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). TDC Database This three- pronged program provided: 1) a free, rapid turnaround confirmation service for positive or equivocal malaria diagnoses from any laboratory in Québec, 2) a biannual malaria-training course for clinical laboratory technolo- gists, and 3) a voluntary proficiency testing program for Québec hospital laboratories, in which once or twice a year they are sent unknown positive and negative smears for identification and receive extensive feedback. From the inception of the quality assurance program, a parallel increase was seen in numbers of specimens being sent to the reference laboratory and to the Québec surveillance program (Figure 5). This fourfold increase represented an epidemic attributable to improved diagnosis and reporting. The second event in Québec was another epidemic, in this case of falciparum malaria, observed in 2000 to 2001 and associated with a large influx of refugees from Tanzanian refugee camps (Figure 4) (29). the increase in the total number of malaria cases contracted in Africa; P. falciparum represented ≈30% of all cases in the early 1980s and increased to 70% in the late 1990s. From 1981 to 2002, 96% of malaria infections acquired in south Asia were non-falciparum malarias, while only 29% of infections from Africa were non-falciparum. None of the 553 cases of malaria originated from China, Malaysia, Saudi Arabia, Peru, or Venezuela, which are frequent trav- el destinations of Québecers. Other common travel destina- tions contributed little to the 20-year malaria case total (e.g., Philippines [1 case], South Africa [1], Costa Rica [2], Mexico [2], and Dominican Republic [3]). Malaria cases from Africa from 1992 to 2000 came predominantly (69%) from the French-speaking African countries, notably high- er than the proportion of U.S. travelers (18%) who acquired malaria in these countries (13–24). Two “epidemics” were observed at TDC during this period (Figures 1 and 2). The first was in 1986 to 1990 and resulted from increasing numbers of P. falciparum infec- tions from Africa, thought to be due to increasing chloro- quine-resistant P. falciparum in chloroquine-prophylaxed travelers (28), and the second was in 1999 through 2000, resulting from increased numbers of P. falciparum infec- tions associated with the arrival in Québec of large num- bers of refugees from Tanzanian refugee camps (29). Both epidemics were recognized and reported in the literature soon after their appearance. Figure 3. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Federal and Provincial Databases A review of the Federal Health Canada databases for the incidence of malaria in Canada, from 1990 through 2002, documents a range from 364 to 1,029 cases per year, with an average of 538 cases per year during the period (or an average of ≈1.8 cases per 100,000 population per year) (6) (Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British Columbia (6,11, Colette Colin [Ministère de la santé et des servic- es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British Columbia (6,11, Colette Colin [Ministère de la santé et des servic- es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British Columbia (6,11, Colette Colin [Ministère de la santé et des servic- es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British Columbia (6,11, Colette Colin [Ministère de la santé et des servic- es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). While all ages were affected, patients were mostly adults from 20 to 59 years of age. A similar pattern of 1197 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 PERSPECTIVES Figure 4. Plasmodium species provincial trends (6,11, Colette Colin [Ministère de la santé et des services sociaux, Québec], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.). known to have important immigration and travel links with North America (Figure 7). Federal and Provincial Databases During the 1990s, France had a 60% increase in malaria in the latter part of the decade (31), reportedly caused by African travel, and Denmark experienced an increase of 68%; Germany, Italy, Spain, Sweden, the Netherlands, and Belgium, however, had sta- ble rates during this time (12,31). None of these countries had the increase in rates of malaria seen in Canada and, to a lesser degree, in the United States from 1995 to 1997. Discussion Malaria importations into Canada can occur by either immigration or travel, and changing malaria attack rates in the countries of exposure are likely to influence the inci- dence of imported disease. Changes in Canadian immigra- tion and refugee patterns from 1990 to 2002 are notable for a threefold increase in annual immigrant numbers from the Indian subcontinent and relatively stable numbers from sub-Saharan Africa. Neither combined nor separate provincial immigration and refugee patterns explain the important swings in annual Canadian malaria rates. Figure 4. Plasmodium species provincial trends (6,11, Colette Colin [Ministère de la santé et des services sociaux, Québec], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.). While the geographic origins of immigrants and refugees do not immediately explain the epidemic changes in P. vivax malaria seen in the mid-1990s, their nonrandom aggregation in certain provinces allows additional insights. African immigrants and refugees have settled all across Canada in every province in numbers that paralleled the province’s population. Immigrants and refugees from the Indian subcontinent did not: 84% settled in Ontario and British Columbia, the provinces with the most pronounced P. vivax epidemics. Canadian travelers to malaria-endemic areas have gradually but steadily increased during the past 15 years, most notably with a threefold increase to Southeast Asia and Central and South America, a twofold increase to the important malarial region of south Asia, and a smaller increase to Africa. Travel patterns did not offer an explanation for either the P. vivax epidemics in British While the geographic origins of immigrants and refugees do not immediately explain the epidemic changes in P. vivax malaria seen in the mid-1990s, their nonrandom aggregation in certain provinces allows additional insights. Figure 4. Plasmodium species provincial trends (6,11, Colette Colin [Ministère de la santé et des services sociaux, Québec], pers. comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.; Lorraine Schiedel [Ontario Ministry of Health and Long Term Care], pers. comm.). International Malaria Surveillance However, the major fluctuations in federal and provincial malaria rates from 1990 to 2002, and, in particular, during the epidemic years, were not found to be directly linked to provincial immigration numbers or to the travel destina- tions of Canadians in general. Unfortunately, no mecha- nism records the destinations of travelers from specific provinces. Ontario and British Columbia are home to 86% of the Punjabi-speaking Canadian population. If provincial travel destination data were available, it would likely show that these provinces were the source of most Canadian travelers to the Indian Punjab (27). Figure 6. Malaria cases/100,000 relative to 1990 (6,12–25,30, Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). Columbia and Ontario in the late 1990s or the P. falci- parum epidemic in Québec from 2000 to 2001. The World Tourism Organization data do not break down Canadian travel by traveler’s province of origin; however, compar- ing U.S. malaria surveillance data with TDC surveillance data, both of which track the likely country of origin of a malaria case, Québec travelers acquire most African malaria in French-speaking African countries (69%), a minor source of malaria for Americans (18%). English- speaking Ontario and British Columbia likely have more “American” travel patterns than francophone Québecers. However, no fluctuations were seen in rates of travel to either East or West Africa or to the Indian subcontinent, the major source of Canada-acquired P. vivax malaria, which would explain the impressive change in Canadian malaria reporting from 1995 through 1997. Working back from individual case data in each province seems to be the most accurate way to identify countries where large numbers of imported malaria may originate. Country of likely origin of the malaria should be indicated on all requisitions for malaria laboratory diagno- sis, and this information and the malaria species should be reported to provincial and then federal surveillance bodies. The fact that the 1995–1997 epidemic was primarily due to P. vivax, the predominant malaria species in India, and that it occurred at the same time as the P. vivax epidemic in the Indian Punjabi states of Punjab and Haryana, is strong evi- dence to conclude that the Canadian epidemic was an extension of the Punjab epidemic. This association is The two surveillance sources of India and the United States were also reviewed for malaria incidence trends. American malaria surveillance includes the likely country of origin of a malaria case. International Malaria Surveillance International Malaria Surveillance National surveillance systems for malaria are far from universal, and compliance with national surveillance instruments, when measured, is low. The stability of the degree of underreporting over time has been not been eval- uated. Despite these limitations, trends in malaria inci- dence over time in different countries can provide useful information. From 1995 to 1997, when parts of Canada were having malaria epidemics, similar but smaller changes in malaria rates were observed in the United States and United Kingdom (Figure 6). An examination of the geographic origin of malaria cases reported in the United States in the mid-1990s showed a more than twofold increase in malaria cases imported from India in 1995 through 1997, with an abrupt drop in these cases in 1998 (12–23). During this same period, a similar epidem- ic of P. vivax malaria occurred in certain states in India Figure 5. Malaria surveillance of Québec province, McGill Centre for Tropical Diseases (TDC), and Québec quality assurance (QA) program (Colette Colin [Ministère de la santé et des services soci- aux, Québec], pers. comm.). Figure 5. Malaria surveillance of Québec province, McGill Centre for Tropical Diseases (TDC), and Québec quality assurance (QA) program (Colette Colin [Ministère de la santé et des services soci- aux, Québec], pers. comm.). 1198 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Malaria Epidemics and Surveillance Systems in Canada Figure 6. Malaria cases/100,000 relative to 1990 (6,12–25,30, Carole Scott [Division of Disease Surveillance, Health Canada], pers. comm.). epidemics in Canada in the last decade. One was a P. vivax epidemic, the epicenter of which was almost certainly in the Punjab, India. The second was a P. falciparum epidem- ic in Québec related to an increased influx of Central African refugees from Tanzanian refugee camps. At the time, neither of these epidemics was brought to the atten- tion of health practitioners in travel clinics through publi- cation or other standard channels. Consequently, possible explanations and potential interventions were not dis- cussed. Trends in immigration do not explain the malaria inci- dence changes seen in Canada. These trends differ for each province both in terms of country of origin and numbers. International Malaria Surveillance 1995;53:518–21. 10. Gyorkos TW, Svenson JE, MacLean JD, Mohamed N, Remondin MH, Franco ED. Compliance with antimalarial chemoprophylaxis and the subsequent development of malaria: a matched case-control study. Am J Trop Med Hyg. 1995;53:511–7. 11. Annual summary of reportable diseases [monograph on the Internet]. British Columbia Centre for Disease Control. 2000 [cited 2003 Aug 20]. Available from: http://www.bccdc.org/downloads/pdf/cdc/ reports/rpt-annual2000.pdf 12. Computerized information system on infectious diseases (CISID) [database on the Internet]. Copenhagen, WHO Regional Office for Europe. 2003 [cited 2003 Aug 3]. Available from: http://data.euro.who.int/cisid/ For surveillance data to be useful and cost-effective, it must be both available in a timely fashion and interpretable. Local surveillance systems have obvious benefits when increased water- and foodborne infections or vaccine-pre- ventable diseases lead to quick public health action. Malaria surveillance differs in two major ways from these classical scenarios. Malaria is an imported disease, and no immediate intervention (e.g., vaccine, chemical disinfec- tant, and handwashing) will affect an epidemic. As with sexually transmitted infections, the control of a malaria epi- demic in Canadian travelers requires public education. In the United States, both malaria speciation and country of likely acquisition of the malaria case are part of surveil- lance. Such information, if part of the Canadian system, would allow rapid appreciation of the etiology of epidemics such as those reviewed here, which would potentially lead to appropriate public health response. 13. Centers for Disease Control and Prevention. Notice to readers: final 2002 reports of notifiable diseases. Morb Mortal Wkly Rep Surveill Summ. 2003;52:741–50. 14. Filler S, Causer LM, Newman RD, Barber AM, Roberts JM, MacArthur J, et al. Malaria surveillance—United States, 2001. Morb Mortal Wkly Rep Surveill Summ. 2003;52:1–14. 15. Causer LM, Newman RD, Barber AM, Roberts JM, Stennies G, Bloland PB, et al. Malaria surveillance—United States, 2000. Morb Mortal Wkly Rep Surveill Summ. 2002;51:9–21. 16. Newman R, Barber AM, Roberts JM, Holtz T, Steketee RW, Parise ME. Malaria surveillance—United States, 1999. Morb Mortal Wkly Rep Surveill Summ. 2002;51:15–28. 17. Holtz TH, Kachur SP, MacArthur JR, Roberts JM, Barber AM, Steketee RW. Malaria surveillance—United States, 1998. Morb Mortal Wkly Rep Surveill Summ. 2001;50;1–18. 18. MacArthur JR, Levin AR, Mungai M, Roberts J, Barber AM, Bloland PB. Malaria surveillance—United States, 1997. Morb Mortal Wkly Rep Surveill Summ. 2001;50:25–44. 19. Mungai M, Roberts J, Barber AM, Williams HA, Ruebush TK, Chang M-H. Malaria surveillance—United States, 1996. Morb Mortal Wkly Rep Surveill Summ. 2001;50:1–22. Dr. International Malaria Surveillance An obvious increase in P. vivax cases from India was seen in the United States, from 150 cases to 371 and down to 123, during 1995 to 1997. This increase paralleled the epidemic peak seen in Canada, pri- marily in Ontario and British Columbia. In India, an epi- demic of P. vivax malaria occurred during this same period (1995–1997) in the Punjabi states of Punjab and Haryana (Figure 7). With negligible changes in travel destination or immigration numbers to explain the 1995–1997 epidemic in Canada, the explanation is probably an increased P. vivax attack rate in Canadians traveling to the Punjab, where a P. vivax epidemic occurred and ended at the same time as the Canadian epidemic. Figure 7. Plasmodium vivax incidence relative to 1998 (6,11, Colette Colin [Ministère de la santé et des services sociaux, Québec], pers comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.). Figure 7. Plasmodium vivax incidence relative to 1998 (6,11, Colette Colin [Ministère de la santé et des services sociaux, Québec], pers comm.; Monica Naus [British Columbia Centre for Disease Control], pers. comm.). Canadian notifiable diseases surveillance data generat- ed by local, provincial, and federal sources provided evi- dence for the occurrence of two as-yet unreported malaria Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 1199 1199 PERSPECTIVES supported by the abrupt halt of both Canadian and Punjabi epidemics in the same year. 7. Last J, editor. Dictionary of epidemiology. 4th ed. Oxford: International Epidemiological Association; 2001. 8. Svenson JE, MacLean JD, Gyorkos TW, Keystone J. Imported malar- ia. Clinical presentation and examination of symptomatic travelers. Arch Intern Med. 1995;155:861–8. The surveillance process for notifiable diseases in Canada and in other countries where malaria is now an imported disease should be reviewed. Specific conditions, such as the frequency of analysis of surveillance data, need to be discussed and agreed on by collectors of these data at each level of government. Without a firm plan in place for analysis and dissemination of results, the validity, not to mention the utility of the entire surveillance system, is placed in jeopardy. One approach could be the American emerging infections programs, a link between public health, academic, and clinical communities (32). 9. Svenson JE, Gyorkos TW, MacLean JD. Diagnosis of malaria in the febrile traveler. Am J Trop Med Hyg. International Malaria Surveillance MacLean is professor of medicine and director of the McGill University Centre for Tropical Diseases. His research interests are parasitic disease outbreak investigation (trichinosis, Metorchis infections, and malaria) and the development of diag- nostic tests for the clinical parasitology laboratory. 20. Williams HA, Roberts J, Kachur SP, Barber AM, Barat LM, Bloland PB, et al. Malaria surveillance—United States, 1995. Morb Mortal Wkly Rep Surveill Summ. 1999;48:1–21. 21. Kachur SP, Reller ME, Barber AM, Barat LM, Koumans EHA, Parise ME. Malaria surveillance—United States, 1994. Morb Mortal Wkly Rep Surveill Summ. 1997;46:1–16. 22. Barat LM, Zucker JR, Barber AM, Parise ME, Paxton LA, Roberts J. Malaria surveillance—United States, 1993. Morb Mortal Wkly Rep Surveill Summ. 1997;46:28–46. Malaria Epidemics and Surveillance Systems in Canada 31. Danis M, Legros F, Thellier M, Caumes E. Données actuelles sur le paludisme en France métropolitaine. Med Trop (Mars). 2002;62:214–8. 27. World Tourist Organization. Madrid [database on the Internet]. [cited 2003 Aug 29]. Available from: http://www.world-tourism.org/frame- set/frame_statistics.html 32. Pinner RW, Rebmann CA, Schuchat A, Hughes JM. Disease surveil- lance and the academic, clinical and public health communities. Emerg Infect Dis. 2003;9:781–7. 28. Yechouron A, Nguyen C, MacLean JD, Keystone J. The changing pattern of imported malaria. Can Dis Wkly Rep. 1988:14;133–6. 29. Ndao M, Bandyayera E, Kokoskin E, Diemert D, Gyorkos T, MacLean JD, et al. Malaria epidemic in Quebec; diagnosis and response to imported malaria. CMAJ. In press. Address for correspondence: J. Dick MacLean, McGill University Centre for Tropical Diseases, Montreal General Hospital, 1650 Cedar Ave, Room D7-153, Montreal, Québec, Canada H3G 1A4; fax: 514-933-9385; email: dick.maclean@mcgill.ca Address for correspondence: J. Dick MacLean, McGill University Centre for Tropical Diseases, Montreal General Hospital, 1650 Cedar Ave, Room D7-153, Montreal, Québec, Canada H3G 1A4; fax: 514-933-9385; email: dick.maclean@mcgill.ca 30. Recommandations sanitaires pour les voyageurs 2003. Bulletin epi- demiologique hebdomadaire. 2003;26:117–24. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 1201 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 1201 References 1. MacLean JD, Ward B. The return of swamp fever: malaria in Canadians. CMAJ. 1999;160:211–2. 23. Zucker JR, Barber AM, Paxton LA, Schultz LJ, Lobel HO, Roberts JM, et al. Malaria surveillance—United States, 1992. Morb Mortal Wkly Rep Surveill Summ. 1995;44:1–15. 2. Carter A. Establishing goals, techniques and priorities for national communicable disease surveillance. Can J Infect Dis. 1991;2:37–40. 24. Centers for Disease Control and Prevention. Malaria surveillance annual summary 1991. Atlanta: U.S. Department of Health and Human Services; 1994. 3. Kain KC, MacPherson DW, Kelton T, Keystone JS, Mendelson J, MacLean JD. Malaria deaths in visitors to Canada and in Canadian travellers: a case series. CMAJ. 2001;164:654–9. 25. U.S. Census Bureau. IDB data access—display mode [database on the Internet]. 2003 Jul 17 [cited 2003 Sep 18]. Available from http://www.census.gov/ipc/www/idbprint.html 4. Slinger R, Giulivi A, Bodie-Collins M, Hindieh F, John RS, Sher G, et al. Transfusion-transmitted malaria in Canada. CMAJ. 2001;164:377–9. 26. Detailed mother tongue, sex, knowledge of official language and age groups for population, for Canada, provinces, territories, census met- ropolitan areas and census agglomerations, 1996 and 2001 Censuses—20% sample data. Ottawa: Statistics Canada; 2002. 2001 census of Canada. Cat. #97F0007XCB01009 and 97F0007XCB01009.B. 5. Statistics Canada. Causes of death 1990–1993 Catalogue #84-208; 1994–1995 Catalogue #84-208-XPB; 1996 Catalogue 84F0208; 1997–1999 Catalogue 84F0208XPB; 2000 Catalogue 84-208-XIE. 6. Notifiable diseases online. Ottawa, population and public health branch, Health Canada; 2000 [database on the Internet]. 2000 [cited 2003 May 28]. 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Characterization of the chloroplast genome of Gleditsia species and comparative analysis
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Characterization of the chloroplast genome of Gleditsia species and comparative analysis Feng Xiao 1, Yang Zhao 1*, Xiurong Wang 1 & Xueyan Jian 2 OPEN Feng Xiao 1, Yang Zhao 1*, Xiurong Wang 1 & Xueyan Jian 2 The genus Gleditsia has significant medicinal and economic value, but information about the chloroplast genomic characteristics of Gleditsia species has been limited. Using the Illumina sequencing, we assembled and annotated the whole chloroplast genomes of seven Gleditsia species (Gleditsia sinensis, Gleditsia japonica var. delavayi (G. delavayi), G. fera, G. japonica, G. microphylla, Fructus Gleditsiae Abnormalis (Zhū Yá Zào), G. microphylla mutant). The assembled genomes revealed that Gleditsia species have a typical circular tetrad structure, with genome sizes ranging from 162,746 to 170,907 bp. Comparative genomic analysis showed that most (65.8–75.8%) of the abundant simple sequence repeats in Gleditsia and Gymnocladus species were located in the large single copy region. The Gleditsia chloroplast genome prefer T/A-ending codons and avoid C/G-ending codons, positive selection was acting on the rpoA, rpl20, atpB, ndhA and ycf4 genes, most of the chloroplast genes of Gleditsia species underwent purifying selection. Expansion and contraction of the inverted repeat (IR)/single copy (SC) region showed similar patterns within the Gleditsia genus. Polymorphism analysis revealed that coding regions were more conserved than non-coding regions, and the IR region was more conserved than the SC region. Mutational hotspots were mostly found in intergenic regions such as “rps16-trnQ”, “trnT-trnL”, “ndhG-ndhI”, and "rpl32-trnL” in Gleditsia. Phylogenetic analysis showed that G. fera is most closely related to G. sinensis,G. japonica and G. delavayi are relatively closely related. Zhū Yá Zào can be considered a bud mutation of the G. sinensis. The albino phenotype of G. microphylla mutant is not caused by variations in the chloroplast genome, and that the occurrence of the albino phenotype may be due to mutations in chloroplast-related genes involved in splicing or localization functions. This study will help us enhance our exploration of the genetic evolution and geographical origins of the Gleditsia genus. The plants in the genus Gleditsia, mainly distributed in central and Southeast Asia and North and South America, they have been used as local and traditional medicines in many regions, particularly in ­China1. The genus rec- ognized 13 ­species2. There are 6 species and 2 varieties of Gleditsia plants in China, including Gleditsia sinensis, G. australis, G. fera, G. japonica, G. microphylla, Gleditsia japonica var. delavayi (G. delavayi), Gleditsia japonica var. velutina (G. velutina), and 1 species (G. triacanthos) which is ­introduced3,4. G. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2024) 14:4262 Plant materials, genomic DNA isolation and genome sequencing Plant materials, genomic DNA isolation and genome sequencing , g g q g We collected various species seed of Gleditsia in China, including G. sinensis (Luodian City, Guizhou Province) (E 106.7379, N 25.2454), G. fera (Ceheng City, Guizhou Province) (E 105.9642, N 24.9327), G. japonica (Guiyang City, Guizhou Province) (E 106.9382, N 26.6969), G. microphylla (Zhijin City, Guizhou Province) (E 106.0246, N 26.5328), G. japonica var. delavayi (G. delavayi) (Xinyi City, Guizhou Province) (E 104.9381, N 25.1755). In addition, there were no seeds of Zhū Yá Zào, we directly collected the leaves of the plant Zhū Yá Zào (Tongren City, Guizhou Province) (E 108.0349; N 27.9852) (Fig. S1). Gymnocladus chinensis (Gen.: Gymnocladus) and Gymnocladus dioicus (Gen.: Gymnocladus) were collected as out group. After bringing various seeds back to the laboratory, then placed in the plant greenhouse incubator for cultivation. In the process of raising seedlings in the greenhouse, a albino mutant plant of G. microphylla was obtained (labeled G. microphylla mutant), which was characterized by albino whole plant, obvious dwarfing, and natural death after 1–1.5 months of growth. Taking single plant leaves of above all species for DNA extraction. Total DNA of each sample was isolated according to the instructions of the DNA extraction kit (EasyPure® Plant Genomic DNA Kit, Beijing Quanshijin Biological Co., Ltd.). Nandrop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to determine the concentration and purity of the DNA. The DNA integrity was assessed by agarose gel electrophoresis. According to the Illumina standard protocol, total DNA was used to generate libraries after DNA extraction which were sequenced using the Illumina NovaSeq 6000 platform, and the sequencing read length was 150 bp. q g q p q g g p All methods were performed on Gleditsia plants that were cultivated for the purposes of the experiments, including the collection of plant material for all analysis, and all relevant institutional, national, and international guidelines and legislation were complied with. Comparative analysish The genome sequences were initially aligned using MAFFT (v7.310)22. The complete chloroplast genomes were compared by the mVISTA program (http://​genome.​lbl.​gov/​vista/​mvista/​submit.​shtml). DnaSP (DNA Sequences Polymorphism)23 was used to calculate the nucleotide diversity (Pi) of coding, non-coding regions and whole complete chloroplast genomes (all chloroplast genome sequences were adjusted to start with LSC, the step size was set to 200 bp with a window length of 600 bp). Analysis of codon usage bias and selective pressures in the evolution y g p Extracted the full-length coding sequences, with an ATG start codon, a stop codon (TGA/TAG/TAA). The nucleotide compositions at the third position (A3s, U3s, C3s and G3s), GC content at third codon positions (GC3s), codon adaptation index (CAI), Codon Bias Index (CBI), effective number of codon (ENC) were deter- mined with ­CodonW20. Relative synonymous codon usage (RSCU) was analyzed with bioPython. KaKs calculator ­program21 with the NG model to calculate the rates of nonsynonymous (Ka), synonymous (Ks), and their ratio (Ka/Ks). When Ks = 0, the value cannot be computed and was represented by *. When Ka = 0 and Ks = 0, the value was represented by NaN. G. sinensis was used as a reference. www.nature.com/scientificreports/ remain relatively independent of species. The use of DNA barcodes of the chloroplast genome will help identify varieties and ­resources12. In recent years, with the development of high-throughput sequencing technology, an increasing number of plant chloroplast genome DNA sequences have been obtained. However, in the Gleditsia genus, only the chloroplast genomes of G. sinensis and G. japonica have been reported so ­far13,14, this limits our understanding of the genetic evolution of Gleditsia. The development of genomic resources for Gleditsia can also assist in molecular breeding studies of this genus. The collected seven Gleditsia species (Gleditsia sinensis, G. delavayi, G. fera, G. japonica, G. microphylla, Zhū Yá Zào, G. microphylla mutant) and two Gymnocladus species (Gymnocladus chinensis, Gymnocladus dioicus) were sequencing, assembled and analyzed, complete chloroplast genomes were obtained. In this study, the seven species chloroplast genome of Gleditsia genus were sequenced, assembled and analyzed to study any common features or differences between species, which helps in the genetic breeding and molecular evolution of Gleditsia. Chloroplast genome assembly, annotation and sequence analysishh p g y, q y The raw data quality control was performed by fastp (v0.12.4)15. The clean paired-end reads were assembled with GetOrganelle (v1.7.6.1)16. The complete chloroplast genome was annotated in ­CPGAVAS217 (http://​www.​herba​ lgeno​mics.​org/​cpgav​as2/). The the transfer RNA (tRNA) genes were verified with tRNAscan-SE18. ­REPuter19 (https://​bibis​erv.​cebit​ec.​uni-​biele​feld.​de/​reput​er/) was used to find the sizes and locations of forward, reverse, pal- indromic, and complementary repeats. Simple sequence repeat (SSRs) were determined using a Perl script MISA (MIcroSAtellite identification tools), including mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, minimum numbers (thresholds) were 10, 5, 4, 3, 3, and 3, respectively. The chloroplast genome sequences were deposited in GenBank (Accession Numbers: OP722579–OP722582). Characterization of the chloroplast genome of Gleditsia species and comparative analysis Feng Xiao 1, Yang Zhao 1*, Xiurong Wang 1 & Xueyan Jian 2 OPEN sinensis (Fam.: Leguminosae; Gen.: Gleditsia), deciduous tree or shrub-like, contains both diploids and having 2n = 28 ­chromosomes5. G. sin- ensis is widely distributed in China and is resistant to drought, cold, pollution, and has strong stress resistance, it integrates medicinal, edible, chemical, material, and ornamental ­purposes6. Fructus Gleditsiae Abnormalis (Zhū Yá Zào), it is the dried and sterile fruit of G. sinensis, there are significant differences in the morphology, structure and composition of G. sinensis and Zhū Yá Zào7, Li et al.8 suggested that it should be a variant of G. sinensis. However, the way in which Zhū Yá Zào came into being is still unknown. y g Identifying the genetic evolutionary relationship between Gleditsia is the key to distinguish varieties. Chlo- roplast gene sequences (ndhF and rpl16) are selected to test biogeographic hypotheses, there is a fundamental division of the genus Gleditsia into three ­clades9. Based on the ITS sequence, ­Schnabel10 conducts a systematic evolutionary study on the 11 species of Gleditsia and the results shows that the Gleditsia and Gymnocladus appear to have originated in eastern Asia during the Eocene. ­Xing11 selected three fragments of chloroplast DNA, MatK, PsbA-trnH, and TrnL-F to establish a phylogenetic tree of Gleditsia, the results shows that the Gymnocladus has a longer evolutionary time than the Gleditsia. The complete chloroplast genome contains a large amount of genetic information and is highly conserved. The self-replication and evolution of its genome 1Institute for Forest Resources and Environment of Guizhou, Key Laboratory of Forest Cultivation in Plateau Mountain of Guizhou Province, College of Forestry, Guizhou University, Guiyang 550025, Guizhou, China. 2College of Continuing Education, Yanbian University, Yanji 133002, Jilin, China. *email: zhy737@126.com | https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 www.nature.com/scientificreports/ Codon bias analysis and selective pressures in the evolutionh y p The GC and GC3s content in the codons of the 9 chloroplast genomes studied were both less than 0.5, indicat- ing a preference for A/T bases and A/T-ending codons in Gleditsia and Gymnocladus chloroplast genomes. We used the CDS of the chloroplast genome to estimate the codon usage frequency of the seven taxa of Gleditsia. All 20 amino acids were encoded by codons in the Gleditsia chloroplast genome and the synonymous codon usage (RSCU value) values were similar. Of the 29 codons with an RSCU value > 1, only one ended with G (TTG). The codons with an RSCU value < 1, except for ATA and CTA ending in A, ended in C or G. Codon pairs ending with C and G in the Gleditsia chloroplast genome had low bias and were non-preferred codons. We analyzed the ka/ks ratio of the 76 unique protein-coding genes in the 9 chloroplast genome (Fig. 3), using G. sinensis as the reference, five genes (rpoA, rpl20, atpB, ndhA, ycf4) were identified under positive selection (Ka/Ks > 1). Ka/Ks ratio of most gene was less than 1. 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Assembly and annotation of the Gleditsia chloroplast genomeh Assembly and annotation of the Gleditsia chloroplast genomeh The seven chloroplast genome of Gleditsia genus and two species from Gymnocladus genus both showed the typical quadripartite structure of angiosperms, consisting of a large single copy (LSC) region (91,203–91,436 bp) and a small single copy (SSC) region (18,845–19,561 bp), which separated by two inverted repeat (IRA and IRB) regions (26,122–26,619 bp); The chloroplast genome sizes of Gleditsia species ranged from 162,746 bp (G. sinensis) to 170,796 (G. delavayi) (Table S1). The GC content of genomes ranged from 33.9 to 35.64%. G. sinensis encoded 84 protein-coding genes, 8 rRNA, 37 tRNA, of which three genes (ycf1, ycf3, clpP) have two introns. G. fera, G. delavayi, Zhū Yá Zào, and G. microphylla encoded 85 protein-coding genes, 8 tRNA genes, and 37 tRNA genes, respectively. G. microphylla mutant encoded 84 protein-coding genes, 8 tRNA genes and 37 tRNA genes. Analysis of the chloroplast genome structure of Gleditsia y p g Using GCview to visualize the sequence alignment between multiple chloroplast genomes, it was found that the sequence between different species of Gleditsia were similar. Through SSR identification, in Gleditsia plas- tomes, the total number of SSRs ranges from 85 to 133 SSRs, while in the Gymnocladus it varied from 109 to 125. Moreover, most (65.8–75.8%) of the SSRs in Gleditsia and Gymnocladus species were located in the LSC. In Gleditsia, the IR regions include between 2.2 and 5.5% SSRs loci, while the SSC region included between 17.9 and 23.3% (Fig. 1a). In the Gymnocladus sequenced here, 69.9–75.3% of the SSRs were situated in the LSC. A total of 89 SSR sites were detected in G. sinensis, including 85 mononucleotide and 4 dinucleotide repeat units. The most abundant repeats were mononucleotide repeats in the Gleditsia genus (Fig. 1b). There were 50 repeats in G. sinensis (Fig. 1c), which included complementary, forward, palindromic, reverse repeats.h The chloroplast genome sequence in Gleditsia was analyzed using the chloroplast genome of G. sinensis as a reference with mVISTA. It was found that the chloroplast genome sequences within the genus Gleditsia were highly similar and conserved, with the coding region being more conserved than the non-coding region, and the IR region being more conserved than the SC region. The IR/SC junctions of the chloroplast genome within Gleditsia showed similar features (Fig. 2). The lengths of the IR regions in the Gleditsia chloroplast genome ranged from 26,122 to 26,619 bp. The rps3 gene was present in the LSC region in G. sinensis, Zhū Yá Zào, G. fera, G. japonica, G. delavayi, G. microphylla, and G. microphylla mut., and all IRs contained a gene rps19, ranging from 61 to 221 bp from the JLB (junction between LSC and IRb) junction. In the sequenced Gleditsia species, the ndhF gene was completely present in the SSC and away from the junction, and the trnH gene was entirely located in the LSC region. These data suggest that the expansion and contraction of the IR/SC region exhibit similar patterns within Gleditsia. Phylogenetic analysis y g y All chloroplast genome sequences were aligned by MAFFT software. The shared CDS genes were extracted by python and aligned using MAFFT. Phylogenetic analysis and tree models was conducted by using the IQTREE software (v2.0.3)24. The phylogenetic tree was visualized using ggtree R package (v3.2.1)25. Scientific Reports | (2024) 14:4262 | https://doi.org/10.1038/s41598-024-54608-6 www.nature.com/scientificreports/ Codon bias analysis and selective pressures in the evolutionh sinensis Zhuyazao G. microphylla mut. G. microphylla G.japonica Gy. dioicus G. fera G. chinensis G. delavayi type IRa SSC IRb LSC shape IRa IRb LSC SSC (a) 10 94 5 14 101 10 5 79 1 5 104 4 97 2 27 101 5 27 101 5 5 84 5 86 0 50 100 G. sinensis zhuyazao G. delavayi G. fera G. japonica G. microphylla G. microphylla mut. Gy. dioicus Gy. chinensi Number of SSRs type di mono tri (b) 39 6 5 2 29 8 11 44 6 2 17 17 14 35 12 3 29 6 15 29 6 15 2 26 15 7 1 31 14 4 0 10 20 30 40 50 G. sinensis zhuyazao G. delavayi G. fera G. japonica G. microphylla G. microphylla mut. Gy. dioicus Gy. chinensi Type of repeat elements type C D F P R (c) Figure 1. Analysis of SSR sites and repetitive sequences in 9 chloroplast genomes. (a): Distribution of SSRs in the Gleditsia and two plastomes from Gymnocladus; (b): Number of different SSRs loci types; (c): Number of different repeats types. Note In a, different shapes represented the position of SSR, and the proportion of text displayed; In (c), F: forward repeats, P: palindromic repeats, R: reverse repeats, C: complementary repeats. 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S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 2.3 (:b R I ) % 2.3 (:b R I ) % 2.3 (:b R I ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 2.3 (:a R I ) % 2.3 (:a R I ) % 2.3 (:a R I ) % 9.7 1 (: C S S ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 2.3 (:b R I ) % 2.3 (:b R I ) % 2.3 (:b R I ) % 8.5 7 (: C S L ) % 8.5 7 (: C S L ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 9.7 1 (: C S S ) % 2.3 (:a R I ) % 2.3 (:a R I ) % 2.3 (:a R I ) % 9.7 1 (: C S S ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 3.3 (:b R I ) % 3.3 (:b R I ) % 3.3 (:b R I ) % 3.3 7 (: C S L ) % 3.3 7 (: C S L ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 0 2 (: C S S ) % 3.3 (:a R I ) % 3.3 (:a R I ) % 3.3 (:a R I ) % 0 2 (: C S S ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 3.5 7 (: C S L ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.2 (:b R I ) % 2.2 (:b R I ) % 3.5 7 (: C S L ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.0 2 (: C S S ) % 2.2 (:a R I ) % 2.2 (:a R I ) % 2.0 2 (: C S S ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 1.7 6 (: C S L ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 5.5 (:b R I ) % 5.5 (:b R I ) % 5.5 (:b R I ) % 5.5 (:b R I ) % 1.7 6 (: C S L ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 5.5 (:a R I ) % 5.5 (:a R I ) % 9.1 2 (: C S S ) % 9.1 2 (: C S S ) % 5.5 (:a R I ) % 5.5 (:a R I ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 9.9 6 (: C S L ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 2.3 (:b R I ) % 2.3 (:b R I ) % 2.3 (:b R I ) % 9.9 6 (: C S L ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S ) % 2.3 (:a R I ) % 2.3 (:a R I ) % 7.3 2 (: C S S ) % 7.3 2 (: C S S IRa:(3.2%) ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 2.2 (:b R I ) % 2.2 (:b R I ) % 7.4 7 (: C S L ) % 7.4 7 (: C S L ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 9.0 2 (: C S S ) % 2.2 (:a R I ) % 2.2 (:a R I ) % 9.0 2 (: C S S G. Codon bias analysis and selective pressures in the evolutionh sinensis Zhuyazao G. microphylla mut. G. microphylla G.japonica Gy. dioicus G. fera G. chinensis G. delavayi type IRa SSC IRb LSC shape IRa IRb LSC SSC (a) 10 94 5 14 101 10 5 79 1 5 104 4 97 2 27 101 5 27 101 5 5 84 5 86 0 50 100 inensis huyazao delavayi G. fera aponica ophylla lla mut. dioicus hinensi Number of SSRs type di mono tri (b) 39 6 5 2 29 8 11 44 6 2 17 17 14 35 12 3 29 6 15 29 6 15 2 26 15 7 1 31 14 4 0 10 20 30 40 50 inensis huyazao delavayi G. fera aponica ophylla lla mut. dioicus hinensi Type of repeat elements typ (c) Figure 1. Analysis of SSR sites and repetitive sequences in 9 chloroplast genomes. (a): Distribution of SSRs in the Gleditsia and two plastomes from Gymnocladus; (b): Number of different SSRs loci types; (c): Number of different repeats types. Note In a, different shapes represented the position of SSR, and the proportion of text displayed; In (c), F: forward repeats, P: palindromic repeats, R: reverse repeats, C: complementary repeats. Codon bias analysis and selective pressures in the evolutionh https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ Gleditsia delavayi rps3 657 bp 574 bp rps19 279 bp 66 bp ycf1 432 bp 39 bp ndhF 2241 bp 93 bp ycf1 5028 bp 432 bp trnN 72 bp 782 bp rps19 279 bp 66 bp trnH 75 bp 14 bp 170796 bp LSC IRb SSC IRa LSC 98889 bp 26173 bp 19561 bp 26173 bp JLB JSB JSA JLA Gymnocladus chinensis rps19 182 bp 97 bp rpl2 1490 bp 184 bp ycf1 807 bp 54 bp ndhF 2238 bp 754 bp ycf1 4677 bp 807 bp trnN 72 bp 1162 bp rpl2 1490 bp 184 bp trnH 75 bp 7 bp 165299 bp LSC IRb SSC IRa LSC 92340 bp 26255 bp 20449 bp 26255 bp JLB JSB JSA JLA Gleditsia fera rps3 657 bp 560 bp rps19 279 bp 69 bp ycf1 432 bp 33 bp ndhF 2241 bp 70 bp ycf1 5028 bp 432 bp trnN 72 bp 782 bp rps19 279 bp 69 bp trnH 75 bp 49 bp 165027 bp LSC IRb SSC IRa LSC 93345 bp 26154 bp 19374 bp 26154 bp JLB JSB JSA JLA Gleditsia triacanthos rps19 182 bp 97 bp rpl2 1490 bp 159 bp ycf1 800 bp 37 bp ndhF 2232 bp 161 bp ycf1 4684 bp 800 bp trnN 72 bp 1128 bp rpl2 1490 bp 159 bp trnH 75 bp 7 bp 157472 bp LSC IRb SSC IRa LSC 87239 bp 25882 bp 18469 bp 25882 bp JLB JSB JSA JLA Gleditsia japonica rps3 657 bp 564 bp rps19 279 bp 86 bp ycf1 432 bp 54 bp ndhF 2241 bp 70 bp ycf1 5028 bp 432 bp trnN 72 bp 782 bp rps19 279 bp 86 bp trnH 75 bp 13 bp 166112 bp LSC IRb SSC IRa LSC 94420 bp 26122 bp 19448 bp 26122 bp JLB JSB JSA JLA Gleditsia microphylla rps3 657 bp 541 bp rps19 279 bp 221 bp ycf1 756 bp 6 bp ndhF 2241 bp 7 bp ycf1 4704 bp 756 bp trnN 72 bp 1106 bp rps19 279 bp 221 bp trnH 75 bp 134 bp 170713 bp LSC IRb SSC IRa LSC 98595 bp 26619 bp 18880 bp 26619 bp JLB JSB JSA JLA Gleditsia microphylla mutant rps3 657 bp 541 bp rps19 279 bp 221 bp ycf1 756 bp 6 bp ndhF 2241 bp 7 bp ycf1 4704 bp 756 bp trnN 72 bp 1106 bp rps19 279 bp 221 bp trnH 75 bp 134 bp 170713 bp LSC IRb SSC IRa LSC 98595 bp 26619 bp 18880 bp 26619 bp JLB JSB JSA JLA Gleditsia sinensis rps3 657 bp 559 bp rps19 279 bp 77 bp trnN 72 bp 782 bp ndhF 2241 bp 51 bp ycf1 4830 bp 219 bp trnN 72 bp 782 bp rps19 279 bp 77 bp trnH 75 bp 10 bp 162746 bp LSC IRb SSC IRa LSC 91203 bp 26136 bp 19271 bp 26136 bp JLB JSB JSA JLA Zhu Ya Zao rps3 657 bp 580 bp rps19 279 bp 61 bp ycf1 432 bp 33 bp ndhF 2241 bp 51 bp ycf1 5028 bp 432 bp trnN 72 bp 782 bp rps19 279 bp 61 bp trnH 75 bp 46 bp 162995 bp LSC IRb SSC IRa LSC 91436 bp 26144 bp 19271 bp 26144 bp JLB JSB JSA JLA Figure 2. Nucleic acid polymorphism analysis Nucleic acid polymorphism analysis We conducted an analysis of Pi values to measure the divergence level in protein-coding genes (Fig. 4a), inter- genic regions (Fig. 4b) and whole chloroplast genome sequences (Fig. 4c) of the 7 Gleditsia species. Taking the common protein coding sequence of Gleditsia as the research object, ycf1 and petL were found as mutational hotspots. Through gene sequence alignment and polymorphism analysis using the chloroplast genome of Gledit- sia as a reference, we found that mutational hotspots occurred in the intergenic regions such as "rps16-trnQ", "trnT-trnL", "ndhG-ndhI", "rpl32-trnL", etc. The IR region was conserved relative to the SC region. Codon bias analysis and selective pressures in the evolutionh Comparison of the border regions of LSC, IR and SSC. Note JLA: junction between LSC and IRa; JLB: junction between LSC and IRb; JSA: junction between SSC and IRa; JSB: junction between SSC and IRb. The numbers above the gene features indicated the distance from the end of the gene to the boundary sites; these features were not scaled. Gymnocladus chinensis 165299 bp Figure 2. Comparison of the border regions of LSC, IR and SSC. Note JLA: junction between LSC and IRa; JLB: junction between LSC and IRb; JSA: junction between SSC and IRa; JSB: junction between SSC and IRb. The numbers above the gene features indicated the distance from the end of the gene to the boundary sites; these features were not scaled. Phylogenetic analysis https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ 0 2 4 6 (a) GCT GCC GCA GCG CGT CGC CGA CGG AGA AGG AAT AAC GAT GAC TGT TGC CAA CAG GAA GAG GGT GGC GGA GGG CAT CAC ATT ATC ATA TTA TTG CTT CTC CTA CTG AAA AAG ATG TTT TTC CCT CCC CCA CCG TCT TCC TCA TCG AGT AGC ACT ACC ACA ACG TGG TAT TAC GTT GTC GTA GTG Ala Arg Asn Asp Cys Gln Glu Gly HIS Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val GCA GCC GCG GCT AGA AGG CGA CGC CGG CGT AAC AAT GAC GAT TGC TGT CAA CAG GAA GAG GGA GGC GGG GGT CAC CAT ATA ATC ATT CTA CTC CTG CTT TTA TTG AAA AAG ATG TTC TTT CCA CCC CCG CCT AGC AGT TCA TCC TCG TCT ACA ACC ACG ACT TGG TAC TAT GTA GTC GTG GTT 0.5 1.0 1.5 Ala Arg Asn Asp Cys Gln Glu Gly HIS Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val RSCU (b) 0.0 0.5 1.0 1.5 2.0 accD atpA atpB atpE atpF atpH atpI ccsA cemA clpP matK ndhA ndhB ndhC ndhD ndhE ndhF ndhG ndhH ndhI ndhJ ndhK petA petB petD petG petL petN psaA psaB psaC psaI psaJ psbA psbB psbC psbD psbE ka/ks 0.0 0.5 1.0 1.5 2.0 psbF psbH psbI psbJ psbK psbM psbN psbT psbZ rbcL rpl14 rpl16 rpl2 rpl20 rpl23 rpl32 rpl33 rpl36 rpoA rpoB rpoC1 rpoC2 rps11 rps12 rps14 rps15 rps16 rps18 rps19 rps2 rps3 rps4 rps7 rps8 ycf1 ycf2 ycf3 ycf4 ka/ks group G. sinensis - Fructus Gleditsiae Abnormalis G. sinensis - G. delavayi G. sinensis - G. fera G. sinensis - G. japonica G. sinensis - G. microphylla G. sinensis - G. microphylla tubianti G. sinensis - Gy. chinensis G. sinensis - Gy. dioicus (c) Figure 3. Codon bias analysis and selective pressures in the evolution. (a): Codon content of 20 amino acids and stop codons in all protein-coding genes of Gleditsia chloroplast genome; (b): Distribution of codon preference in Gleditsia; (c): Ka/Ks values of protein-coding genes of the seven comparative combinations. Note In the a, the top panel shows the RSCU for the corresponding amino acids, the colored block which are shown in the below represent different codons; In (c), Ka: nonsynonymous; Ks: synonymous. Phylogenetic analysis 0 2 4 6 (a) GCT GCC GCA GCG CGT CGC CGA CGG AGA AGG AAT AAC GAT GAC TGT TGC CAA CAG GAA GAG GGT GGC GGA GGG CAT CAC ATT ATC ATA TTA TTG CTT CTC CTA CTG AAA AAG ATG TTT TTC CCT CCC CCA CCG TCT TCC TCA TCG AGT AGC ACT ACC ACA ACG TGG TAT TAC GTT GTC GTA GTG Ala Arg Asn Asp Cys Gln Glu Gly HIS Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val GCA GCC GCG GCT AGA AGG CGA CGC CGG CGT AAC AAT GAC GAT TGC TGT CAA CAG GAA GAG GGA GGC GGG GGT CAC CAT ATA ATC ATT CTA CTC CTG CTT TTA TTG AAA AAG ATG TTC TTT CCA CCC CCG CCT AGC AGT TCA TCC TCG TCT ACA ACC ACG ACT TGG TAC TAT GTA GTC GTG GTT 0.5 1.0 1.5 Ala Arg Asn Asp Cys Gln Glu Gly HIS Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val RSCU (b) 0.0 0.5 1.0 1.5 2.0 accD atpA atpB atpE atpF atpH atpI ccsA cemA clpP matK ndhA ndhB ndhC ndhD ndhE ndhF ndhG ndhH ndhI ndhJ ndhK petA petB petD petG petL petN psaA psaB psaC psaI psaJ psbA psbB psbC psbD psbE ka/ks 0.0 0.5 1.0 1.5 2.0 psbF psbH psbI psbJ psbK psbM psbN psbT psbZ rbcL rpl14 rpl16 rpl2 rpl20 rpl23 rpl32 rpl33 rpl36 rpoA rpoB rpoC1 rpoC2 rps11 rps12 rps14 rps15 rps16 rps18 rps19 rps2 rps3 rps4 rps7 rps8 ycf1 ycf2 ycf3 ycf4 ka/ks group G. sinensis - Fructus Gleditsiae Abnormalis G. sinensis - G. delavayi G. sinensis - G. fera G. sinensis - G. japonica G. sinensis - G. microphylla G. sinensis - G. microphylla tubianti G. sinensis - Gy. chinensis G. sinensis - Gy. dioicus (c) Figure 3. Codon bias analysis and selective pressures in the evolution. (a): Codon content of 20 amino acids and stop codons in all protein-coding genes of Gleditsia chloroplast genome; (b): Distribution of codon preference in Gleditsia; (c): Ka/Ks values of protein-coding genes of the seven comparative combinations. Note In the a, the top panel shows the RSCU for the corresponding amino acids, the colored block which are shown in the below represent different codons; In (c), Ka: nonsynonymous; Ks: synonymous. 0 2 4 6 (a) RSCU G. sinensis - G. fera G. sinensis - G. Phylogenetic analysis japonica Figure 3. Codon bias analysis and selective pressures in the evolution. (a): Codon content of 20 amino acids and stop codons in all protein-coding genes of Gleditsia chloroplast genome; (b): Distribution of codon preference in Gleditsia; (c): Ka/Ks values of protein-coding genes of the seven comparative combinations. Note In the a, the top panel shows the RSCU for the corresponding amino acids, the colored block which are shown in the below represent different codons; In (c), Ka: nonsynonymous; Ks: synonymous. Phylogenetic analysis Fourteen chloroplast genome sequences were used for constructing the systematic evolutionary tree, nine of which were provided by this study and five were provided by other studies, the accession numbers can be found at the end of each branch. Based on the full-length chloroplast genome and shared CDS sequences, the optimal model TVM + F + I was calculated by IQTREE according to BIC. Phylogenetic analysis was conducted using the maximum likelihood (ML) based on the full-length chloroplast genome and shared CDS sequences, and the resulting trees (Fig. 5) showed the two datasets produced similar phylogenetic trees with high support and only differed for some nodes’ supporting values, the topologies of the ML based on the full-length chloroplast genome and ML based on the shared CDS sequence were essentially consistent. G. sinensis and Zhū Yá Zào clustered into a subclade. G.fera was most closely related to G. sinensis. Two G. microphylla (OP722576.1, NC_047369.1) and G. microphylla mutant formed a single branch. Discussionh australis seed implantation site is obviously swollen, few fruitless ­necks40. G. velutina is endemic to Hunan Province, China, and is a rare and endangered plant under national key ­protection41. Stress-related genes had been positively selected during the evolution through comparative transcriptome analysis of Gleditsia ­genus42. In this study, positive selection was acting on five genes (rpoA, rpl20, atpB, ndhA, ycf4), which were identified under positive selection (Ka/Ks > 1), Ka/Ks ratio of most gene were less than 1, pairwise Ka/Ks analysis showed that most of the chloroplast genes of Gleditsia species underwent purifying selection, the purifying selection on most chloroplast genes within Gleditsia would be evolutionary result of the preservation of the adaptive characteristics of Gleditsia species.h the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong ­purification37. The average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec- tion ­pressures38. Purifying selection constantly sweeps away deleterious mutations in population, the purifying selection on most chloroplast genes within Chrysosplenium would be evolutionary result of the preservation of the adaptive characteristics of Chrysosplenium ­species37. G. microphylla is used currently for food, health care products, and cosmetics, as well as for the treatment of various cancers and heart, vascular, and infec- tious ­diseases39. G. japonica pod flat, irregularly twisted; G. delavayi distributed only in Yunnan and Guizhou Province, China; G. fera distributed gentle slopes, mountain valleys, forests, beside villages, near roads, sunny places, occasionally cultivated, among the species studied, G. fera can be divided into fast-growing ­genotype5; G. australis seed implantation site is obviously swollen, few fruitless ­necks40. G. velutina is endemic to Hunan Province, China, and is a rare and endangered plant under national key ­protection41. Stress-related genes had been positively selected during the evolution through comparative transcriptome analysis of Gleditsia ­genus42. Discussionh The chloroplast genome generally ranges in size from 120 to 160 kb and exhibits a highly conserved ­structure26. The sequencing, assembly, and analysis of chloroplast genomes can identify common features or differences between species, which can be used as DNA barcodes. Seven Gleditsia species and two Gymnocladus species both have a circular tetrad structure, consisting of one LSC and SSC region, separated by two IR inverted repeat regions, the size of the Gleditsia chloroplast genome ranged from 162,746 to 170,907 bp. Most of the SSRs were located in the intergenic ­areas27. Based on SSR identification and examination of their location on the chloroplast genome, it was found that Mononucleotide repeats were the most abundant SSR type in the Gleditsia genus. The majority of SSRs (65.8–75.8%) in Gleditsia and Gymnocladus species were located in the LSC region, which is consistent with previous reports on chloroplast SSRs in other ­plants28,29.fi Codon usage of highly expressed genes was selected in evolution to maintain the efficiency of global protein ­translation30. The RSCU values of the CDSs of Gleditsia in the present study were similar, the RSCU values of tryptophan and methionine amino acids were 1, they were the only amino acids with no codon bias. There were 29 codons with an RSCU value > 1, only one of which ended with G (TTG); The codons with an RSCU value < 1, except for ATA and CTA ending in A, ended in C or G, the codon pairs ending with C and G in the Gleditsia chloroplast genome had low bias, and they were nonpreferred codons. The codons with an RSCU value > 1 were prefer A/T-ending codons in Gleditsia genus (Fig. 3b). Six Euphorbiaceae plant ­species31 and seven Miscanthus ­species32 were biased towards A/T bases and A/T-ending codons. Quercus chloroplast genomes prefer A/T-ending codons and avoid C/G-ending ­codons33. Additionally, the Ka/Ks revealed selection pressure on protein-coding ­genes34, Ka/Ks ratios > 1, close to 1, or < 1 indicate that the gene has undergone positive selection, neutral selec- tion, or purifying selection, ­respectively35. The Ka/Ks ratios for the majority (74 of 79) genes were below 1 for https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong ­purification37. Discussionh The average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec- ycf1 petL psbM psbT rps15 ndhF psbK ccsA ycf4 ndhH 0.000 0.003 0.006 0.009 Nucleotide diversity (a) accD_-_--psaI_+_ rpl36_-_--rps11_+_ psbK_+_--rps16__exon1_+_ rpl2__exon2_-_--rps19_+_ psbZ_-_--rps14_-_ ndhG_+_--ndhI_-_ rpl14_-_--rps8_+_ rpl33_-_--rps18_+_ atpF__exon1_+_--atpH_-_ ndhD_+_--psaC_-_ 0.00 0.02 0.04 0.06 0.08 Nucleotide diversity (b) rps16-trnQ trnT-trnL psbZ-trnG rps4-ndhj 0.00 0.05 0.10 0 6500 13000 19500 26000 32500 39000 45500 52000 58500 65000 71500 78000 84500 91000 97500 104000 110500 117000 123500 130000 136500 143000 149500 156000 162500 Nucleotide diversity (Pi) LSC IRb SSC IRa (c) Figure 4. Nucleotide diversity of chloroplast genomes in Gleditsia. (a): Pi in CDS; (b): Pi in intergenic regions; (c): chloroplast genome Pi values. Note G. sinensis was used as a reference genome for comparison, window length: 300 bp, step length: 200 bp; X axis: position of the midpoint of each window; Y axis: Pi of each window. ycf1 petL psbM psbT rps15 ndhF psbK ccsA ycf4 ndhH 0.000 0.003 0.006 0.009 Nucleotide diversity (a) accD_-_--psaI_+_ rpl36_-_--rps11_+_ psbK_+_--rps16__exon1_+_ rpl2__exon2_-_--rps19_+_ psbZ_-_--rps14_-_ ndhG_+_--ndhI_-_ rpl14_-_--rps8_+_ rpl33_-_--rps18_+_ atpF__exon1_+_--atpH_-_ ndhD_+_--psaC_-_ 0.00 0.02 0.04 0.06 0.08 Nucleotide diversity (b) ycf1 petL psbM psbT rps15 ndhF psbK ccsA ycf4 ndhH 0.000 0.003 0.006 0.009 Nucleotide diversity (a) accD_-_--psaI_+_ rpl36_-_--rps11_+_ psbK_+_--rps16__exon1_+_ rpl2__exon2_-_--rps19_+_ psbZ_-_--rps14_-_ ndhG_+_--ndhI_-_ rpl14_-_--rps8_+_ rpl33_-_--rps18_+_ atpF__exon1_+_--atpH_-_ ndhD_+_--psaC_-_ 0.00 0.02 0.04 0.06 0.08 Nucleotide diversity (b) rps16-trnQ trnT-trnL psbZ-trnG rps4-ndhj 0.00 0.05 0.10 0 6500 13000 19500 26000 32500 39000 45500 52000 58500 65000 71500 78000 84500 91000 97500 104000 110500 117000 123500 130000 136500 143000 149500 156000 162500 Nucleotide diversity (Pi) LSC IRb SSC IRa (c) (c) Nucleotide diversity (Pi) Figure 4. Nucleotide diversity of chloroplast genomes in Gleditsia. (a): Pi in CDS; (b): Pi in intergenic regions; (c): chloroplast genome Pi values. Note G. sinensis was used as a reference genome for comparison, window length: 300 bp, step length: 200 bp; X axis: position of the midpoint of each window; Y axis: Pi of each window. the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong ­purification37. The average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec- tion ­pressures38. Discussionh Purifying selection constantly sweeps away deleterious mutations in population, the purifying selection on most chloroplast genes within Chrysosplenium would be evolutionary result of the preservation of the adaptive characteristics of Chrysosplenium ­species37. G. microphylla is used currently for food, health care products, and cosmetics, as well as for the treatment of various cancers and heart, vascular, and infec- tious ­diseases39. G. japonica pod flat, irregularly twisted; G. delavayi distributed only in Yunnan and Guizhou Province, China; G. fera distributed gentle slopes, mountain valleys, forests, beside villages, near roads, sunny places, occasionally cultivated, among the species studied, G. fera can be divided into fast-growing ­genotype5; G. australis seed implantation site is obviously swollen, few fruitless ­necks40. G. velutina is endemic to Hunan Province, China, and is a rare and endangered plant under national key ­protection41. Stress-related genes had been positively selected during the evolution through comparative transcriptome analysis of Gleditsia ­genus42. In this study, positive selection was acting on five genes (rpoA, rpl20, atpB, ndhA, ycf4), which were identified under positive selection (Ka/Ks > 1), Ka/Ks ratio of most gene were less than 1, pairwise Ka/Ks analysis showed that most of the chloroplast genes of Gleditsia species underwent purifying selection, the purifying selection on most chloroplast genes within Gleditsia would be evolutionary result of the preservation of the adaptive characteristics of Gleditsia species. d h d b 43 Th h hl bl the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong ­purification37. The average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec- tion ­pressures38. Purifying selection constantly sweeps away deleterious mutations in population, the purifying selection on most chloroplast genes within Chrysosplenium would be evolutionary result of the preservation of the adaptive characteristics of Chrysosplenium ­species37. G. microphylla is used currently for food, health care products, and cosmetics, as well as for the treatment of various cancers and heart, vascular, and infec- tious ­diseases39. G. japonica pod flat, irregularly twisted; G. delavayi distributed only in Yunnan and Guizhou Province, China; G. fera distributed gentle slopes, mountain valleys, forests, beside villages, near roads, sunny places, occasionally cultivated, among the species studied, G. fera can be divided into fast-growing ­genotype5; G. Discussionh DNA barcodes derived from chloroplast genomes will be useful for identifying varieties and ­resources12. DNA barcodes with the largest nucleotide diversity are considered to be the focus of phylogenetic analysis and plant ­identification47. Chloroplast gene sequences (ndhF and rpl16) are selected to test biogeographic hypotheses, there is a fundamental division of the genus Gleditsia into three ­clades9. According to sliding win- dow analysis, rps16-trnQ, rpl32-trnL, ndhD-psaC and ycf1 showed the greatest variations in Ilex48. The several non-coding sites (psbI–atpA, atpH–atpI, rpoB–petN, psbM–psbD, ndhf–rpl32, and ndhG–ndhI) and three genes (ycf1, ycf2, and accD) showed significant ­variation49. Positive selection is observed in 14 protein coding genes (accD, ccsA, ndhA, ndhB, psbJ, rbcL, rpl20, rpoC1, rpoC2, rps12, rps18, ycf1, ycf2 and ycf4) in nine species of subfamily ­Zingiberoideae50. Ka/Ks values of three genes petL, rpl20, and ycf4 were higher than one in the pair- wise comparation of Galegeae officinalis and other three Galegeae ­species51. Mutational hotspots of shared genes and intergenic spacers of the chloroplast genomes of the Gleditsia species were identified. Taking the common protein coding sequence of Gleditsia as the research object, ycf1 and petL were found as mutational hotspots. ycf1 encodes unknown function proteins. The petL gene encodes the 3.5 kDa subunit of cytochrome b6/f ­complex52. In other studies, two regions of the plastid gene ycf1, ycf1a and ycf1b, were the most variable loci and of 420 tree species, 357 species could be distinguished using ycf1b53. The polymorphism of chloroplast genome is useful for evolutionary analysis of Gleditsia. Mutational hotspots in Gleditsia were found in the intergenic regions such as "rps16-trnQ", "trnT-trnL", "ndhG-ndhI", and "rpl32-trnL". trnK-rps16 (exon2-intron), trnT-trnL and ycf1 are also reported in Allium54. These hypervariable regions as potential DNA barcode regions for Gleditsia. ph yp g p g A genetic distance analysis based on the ISSR genetic diversity revealed that G. japonica and G. delavayi had a closer genetic ­relationship55. By using the complete chloroplast genomes and shared CDS genes, phylogenetic analysis was performed. The results showed that the two datasets produced similar phylogenetic trees, the rela- tionships of genus were consistent with high support and only differed for some nodes’ supporting values. Based on morphology and phylogenetic analysis, G. japonica and G. delavayi appear most closely related. Zhū Yá Zào is derived from the plant G. Discussionh In this study, positive selection was acting on five genes (rpoA, rpl20, atpB, ndhA, ycf4), which were identified under positive selection (Ka/Ks > 1), Ka/Ks ratio of most gene were less than 1, pairwise Ka/Ks analysis showed that most of the chloroplast genes of Gleditsia species underwent purifying selection, the purifying selection on most chloroplast genes within Gleditsia would be evolutionary result of the preservation of the adaptive characteristics of Gleditsia species.h p IR region can indicate the distance between species to a certain ­extent43. The highly variable regions can provide useful plastid markers for studies regarding the identification, phylogeny, and population ­genetics44. Using mVISTA to analyze chloroplast genome sequences within the genus Gleditsia, coding regions were more conserved than non-coding regions, and IR regions were more conserved than SC regions. Analysis of IR ampli- fication data indicates that expansion and contraction of IR/SC regions show similar patterns within the genus, which is also proved from the polymorphism analysis, in which the IR regions were conserved relative to the https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ Gleditsia delavayi(OP722574.1) Gleditsia fera(OP722575.1) Gleditsia velutina(OP764692.1) Gleditsia japonica(OP722577.1) Gleditsia sinensis(OP722579.1) Gleditsia sinensis zhuyazao(OP722580.1) Gleditsia sinensis(MT483984.1) Gleditsia sinensis(NC_047282.1) Gleditsia sinensis(MZ636527.1) Gleditsia microphylla(OP722576.1) Gleditsia microphylla var(OP722581.1) Gleditsia microphylla(NC_047369.1) Gymnocladus chinensis(OP722582.1) Gymnocladus dioicus(OP722578.1) (a) Gleditsia delavayi(OP722574.1) Gleditsia fera(OP722575.1) Gleditsia velutina(OP764692.1) Gleditsia japonica(OP722577.1) Gleditsia sinensis zhuyazao(OP722580.1) Gleditsia sinensis(MT483984.1) Gleditsia sinensis(NC_047282.1) Gleditsia sinensis(OP722579.1) Gleditsia sinensis(MZ636527.1) Gleditsia microphylla var(OP722581.1) Gleditsia microphylla(OP722576.1) Gleditsia microphylla(NC_047369.1) Gymnocladus chinensis(OP722582.1) Gymnocladus dioicus(OP722578.1) (b) Figure 5. Gleditsia phylogenetic tree analysis using the maximum likelihood (ML). (a): Phylogenetic analysis based on chloroplast genome sequence; (b): Phylogenetic analysis based on shared CDS sequence. Gleditsia delavayi(OP722574.1) Gleditsia fera(OP722575.1) Gleditsia velutina(OP764692.1) Gleditsia japonica(OP722577.1) Gleditsia sinensis(OP722579.1) Gleditsia sinensis zhuyazao(OP722580.1) Gleditsia sinensis(MT483984.1) Gleditsia sinensis(NC_047282.1) Gleditsia sinensis(MZ636527.1) Gleditsia microphylla(OP722576.1) Gleditsia microphylla var(OP722581.1) Gleditsia microphylla(NC_047369.1) Gymnocladus chinensis(OP722582.1) Gymnocladus dioicus(OP722578.1) (a) Gleditsia delavayi(OP722574.1) Gleditsia fera(OP722575.1) Gleditsia velutina(OP764692.1) Gleditsia japonica(OP722577.1) Gleditsia sinensis zhuyazao(OP722580.1) Gleditsia sinensis(MT483984.1) Gleditsia sinensis(NC_047282.1) Gleditsia sinensis(OP722579.1) Gleditsia sinensis(MZ636527.1) Gleditsia microphylla var(OP722581.1) Gleditsia microphylla(OP722576.1) Gleditsia microphylla(NC_047369.1) Gymnocladus chinensis(OP722582.1) Gymnocladus dioicus(OP722578.1) (b) (b) (a) Figure 5. Gleditsia phylogenetic tree analysis using the maximum likelihood (ML). (a): Phylogenetic analysi based on chloroplast genome sequence; (b): Phylogenetic analysis based on shared CDS sequence. SSC and LSC regions, similar to studies in other ­plants45. Mutation hotspots can be used as suitable loci for population genetics and phylogenetic studies. Hypervariable regions can be as candidates for DNA barcode ­development46. Conclusion h d In this study, we sequenced and compared the complete chloroplast genomes of seven genotypes from Gleditsia. Assembly and annotation of the chloroplast genomes found that Gleditsia species chloroplast genomes have a typical circular tetrad structure, the size of the chloroplast genomes ranged from 162,746 to 170,907 bp. Through comparative genomic analysis, most (65.8–75.8%) of the SSRs in Gleditsia and Gymnocladus species are located in the LSC. The codon pairs ending with C and G in the Gleditsia chloroplast genome have low bias which are nonpreferred codons, the genus Gleditsia prefer T/A-ending codons and avoid C/G-ending codons. The selec- tion pressure estimation (Ka/Ks ratios) of genes in the Gleditsia species showed that rpoA, rpl20, atpB, ndhA and ycf4 were subjected to positive selection, most of the chloroplast genes of Gleditsia species underwent purifying selection. The genus Gleditsia face relatively weak selection pressure. Mutational hotspots mostly occurred in "rps16-trnQ", "trnT-trnL", "ndhG-ndhI", "rpl32-trnL" and other intergenic regions in Gleditsia. Phylogenetic analysis shows that G. fera was most closely related to G. sinensis, G. japonica and G. delavayi were relatively close, Zhū Yá Zào can be considered a bud mutation of the G. sinensis. Data availability Th d d y The datasets generated and analyzed in this study are available in the GenBank of NCBI, and the complete chlo- roplast genome sequence were available under the accession Number OP722579-OP722582. Received: 15 July 2023; Accepted: 14 February 2024 Received: 15 July 2023; Accepted: 14 February 2024 Discussionh sinensis, produced by old or injured plants, there was no significant difference in the contents of saponin compounds between Fructus Gleditsiae abnormalis and Fructus Gleditsiae sinensis by LC-ELSD7,56. Li et al.8 suggested that Zhū Yá Zào should be a variant of G. sinensis. The evolutionary relationship between G. sinensis and Zhū Yá Zào was the closest, G. sinensis and Zhū Yá Zào clustered into a subclade (Fig. 5). Zhū Yá Zào can be considered a bud mutation of the G. sinensis.t Albino phenotypes often occur in nature. In the process of raising seedlings in the greenhouse, a albino mutant plant of G. microphylla was obtained (labeled G. microphylla mutant), which was characterized by albino whole plant, obvious dwarfing, and natural death after 1–1.5 months of growth. OsSLC1 is responsible for the seedling-lethal chlorosis phenotype in the rice seedling-lethal chlorosis 1 mutant, loss-of-function of OsSLC1 affected the intron splicing of multiple group II introns, and especially precluded the intron splicing of rps1657. https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ The albinism of Camellia sinensis cv. Baiye1 was due to chloroplast dysplasiaand the blocking synthesis of Pchlide a from Mg-proto ­IX58. Deficiency in grana stacking in chloroplasts and inhibition of gene expression related to chloroplast localization may also lead to the production of albino ­seedlings59. By assembling and comparing the chloroplast genomes of the G. microphylla mutant and G. microphylla, we found that their sequences were com- pletely identical. This suggests that the albino phenotype is not caused by variations in the chloroplast genome, and that the occurrence of the albino phenotype may be due to mutations in chloroplast-related genes involved in splicing or localization functions. This requires further experimental validation in the future. References 1. Zhang, J.-P. et al. Gleditsia species: An ethnomedical, phytochemical and pharmacological review. J. Ethnopharmacol. 178, 155–171 (2016). ( ) 2. Gordon, D. A revision of the genus Gleditsia (Leguminosae) (1967).h 2. Gordon, D. A revision of the genus Gleditsia (Leguminosae) (1967). Th d h f h d d f 3. Rong, L. F., Bin, L., Wei, L. & Qi, Z. Y. The Academic Exchange Meeting of the Tree Introduction and Domestication Committee o the Chinese Society of Forestry y f y 4. Wanchun, G., Cuiling, S. & Yanping, L. 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Plant Commun. https://​doi.​or 1016/j.​xplc.​2023.​100611 (2023). https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | Author contributions Conceptualization, X.F. and Z.Y.; methodology, X.F.; software, X.F., Z.Y.; validation, X.F., Z.Y.; formal analy- sis, W.X.; investigation, X.F.; resources, X.F., J.X.Y.; data curation, Z.Y.; writing-original draft preparation, X.F.; writing-review and editing, X.F., Z.Y., J.X.Y.; visualization, X.F., Z.Y.; supervision, Z.Y.; project administration, Wang X.; funding acquisition, W.X. All authors have read and agreed to the published version of the manuscript. Fundingh g This research was funded by the Science and Technology Plan Project of Guizhou Province ([2022] general 102), the characteristic forestry industry research project of guizhou province (GZMC-ZD20202102), the Science and Technology Plan Project of Guizhou Province ([2020]1Y056). www.nature.com/scientificreports/ ( ) 53. 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Exploring the anti-inflammatory effect of Fructus Gleditsia sinensis Lam., Fructus Gleditsiae abnormalis, and Gym- nocladus chinensis Baill. using SPME-GC-MS, network pharmacology, and molecular docking. Arab. J. Chem. 15, 103859 (2022). 57. Lv, J. et al. OsSLC1 encodes a pentatricopeptide repeat protein essential for early chloroplast development and seedling survival. Rice 13 1–13 (2020) 7. Lv, J. et al. OsSLC1 encodes a pentatricopeptide repeat protein essential for early chloroplast development and seedling survival Rice 13, 1–13 (2020). 58. Ying, Y., Li-tang, L. & De-gang, Z. Comparison of ultrastructure, chlorophyll and precursor contents of chloroplasts between al- bino leaves and green leaves in Camellia sinensis ’Baiyel 1’. J. Mt. Agric. Biol. (2019).i g y g 9. Li, N.-N. et al. Dissection of chemical composition and associated gene expression in the pigment-deficient tea cultivar ‘Xiaoxueya reveals an albino phenotype and metabolite formation. Front. Plant Sci. 10, 1543 (2019). Competing interests h p g The authors declare no competing interests. Scientific Reports | (2024) 14:4262 | https://doi.org/10.1038/s41598-024-54608-6 www.nature.com/scientificreports/ Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​024-​54608-6. Correspondence and requests for materials should be addressed to Y.Z. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2024 https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 |
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ANALISIS NILAI PERUSAHAAN BANK UMUM SYARIAH DI INDONESIA
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ANALISIS NILAI PERUSAHAAN BANK UMUM SYARIAH DI INDONESIA ALFIAN HALID SOFIAN Alfianhalid@yahoo.com Keyword : CSR, profitability, liquidity, leverage, growth, firm value ISSN 1412-2936 EISSN 2549-7308 ANALISIS NILAI PERUSAHAAN BANK UMUM SYARIAH DI INDONESIA ISSN 1412-2936 EISSN 2549-7308 ISSN 1412-2936 EISSN 2549-7308 Abstract Company value may reflect the assets owned by a company in the form of securities of one of them in the form of shares. The stock price traded on the stock exchange is an indicator of corporate value. Corporate value is influenced by many factors. This study aims examine the effect csr, profitability, liquidity, leverage and growth for the value of the company. This research is a research using quantitative approach. The population in this study were all companies of Sharia Banks registered in Bank Indonesia, from the population used the total sampling method and the total population of 11 Sharia Commercial Banks became the sample. The data used is secondary data. Data analysis used is path analysis with SPSS 23 The results show that CSR can contribute to corporate value, profitability can not contribute to corporate value, liquidity can contribute to firm value, leverage can contribute to firm value, and growth can not contribute to firm value. 193 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 ISSN 1412-2936 EISSN 2549-7308 Syariah 7. PT. Bank Panin Syariah 29,93 32,90 23,53 20,91 8. PT. Bank Bukopin Syariah 31,74 33,47 34,26 32,87 9. PT. Bank Victoria Syariah 25,28 26,76 27,85 29,34 10. PT. BCA Syariah 24,72 29,78 31,75 34,87 11. PT. Maybank Indonesia Syariah 27,38 29,83 35,47 34,54 PENDAHULUAN Nilai perusahaan dapat mencerminkan nilai asset atau aktiva yang dimiliki perusahaan seperti surat- surat berharga salah satunya berupa saham. Kemampuan perusahaan membayar dividen merupakan salah satu faktor yang mempengaruhi harga saham. Bagi perusahaan yang menerbitkan saham di pasar modal, harga saham yang diperjualbelikan di bursa efek merupakan indikator nilai perusahaan. Memaksimumkan nilai pasar perusahaan sama dengan memaksimumkan harga pasar saham. Perusahaan yang memiliki total aset dengan jumlah besar akan lebih banyak mendapat perhatian investor, kreditor maupun para pemakai informasi keuangan lainnya, serta pihak manajemen akan lebih leluasa dalam menggunakan aset yang ada untuk meningkatkan nilai perusahaan. Kondisi perekonomian yang semakin tidak menentu atau unpredictable perusahaan bersaing dalam mencapai keberhasilannya. Hal ini tentunya dapat dilihat dengan nilai perusahaan itu sendiri. Nilai perusahaan dapat menjadi pedoman atau acuan bagi investor atau stakeholders lainnya di dalam menilai keberlangsungan (going concern) masing-masing perusahaan. Pergerakan saham bank umum syariah juga dapat menunjukkan eksistensi dari nilai perusahaan itu sendiri. Adapun nilai perusahaan Bank Umum Syariah dapat dilihat dalam tabel 1 berikut: Tabel. 1 Nilai Perusahaan Bank Umum Tahun 2012-2015 (persen) No. Nama Perusahaan Tahun 2012 2013 2014 2015 1. PT. Bank Syariah Mandiri 46,27 40,80 37,34 49,52 2. PT. Bank Muamalat Indonesia 49,93 50,07 57,12 58,59 3. PT. Bank BNI Syariah 35,20 37,03 40,76 44,22 4. PT. Bank BRI Syariah 29,35 34,31 38,80 39,67 5. PT. Bank Mega Syariah 20,73 22,76 24,89 30,43 6. PT. Bank Jabar Banten 26,74 29,77 31,43 33,79 Beberapa penelitian menyebutkan ada banyak faktor yang dapat mempengaruhi nilai perusahaan. Diantaranya yaitu CSR, yang disebutkan bahwa CSR berpengaruh signifikan terhadap nilai perusahaan (Arafat et al, 2012; Kusumadilaga, Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 194 ISSN 1412-2936 EISSN 2549-7308 Kadek Ayu dan Ni Putu Ayu Darmayanti. 2015), dengan variabel independen yang digunakan yaitu Profitabilitas, Likuiditas, Pertumbuhan, dan Investment Oppurtunity Set Terhadap variabel dependen yaitu Nilai Perusahaan, sedangkan pada penelitian ini variabel independen yang digunakan CSR, Profitabilitas, Likuiditas, Leverage dan Growth, terhadap variabel dependen yaitu nilai perusahaan. Perbedaan lain dengan penelitian sebelumnya adalah objek penelitian. Bank Umum Syariah menjadi objek dalam penelitian ini, hal ini dikarenakan Bank umum syariah merupakan lembaga keuangan yang berasaskan prinsip-prinsip syariah, yang telah dijelaskan di dalam Undang-undang No.10/1998 tentang perbankan Islam dan disempurnakan dalam Undang- undang No. 7 tahun 1992, serta diikuti pula dengan Undang-undang No.23/1999 mengenai Bank Indonesia yang memberikan peluang kepada Bank Umum Nasional untuk membuka Unit Usaha Syariah (UUS). Di samping itu, Bank Umum Syariah semakin mengalami perkembangan dan ikut serta dalam memajukan perekonomian dunia, khususnya di Indonesia. Perkembangan Bank Umum syariah ISSN 1412-2936 EISSN 2549-7308 ISSN 1412 2936 EISSN 2549-7308 Kadek Ayu dan Ni Putu Ayu Darmayanti. 2015), dengan variabel independen yang digunakan yaitu Profitabilitas, Likuiditas, Pertumbuhan, dan Investment Oppurtunity Set Terhadap variabel dependen yaitu Nilai Perusahaan, sedangkan pada penelitian ini variabel independen yang digunakan CSR, Profitabilitas, Likuiditas, Leverage dan Growth, terhadap variabel dependen yaitu nilai perusahaan. Perbedaan lain dengan penelitian sebelumnya adalah objek penelitian. Bank Umum Syariah menjadi objek dalam penelitian ini, hal ini dikarenakan Bank umum syariah merupakan lembaga keuangan yang berasaskan prinsip-prinsip syariah, yang telah dijelaskan di dalam Undang-undang No.10/1998 tentang perbankan Islam dan disempurnakan dalam Undang- undang No. 7 tahun 1992, serta diikuti pula dengan Undang-undang No.23/1999 mengenai Bank Indonesia yang memberikan peluang kepada Bank Umum Nasional untuk membuka Unit Usaha Syariah (UUS). Di samping itu, Bank Umum Syariah semakin mengalami perkembangan dan ikut serta dalam memajukan perekonomian dunia, khususnya di Indonesia. ISSN 1412-2936 EISSN 2549-7308 ISSN 1412-2936 EISSN 2549-7308 akan dengan sengaja memberikan signal ke pasar, agar pasar dapat membedakan kualitas perusahaan tersebut dengan perusahaan lainnya (Hartono, 2005) dalam Adnantara (2013). Signal ini dapat berupa promosi atau informasi lainnya, salah satunya adalah informasi tentang Corporate Social Responsibility (CSR), dengan harapan dapat meningkatkan nilai perusahaan (Adnantara, 2013). saham bank umum syariah dari tahun ke tahun. Berdasarkan latar belakang yang telah diuraikan di atas, maka rumusan masalah dalam penelitian ini sebagai berikut: 1. Apakah Corporate Social Responsibility berpengaruh terhadap nilai perusahaan? 2. Apakah Profitabilitas berpengaruh terhadap nilai perusahaan? Rustiarini (2010) dalam Umro dan Fidiana (2016) teori sinyal membahas mengenai dorongan perusahaan untuk memberikan informasi kepada pihak eksternal maupun pihak internal. Dorongan tersebut disebabkan karena terjadinya asimetri informasi antara pihak manajemen dan pihak eksternal. Untuk mengurangi asimetri informasi maka perusahaan harus mengungkapkan informasi yang dimiliki, baik informasi keuangan maupun non keuangan. 3. Apakah leverage berpengaruh terhadap nilai perusahaan? 4. Apakah likuiditas berpengaruh terhadap nilai perusahaan? 5. Apakah Growth berpengaruh terhadap nilai perusahaan? Teori Sinyal (Signaling Theory) Teori ini menjelaskan mengapa perusahaan mempunyai dorongan untuk memberikan informasi laporan keuangan pada pihak eksternal. Dorongan perusahaan untuk memberikan informasi karena terdapat asimetri informasi antara perusahaan dan pihak luar karena perusahaan mengetahui lebih banyak mengenai perusahaan dan prospek yang akan datang daripada pihak luar. Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 19 perusahaan mempunyai dorongan untuk memberikan informasi laporan keuangan pada pihak eksternal. Dorongan perusahaan untuk memberikan informasi karena terdapat asimetri informasi antara perusahaan dan pihak luar karena perusahaan mengetahui lebih banyak mengenai perusahaan dan prospek yang akan datang daripada pihak luar. Teori Signal menyatakan bahwa perusahaan yang memiliki kualitas baik informasi yang dimiliki, baik informasi keuangan maupun non keuangan. Teori Stakeholder Secara umum dalam teori stakeholder menunjukkan bahwa perusahaan tidak hanya bertanggung jawab pada kesejahteraan perusahaan saja, melainkan harus memiliki tanggug jawab sosial dengan mempertimbangkan kepentingan semua pihak yang terkena dampak dari tindakan atau kebijakan strategi Tabel. 1 Nilai Perusahaan Bank Umum Perkembangan Bank Umum syariah juga ditunjukkan dengan naik turunnya 2010), namun dalam penelitian yang dilakukan oleh (Agustine, 2012; Maspupah, 2014) menyebutkan bahwa CSR tidak berpengaruh terhadap nilai perusahaan. Sebagian besar penelitian menunjukkan hasil yang beragam. Menurut (Umro, Madinatul dan Fidiana, 2016) Corporate Social Responsibility (CSR) berpengaruh positif terhadap nilai perusahaan dan profitabilitas tidak berpengaruh terhadap nilai perusahaan. Adapun menurut Agustine (2010) dalam Cecilia (2014), profitabilitas sebagai indikator kinerja perusahaan berpengaruh positif terhadap nilai perusahaan. Fandini (2013) dan Hermunigsih (2013) juga melakukan penelitian tentang nilai perusahaan tetapi dengan variabel independen yang berbeda, yang menunjukkan bahwa profitabilitas berpengaruh positif terhadap nilai perusahaan. Hasil penelitian ini sejalan dengan penelitian yang dilakukan Kurnia (2015), Pangulu (2014), dan Hemastuti (2014) yang menunjukkan bahwa profitabilitas berpengaruh positif terhadap nilai perusahaan. Perbedaan penelitian ini dengan penelitian sebelumnya yaitu pada variabel yang digunakan dalam mengukur nilai perusahaan, seperti halnya yang dilakukan oleh (Sudiani, Ni 195 Corporate Social Responsibility (CSR) Corporate Social Responsibility (CSR) dicetuskan sebagai suatu visi akuntabilitas suatu bisnis. Kuncinya adalah terfokus pada proteksi lingkungan dan keselamatan kerja para pekerja, dan pengembangan komunitas dan masyarakat secara umum baik saat ini maupun masa depan (Lilis, 2012). Menurut Djajadiningrat dan Melia, 2004: 35,36 dalam Lilis (2012), Konsep CSR ini mengarahkan bahwa suatu perusahaan tidak akan dapat bertahan lama apabila dia mengisolasikan dan membatasi dirinya dari masyarakat sekitarnya. Teori Stakeholder │ Secara umum dalam teori stakeholder menunjukkan bahwa perusahaan tidak hanya bertanggung jawab pada kesejahteraan perusahaan saja, melainkan harus memiliki tanggug jawab sosial dengan mempertimbangkan kepentingan semua pihak yang terkena dampak dari tindakan atau kebijakan strategi Teori Signal menyatakan bahwa perusahaan yang memiliki kualitas baik 196 ISSN 1412-2936 EISSN 2549-7308 (Cecilia, dkk, 2014). Dengan menerapkan CSR, diharapkan perusahaan akan memperoleh legitimasi sosial dan memaksimalkan kekuatan keuangannya dalam jangka panjang (Kiroyan, 2006). Hal ini mengindikasikan bahwa perusahaan yang menerapkan CSR mengharapkan akan direspon positif oleh para pelaku pasar. ISSN 1412-2936 EISSN 2549-7308 (Cecilia, dkk, 2014). Dengan menerapkan CSR, diharapkan perusahaan akan memperoleh legitimasi sosial dan memaksimalkan kekuatan keuangannya dalam jangka panjang (Kiroyan, 2006). Hal ini mengindikasikan bahwa perusahaan yang menerapkan CSR mengharapkan akan direspon positif oleh para pelaku pasar. perusahaan. Kesuksesan suatu perusahaan sangat tergantung pada kemampuannya dalam menyeimbangkan beragam kepentingan dari para stakeholder atau pemangku kepentingan (Lako, 2011). ISSN 1412-2936 EISSN 2549-7308 ISSN 1412-2936 EISSN 2549-7308 Dengan rasio profitabilitas yang tinggi yang dimiliki sebuah perusahaan akan menarik minat investor untuk menanamkan modalnya diperusahaan (Dewi dan Wirajaya, 2013) dalam (I Gusti Ngurah Agung Dwi Pramana, 2016). Growth Wahyudi dan Hartini (2006) dalam Ni Kadek Ayu Sudiani dan Ni Putu Ayu Darmayanti (2016) menyebutkan bahwa investasi perusahaan dalam aktiva tetap adalah potensi keuntungan bagi perusahaan di masa depan, sehingga growth perusahaan adalah hal positif yang dapat meningkatkan nilai perusahaan. Semakin besar tingkat growth perusahaan maka akan semakin tinggi potensi profitabilitas perusahaan di masa depan sehingga semakin tinggi pula nilai perusahaan. LIkuiditas Nilai likuiditas yang tinggi mencerminkan kemampuan perusahaan yang tinggi untuk memenuhi kewajiban jangka pendeknya. Perusahaan yang memiliki nilai likuiditas yang baik akan dianggap memiliki kinerja yang baik oleh investor. Menurut Sartono (2010:114) dalam Sudiyani dan Darmayanti (2016) Likuiditas merupakan kemampuan perusahaan untuk memenuhi kewajiban finansial yang berjangka pendek tepat pada waktunya. Ukuran likuiditas perusahaan yang diproksikan dengan current ratio (CR) yang merupakan perbandingan antara aktiva lancar (current asset) dengan hutang lancar (current liabilities). Likuiditas yang tinggi menunjukkan kekuatan perusahaan dari segi kemampuan untuk memenuhi hutang lancar dari harta lancar yang dimiliki sehingga hal ini meningkatkan kepercayaan pihak luar terhadap perusahaan tersebut. Profitabilitas Profitabilitas adalah kemampuan perusahaan dalam menghasilkan laba (Suharli, 2007). Perusahaan yang dapat mengelola asetnya dengan baik, cenderung menghasilkan kinerja keuangan yang baik pula, yang diwujudkan dengan adanya laba yang tinggi (ROA yang tinggi). Profitabilitas digunakan untuk mengukur sampai sejauh mana keefektifan dari keseluruhan manajemen dalam menciptakan keuntungan bagi perusahaan setelah bunga dan pajak (Sianturi, 2015)dalam (Mariani, 2016). Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 19 sekitarnya. Pengungkapan atas kegiatan CSR adalah sinyal yang bagus bagi investor dan stakeholder bahwa perusahaan aktif dalam melakukan kegiatan CSR, serta nilai pasar perusahaan berada dalam posisi yang bagus. Kinerja sosial perusahaan yang bagus membantu perusahaan untuk memperoleh reputasi dari pasar modal dan pasar utang sehingga membuat nilai suatu perusahaan meningkat menciptakan keuntungan bagi perusahaan setelah bunga dan pajak (Sianturi, 2015)dalam (Mariani, 2016). Profitabilitas yang tinggi mencerminkan kemampuan perusahaan dalam menghasilkan keuntungan yang tinggi bagi pemegang saham. Semakin besar keuntungan yang diperoleh semakin besar pula kemampuan perusahaan untuk membayarkan devidennya, dan hal ini berdampak pada kenaikan nilai perusahaan. Pengungkapan atas kegiatan CSR adalah sinyal yang bagus bagi investor dan stakeholder bahwa perusahaan aktif dalam melakukan kegiatan CSR, serta nilai pasar perusahaan berada dalam posisi yang bagus. Kinerja sosial perusahaan yang bagus membantu perusahaan untuk memperoleh reputasi dari pasar modal dan pasar utang sehingga membuat nilai suatu perusahaan meningkat Profitabilitas yang tinggi mencerminkan kemampuan perusahaan dalam menghasilkan keuntungan yang tinggi bagi pemegang saham. Semakin besar keuntungan yang diperoleh semakin besar pula kemampuan perusahaan untuk membayarkan devidennya, dan hal ini berdampak pada kenaikan nilai perusahaan. 197 ISSN 1412-2936 EISSN 2549-7308 perusahaan. Leverage dihitung dari total utang dibagi dengan total aset. Perusahaan dengan tingkat leverage yang tinggi menunjukkan bahwa perusahaan lebih banyak bergantung pada utang dalam membiayai aset perusahaan. Utang bagi perusahaan memiliki beban tetap yang berupa beban bunga. Semakin besar utang yang dimiliki perusahaan maka beban bunga yang harus dibayarkan juga semakin tinggi. Perusahaan yang memiliki utang tinggi akan mendapatkan insentif pajak berupa potongan atas bunga pinjaman sehingga perusahaan yang memiliki beban pajak tinggi dapat melakukan penghematan pajak dengan cara menambah utang perusahaan (Suyanto dan Suparmono, 2012). Pengaruh Likuiditas Terhadap Nilai Perusahaan Likuiditas dalam teori berhubungan positif dengan nilai perusahaan. Semakin tinggi likuiditas maka nilai perusahaan tinggi dan semakin rendah likuiditas maka nilai perusahaan rendah. Kemampuan kas yang tinggi akan berdampak terhadap kemampuan kewajiban jangka pendek perusahaan dan berdampak positif terhadap nilai perusahaan (Mahendra, 2012). H 1 : Ada pengaruh corporate sosial responsibility (CSR) terhadap nilai perusahaan ISSN 1412-2936 EISSN 2549-7308 ISSN 1412-2936 EISSN 2549-7308 ISSN 1412-2936 EISSN 2549-7308 ISSN 1412 2936 EISSN 2549-7308 maka nilai perusahaan rendah. Semakin baik perusahaan membayar return terhadap pemegang saham akan meningkatkan nilai perusahaan. Nilai perusahaan dapat pula dipengaruhi oleh besar kecilnya profitabilitas yang dihasilkan oleh perusahaan. Weston dan Copeland (1992) dalam Prasetyorini (2013) mendefinisikan profitabilitas sejauh mana perusahaan menghasilkan laba dari penjualan dan investasi perusahaan. Apabila profitabilitas perusahaan baik maka para stakeholders yang terdiri dari kreditur, supplier, dan juga investor akan melihat sejauh mana perusahaan dapat menghasilkan laba dari penjualan dan investasi perusahaan. Dengan baiknya kinerja perusahaan akan meningkatkan pula nilai perusahaan. EISSN 2549-7308 maka nilai perusahaan rendah. Semakin baik perusahaan membayar return terhadap pemegang saham akan meningkatkan nilai perusahaan. Nilai perusahaan dapat pula dipengaruhi oleh besar kecilnya profitabilitas yang dihasilkan oleh perusahaan. Weston dan Copeland (1992) dalam Prasetyorini (2013) mendefinisikan profitabilitas sejauh mana perusahaan menghasilkan laba dari penjualan dan investasi perusahaan. Apabila profitabilitas perusahaan baik maka para stakeholders yang terdiri dari kreditur, supplier, dan juga investor akan melihat sejauh mana perusahaan dapat menghasilkan laba dari penjualan dan investasi perusahaan. Dengan baiknya kinerja perusahaan akan meningkatkan pula nilai perusahaan. Pengembangan Hipotesis Pengaruh Pengungkapan Corporate Sosial Responsibility Terhadap Nilai Perusahaan Frekuensi dilakukannya Corporate Social Responsibility oleh perusahaan akan mendongkrak citra perusahaan. Investor lebih berminat pada perusahaan yang memiliki citra baik di masyarakat karena semakin baik citra perusahaan, loyalitas konsumen semakin tinggi sehingga dalam waktu lama penjualan perusahaan akan meningkat dan profitabilitas perusahaan juga akan meningkat (Retno dan Priantinah, 2012) dalam (Mariani,dkk, 2016). Pengungkapan Corporate Social Responsibility perusahaan diharapkan dapat memberikan sinyal yang positif kepada investor, sehingga dapat meningkatkan nilai perusahaan (Cheng dan Christiawan, 2011).Berdasarkan uraian di atas dapat dirumuskan hipotesis sebagai berikut. Pengembangan Hipotesis Pengaruh Pengungkapan Corporate Sosial Responsibility Terhadap Nilai Perusahaan Leverage menggambarkan yang dimiliki oleh Leverage menggambarkan struktur modal yang dimiliki oleh 198 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Pengaruh ProfitabilitasTerhadap Nilai Perusahaan Profitabilitas dalam teori berhubungan positif dengan nilai perusahaan. Semakin tinggi profitabilitas maka nilai perusahaan tinggi dan semakin rendah profitabilitas Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 199 EISSN 2549-7308 Leverage dalam teori berhubungan negatif dengan nilai perusahaan. Semakin tinggi leverage maka nilai perusahaan rendah dan semakin rendah leverage maka nilai perusahaan tinggi. Penggunaan hutang harus hati-hati oleh pihak manajemen, karena semakin besar hutang akan menurunkan nilai perusahaan. Leverage yang parsial dalam hasil penelitian yang dilakukan (Mahendra, dkk, 2012) menemukan memiliki pengaruh negatif tidak signifikan terhadap nilai perusahaan. Berdasarkan uraian di atas dapat dirumuskan hipotesis sebagai berikut. Likuiditas menunjukkan kemampuan perusahaan untuk memenuhi kewajiban jangka pendeknya. Tingkat likuiditas yang tinggi akan menunjukkan bahwa perusahaan berada dalam kondisi yang baik sehingga akan menambah permintaan saham dan tentunya akan menaikkan harga saham (Mahendra, 2011) dalam (Mariani, dkk, 2016). Kemampuan kas yang tinggi akan berdampak pada kemampuan memenuhi kewajiban jangka pendek perusahaan dan berdampak positif pada perusahaan (Mahendra, 2012) dalam (Mariani, dkk, 2016). Semakin tinggi likuiditas perusahaan, semakin banyak dana yang tersedia bagi perusahaan untuk membayar dividen, membiayai operasi dan investasinya, sehingga persepsi investor pada kinerja perusahaan akan meningkat (Nurhayati, 2013) dalam (Mariani, dkk, 2016). H 3 : Ada pengaruh leverage terhadap nilai perusahaan Pengaruh Growth Terhadap Nilai Perusahaan Pertumbuhan perusahaan pada penelitian ini diukur dengan growth (pertumbuhan asset). Pertumbuhan perusahaan adalah selisih total aset yang dimiliki oleh perusahaan pada periode sekarang dengan periode sebelumnya terhadap total aset periode sebelumnya pada perusahaan Bank Umum Syariah di Bank Indonesia periode 2012-2015. ISSN 1412-2936 EISSN 2549-7308 Populasi dan Sampel Penelitian Populasi perusahaan dalam aktiva tetap adalah potensi keuntungan bagi perusahaan di masa depan, sehingga pertumbuhan perusahaan adalah hal positif yang dapat meningkatkan nilai perusahaan. Berdasarkan uraian di atas dapat dirumuskan hipotesis sebagai berikut. Populasi menurut Iqbal dalam Aulia (2008:45) adalah totalitas dari semua objek atau individu yang memiliki karakteristik tertentu, jelas, dan lengkap yang akan diteliti (bahan penelitian). Populasi dalam penelitian ini adalah semua peruasahaan Bank Umum Syariah yang terdaftar di Bank Indonesia sebanyak 11 perusahaan. H 5 : Ada pengaruh growth terhadap nilai perusahaan Pengaruh Leverage Terhadap Nilai Perusahaan Menurut Jensen (1976) dalam Febrianti (2012), nilai perusahaan dapat meningkat dengan adanya hutang apabila hutang tersebut digunakan bukan untuk investasi yang tidak menguntungkan, melainkan digunakan untuk mengendalikan penggunaan arus kas yang berlebihan oleh pihak manajemen perusahaan. Wahyudi dan Hartini (2006) dalam Ni Kadek Ayu Sudiani dan Ni Putu Ayu Darmayanti (2016) menyebutkan bahwa investasi 200 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 ISSN 1412-2936 EISSN 2549-7308 ISSN 1412-2936 EISSN 2549-7308 TABEL 2. Daftar total sampling Bank Umum Syariah di Indonesia No Nama Bank 1 PT. Bank Syariah Mandiri 2 PT. Bank Syariah Muamalat Indonesia 3 PT. Bank Syariah BNI 4 PT. Bank Syariah BRI 5 PT. Bank Syariah Mega Indonesia 6 PT. Bank Jabar Banten Syariah 7 PT. Bank Panin Syariah 8 PT. Bank Syariah Bukopin 9 PT. Bank Victoria Syariah 10 PT. BCA Syariah 11 PT. Maybank Indonesia Syariah TABEL 2. Daftar total sampling Bank Umum Syariah di Indonesia TABEL 2. Daftar total sampling untuk mencari adanya hubungan antara variabel dependen dengan satu atau lebih variabel independen. Persamaan yang digunakan untuk menguji hipotesa adalah: Y = a +b 1X 1+ b 2 X 2 + b 3 X 3 + b 4 X 4 + e Y = a +b 1X 1+ b 2 X 2 + b 3 X 3 + b 4 X 4 + e Keterangan : Y = a +b 1X 1+ b 2 X 2 + b 3 X 3 + b 4 X 4 + e Keterangan : Keterangan : Keterangan : Y : Nilai perusahaan a : Intersep (atau nilai Y pada saat X=0) b : Koefisien regresi X 1 : Corporate Social Responsibility (CSR) X 2 : Profitabilitas X 3 : Leverage X 4 : Likuiditas e : Galat / standart error Y : Nilai perusahaan a : Intersep (atau nilai Y pada saat X=0) b : Koefisien regresi X 1 : Corporate Social Responsibility (CSR) Y : Nilai perusahaan a : Intersep (atau nilai Y pada saat X=0) Y : Nilai perusahaan b : Koefisien regresi X 1 : Corporate Social Responsibility (CSR) Uji Asumsi Klasik Uji asumsi klasik dilakukan untuk mengetahui data yang layak untuk dianalisis. Pengujian data dalam penelitian ini yaitu:Uji Normalitas, Uji Multikolinearitas, Uji Autokolerasi, dan Uji Heterokedastisitas. e : Galat / standart error Untuk kepentingan analisis data tersebut, dipergunakan program komputer SPSS 23. Pengujian hipotesis dilakukan dengan uji t, namun sebelum meregresi data, dilakukan uji asumsi klasik regresi. Uji Hipotesis Penelitian ini menggunakan regresi berganda. Bentuk persamaan dalam penelitian ini adalah: Teknik Analisis Data dan Uji Statistik Statistik Deskriptif Statistik deskriptif digunakan untuk menjelaskan deskripsi data dari seluruh variabel yang akan dimasukkan dalam model penelitian yang dilihat dari nilai minimum, nilai maksimum, nilai rata-rata dan standar deviasi. Jenis dan Desain Penelitian Sampel adalah bagian dari jumlah karakteristik yang dimiliki oleh populasi tersebut (Sugiyono, 2013:116). Teknik penentuan sampel yang digunakan dalam penelitian ini adalah total sampling dimana pemilihan elemen populasi dilakukan sedemikian rupa sehingga setiap elemen tersebut mempunyai kesempatan yang sama untuk terpilih. Sampel akhir adalah 11 perusahaan Bank Umum Syariah yang terdaftar di Bank Indonesia tahun 2012- 2015. Jenis penelitian yang digunakan dalam penelitian ini adalah penelitian kuantitatif. Analisis data dalam penelitian kuantitatif yaitu menggunakan statistik deskriptif. Variabel dependen penelitian ini adalah nilai perusahaan. Nilai perusahaan merupakan persepsi investor terhadap perusahaan yang sering dikaitkan harga saham. Harga saham yang tinggi membuat nilai perusahaan juga tinggi. Harga saham merupakan harga yang terjadi pada saat saham diperdagangkan di pasar. Variabel independen yang digunakan dalam penelitian ini yaitu Corporate Social Reponsibility (CSR), profitabilitas, likuiditas, leverage dan growth. 201 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Uji multikolinearitas Uji multikolinearitas bertujuan menguji apakah model regresi ditemukan adanya korelasi antarvariabel bebas atau independen (Ghozali, 2006:91). Jika hasil menunjukkan nilai Variance Inflation Factor (VIF) ≥ 10 berarti ada multikolinearitas, sebaliknya jika nilai VIF < 10 berarti tidak ada multikolinearitas. PEMBAHASAN Metode analisis data yang digunakan dalam penelitian ini menggunakan path analysis dengan SPSS 23. e : Error Penelitian ini menggunakan analisis regresi linier berganda karena untuk mengetahui pengaruh variabel independen (CSR, profitabilitas, leverage, dan likuiditas) terhadap variabel dependen (nilai perusahaan). Alasan lain peneliti menggunakan analisis regresi linier berganda karena variabel independennya lebih dari satu. Gambar 1. Hasil Pengujian Normalitas Pada gambar 1 grafik normal plot terlihat titik-titik menyebar di sekitar garis diagonal dan mengikuti arah garis diagonal. Dengan demikian dapat diketahui bahwa variabel independen dan dependen memiliki distribusi normal sehingga dari segi normalitas, model regresi layak dipakai untuk menganalisis pengaruh variabel independen terhadap variabel dependen. ISSN 1412-2936 EISSN 2549-7308 ISSN 1412-2936 EISSN 2549-7308 X2 : Profitabilitas (Return on Total Leverage/ROA) X3 : Leverage X4 : Likuiditas (Current Ratio/CR) b1,…,b5 : Koefisien regresi Gambar 1. Hasil Pengujian Normalitas X4 : Likuiditas (Current Ratio/CR) b1,…,b5 : Koefisien regresi Uji Regresi Y = a + b1+ b2X2 + b3X3 + b4D1+e Berdasarkan pada hipotesis yang diajukan, metode analisis yang digunakan untuk membuktikan hipotesa adalah metode statistik regresi linier berganda. Analisis regresi bertujuan Keterangan: Y : Nilai Perusahaan X1 : Corporate Social Responsibility Disclosure (CSRD) 202 Tabel 4. Hasil Uji Regresi Tabel 4. Hasil Uji Regresi Coefficientsa Model Unstandardized Coefficients T Sig. B Std. Error 1 (Constant) 30.544 2.448 12.476 .000 CSR 1.831E-9 .000 6.925 .000 Profitabilitas 3.563 18.263 .195 .846 Likuiditas .039 .018 2.218 .033 Leverage -2.437 1.038 -2.347 .024 Growth -2.054 1.728 -1.189 .242 a. Dependent Variable: NP Uji Normalitas Uji normalitas dilakukan untuk menguji apakah dalam model regresi variabel pengganggu atau residual memiliki distribusi normal. Uji normalitas data menggunakan analisis statistik non-parametrik uji Kolmogorov-Smirnov. Dalam uji ini variabel yang mempunyai asymp. Sig < 0,05 maka diartikan bahwa variabel-variabel tersebut memiliki distribusi tidak normal (Ghozali, 2006:110). Hasil uji normalitas berikut ini. Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 203 ISSN 1412-2936 EISSN 2549-7308 independen. Model regresi yang baik adalah yang homokedastisitas atau tidak terjadi heteroskedastisitas. Penelitian ini menggunakan Grafik Plot, dasar analisis: ISSN 1412-2936 EISSN 2549-7308 Tabel 3. Hasil Uji multikolinearitas Tabel 3. Hasil Uji multikolinearitas No Variabel Independen VIF 1 CSR 1.181 2 Profitabilitas 1.072 3 Likuiditas 1.108 4 Leverage 1.152 5 Growth 1.063 independen. Model regresi yang baik adalah yang homokedastisitas atau tidak terjadi heteroskedastisitas. Penelitian ini menggunakan Grafik Plot, dasar analisis: Gambar 2. Hasil Uji Heterokedastisitas Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Uji Autokolerasi Uji autokolerasi bertujuan menguji apakah dalam model regresi linear ada korelasi antara kesalahan pengganggu pada periode t dengan kesalahan pengganggu pada periode t-1 (sebelumnya). Deteksi adanya autokorelasi dapat dilihat dari angka DW (Durbin-Watson). Angka D-W dibawah - 2 berarti ada autokorelasi positif. Berikut hasil pengujian autokorelasi. Gambar 2. Hasil Uji Heterokedastisitas Berdasarkan gambar 2 grafik scatterplots terlihat bahwa titik-titik menyebar secara acak serta tersebar baik di atas maupun di bawah angka 0 pada sumbu y dan titik membentuk pola tertentu. Dengan demikian dapat disimpulkan bahwa tidak terjadi heterogenitas pada model persamaan regresi yang digunakan. Tabel 4. Hasil Uji Autokorelasi Tabel 4. Hasil Uji Autokorelasi Model Summary b Model R R Square Adjusted R Square Std. Error of the Estimate Durbin- Watson 1 .845 a .714 .676 5.11482 1.704 SARAN Bagi perusahaan khususnya Bank Umum Syariah yang memiliki tingkat profitabilitas yang rendah, sebaiknya lebih baik dalam pengelolaan asetnya dan kebijakan hutangnya . Hal ini dikarenakan ROE adalah fungsi dari profit margin, assets turnover dan equity multiplier. Adapun langkah-langkah yang dilakukan untuk meningkatkan profitabilitas yaitu mengurangi harga pokok penjualan atau beban operasi perusahaan, meningkatkan penjualan tanpa adanya peningkatan beban atau biaya secara proposional, dan meningkatkan penggunaan utang relative terhadap ekuitas sampai titik yang tidak membahayakan keuangan perusahaan. Tabel tersebut juga menunjukkan signifikansi antar variabel, yaitu CSR, likuiditas dan leverage memiliki signifikansi sebesar 0.000 0.033 dan 0.024 yang artinya CSR, likuiditas dan leverage berpengaruh positif terhadap nilai perusahaan, sedangkan profitabilitas dan growth berpengaruh negatif terhadap nilai perusahaan dengan signifikansi sebesar 0.846 dan 0.242. SIMPULAN Hasil penelitian menunjukkan bahwa CSR, likuiditas, dan leverage memberikan kontribusi dalam meningkatkan nilai perusahaan. Hasil penelitian ini juga menunjukkan profitabilitas dan growth tidak memberikan kontribusi dalam meningkatkan nilai perusahaan. Bagi perusahaan khususnya Bank Umum Syariah yang memiliki tingkat growth yang rendah, sebaiknya memilih strategi pertumbuhan yang tepat, fokus pada penetrasi pasar, perkuat pada segmen mikro, dan inovasi produk. Uji Heterokedastisitas Uji heteroskedastisitas bertujuan menguji apakah dalam model regresi terjadi ketidaksamaan variance dari residual satu pengamatan ke pengamatan yang lain. Pengujian ini juga bertujuan untuk menguji apakah dalam model regresi terjadi ketidaksamaan deviasi standar nilai variabel dependen pada setiap variabel Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 204 ISSN 1412-2936 EISSN 2549-7308 sehingga ada perusahaan yang mengalami kerugian. Berdasarkan persamaan tersebut nilai 30.544 merupakan intersep/konstanta, nilai tersebut merupakan nilai dari variabel nilai perusahaan apabila CSR, profitabilitas, likuiditas, leverage dan growth ditetapkan sama dengan nol, maka nilai perusahaan diproksikan dengan return saham sebesar 30.544. 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Modulation of Tonically Active Neurons of the Monkey Striatum by Events Carrying Different Force and Reward Information
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Systems/Circuits Modulation of Tonically Active Neurons of the Monkey Striatum by Events Carrying Different Force and Reward Information Simon Nougaret and Sabrina Ravel Institut de Neurosciences de la Timone, UMR7289, CNRS, Aix-Marseille Universite´, 13005 Marseille, France Simon Nougaret and Sabrina Ravel Institut de Neurosciences de la Timone, UMR7289, CNRS, Aix-Marseille Universite´, 13005 Marseille, France The role of basal ganglia in motivational processes has been under scrutiny in recent decades, with increasing evidence from clinical studies of cognitive and motivational deficits in patients with basal ganglia lesions. Tonically active neurons (TANs), the presumed striatal cholinergic interneurons, could be important actors in integrating and relaying motivational information arising from various modalities. Their multiphasic responses to rewards and to conditioned stimuli associated with reward conferred them a role in limbic processes.Theyarealsomodulatedbyatask’smotoraspect.Recentstudiessuggesttheyareinfluencedbythecontextinwhichbehavioral responses are expressed. To investigate the role of TANs in motor-limbic interaction processes, we recorded 169 TANs in the striatum of two monkeys performing a motivational task, in which they had to develop a variable force to receive different amounts of reward in response to visual stimuli. Our results reveal new features of TANs response properties. First, TANs usually responded either by a pause or an elevation of discharge rate to the visual cues and the reward, with few neurons combining both pause and rebound. Second, the elevations of discharge rate after the cues were most sensitive to the least valuable (high force or small reward) task conditions. Finally, the responses of TANs to the visual cues were time locked on the onset of the animal’s movement. TANs’ population and responses could thus play a role in signaling less attractive situations, those with either a high motor demand and/or small reward. ey words: basal ganglia; cholinergic interneurons; effort; electrophysiology; motivation; nonhuman primat Tonically active neurons (TANs) are known for their responses to unpredictable positive or negative events. However, here we show that TANs respond by a pause or an increase in their activity to all rewarding events in a task in which combined visual cues indicate to the monkeysthelevelsofforcetoproduceandtheupcomingreward.Unlikethepause,theincreaseinactivityismodulated by task parameters and is most sensitive to the least attractive task conditions (high force and/or small reward). TANs’ responses triggered by cue occurrence are also modulated by movement-related information (movement onset). We therefore propose here that TANscouldplayarole,viatheiractiononstriatalprojectionsneurons,inmaintaininghighcost/lowbenefitratiobehaviors. Clinical studies have reported that basal ganglia disorders not only trigger motor, but also cognitive and motivational deficits (Brown et al., 1997; Pessiglione et al., 2006). 15214 • The Journal of Neuroscience, November 11, 2015 • 35(45):15214–15226 15214 • The Journal of Neuroscience, November 11, 2015 • 35(45):15214–15226 Systems/Circuits Systems/Circuits Modulation of Tonically Active Neurons of the Monkey Striatum by Events Carrying Different Force and Reward Information Tonically active neurons (TANs), presumed to be cholinergic interneurons, are distributed throughout the striatum, and their responses have generally been described as homogeneous (Aosaki et al., 1995; Apicella, 2002; Adler et al., 2012). TANs are known to be involved in reward-related processes and associative learning. Most stud- ies have shown that their activity is modulated by the temporal prediction of a conditioned stimulus or a reward, its probability of occurrence, and the affective significance of stimuli. Their re- sponses are also modulated by the general context in which learn- ing or the expression of the learned behavior take place (Aosaki et al., 1994; Apicella et al., 1997, 2009, 2011; Sardo et al., 2000; Shimo and Hikosaka, 2001; Ravel et al., 2003; Morris et al., 2004; Received Jan. 6, 2015; revised Sept. 12, 2015; accepted Oct. 6, 2015. Authorcontributions:S.R.designedresearch;S.N.andS.R.performedresearch;S.N.andS.R.analyzeddata;S.N. and S.R. wrote the paper. This work was supported by Centre National de la Recherche Scientifique and the Fondation de France (Grant 2008 005902). We thank Drs. Paul Apicella, Christelle Baunez, and Janine Simmons for helpful comments and discussions. The authors declare no competing financial interests. Correspondence should be addressed to Sabrina Ravel, Institut de Neurosciences de la Timone, UMR 7289, Aix-Marseille Université and CNRS, Campus Santé Timone, 27 bd Jean Moulin, 13385 Marseille, France. E-mail: Sabrina.Ravel@univ-amu.fr. DOI:10.1523/JNEUROSCI.0039-15.2015 Copyright © 2015 the authors 0270-6474/15/3515214-13$15.00/0 Authorcontributions:S.R.designedresearch;S.N.andS.R.performedresearch;S.N.andS.R.analyzeddata;S.N. and S.R. wrote the paper. Received Jan. 6, 2015; revised Sept. 12, 2015; accepted Oct. 6, 2015. Introduction In the same way, a red filled circle indicated to the animals that the reward delivered would be small [0.3 ml of water; “small reward” (r)]; a filled red square indicated that it would be large [1.2 ml of water; “large reward” (R)]. The four possible combinations of visual stimuli (fr, fR, Fr, FR) set the four different conditions of the task. In response to these stimuli, monkeys had to increase their pressing force on the lever to reach the required force in a period shorter than 1 s [maximal reaction time (RT)] and hold this force for 1 s (holding time) to receive the reward. For each correct trial, monkeys were rewarded with the small or large reward according to the shape of the red stimuli. Both visual stimuli were extinguished as soon as the reward was delivered. To monitor force, monkeys were helped by visual feedback, a vertical rectangle representing the range of the required force located just below the visual stimuli. In this rectangle, a white cursor indicated in real time the force developed on the lever when in the required force range. After receiving the reward, the monkeys returned to a basal pressing force in preparation for the next trial, which did not begin until the total duration of the current trial (4.5 s) had elapsed. The responses of TANs classically consist of a pause followed by a rebound in activity, sometimes preceded by a short initial increase. TANs could, in turn, modulate the activity of phasically active neurons (PANs), the striatal projection neurons, by a mod- ification of their excitability state during the pause displayed by TANs in relation with stimuli associated with reward or reward itself, resulting in a time window during which other afferences could be expressed (Akins et al., 1990). PAN responses show various modulations in relation to movement, reward, or reward expectancy, depending on their location in the striatum (Hiko- saka et al., 1989; Crutcher and Alexander, 1990; Apicella et al., 1991). Thus, TANs could potentially directly, or indirectly by acting on presynaptic afferences on PANs, participate in motor, cognitive, and/or motivational processes, via the PANs. Introduction The role of TANs in motivational processes has been mainly studied in the context of classical conditioning or instrumental tasks in which the reward has been manipulated, in its occurrence or size for instance, but very little attention has been devoted to their response properties in more complex tasks modulating both motor and reward aspects. However, in day to day life, animals and humans must often apply different levels of effort in response to environmental stimuli to achieve variable quantities of reward. To investigate this situation, we studied the activity of TANs in a single experimental protocol in which visual cues provide infor- mation about both required effort and expected reward. If TANs can encode complex motivational value, we thus expect that they would respond to visual cues predictive of the reward as a func- tion of their rewarding value (computed from the effort required and the expected reward). Moreover, the development of the required force is necessary to succeed in our task. Consequently, the activity of TANs, in this context, could also be modulated by the movement as it has been reported previously. There were three different cases in which a trial was considered as failed and no reward given. First, trials in which the required force was not reached within a 1 s period were considered omission errors. Second, trials in which the required force was not held for at least 1 s (holding time) were considered holding errors. Last, trials in which the force de- veloped was greater than the upper limit of the required force (495 and 810 g, respectively, for the low and the high forces) were considered threshold errors. Both holding and threshold errors were considered as execution errors. After an error, a correction trial ensued; the same asso- ciation of visual stimuli was presented again to the monkeys until they performed the trial correctly. Moreover, trials in which the monkeys began to increase their pressing force on the lever within 100 ms after the occurrence of the visual stimuli were considered as anticipations and were excluded from analysis. Before the electrophysiological recordings began, the monkeys were extensively trained (4 to 6 months) until a performance threshold of 80% correct trials was achieved, in which the preparatory period, the maximal reaction time, and the holding time were all of 1 s. Introduction Basal ganglia, and more specifically one of their input structures, the striatum, have been considered to be important for perform- ing and maintaining adapted motivated behaviors. Authorcontributions:S.R.designedresearch;S.N.andS.R.performedresearch;S.N.andS.R.analyzeddata;S.N. and S.R. wrote the paper. This work was supported by Centre National de la Recherche Scientifique and the Fondation de France (Grant 2008 005902). We thank Drs. Paul Apicella, Christelle Baunez, and Janine Simmons for helpful comments and discussions. The authors declare no competing financial interests. Correspondence should be addressed to Sabrina Ravel, Institut de Neurosciences de la Timone, UMR 7289, Aix-Marseille Université and CNRS, Campus Santé Timone, 27 bd Jean Moulin, 13385 Marseille, France. E-mail: Sabrina.Ravel@univ-amu.fr. DOI:10.1523/JNEUROSCI.0039-15.2015 DOI:10.1523/JNEUROSCI.0039-15.2015 Copyright © 2015 the authors 0270-6474/15/3515214-13$15.00/0 J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15215 Nougaret and Ravel • TANs’ Modulation by Force and Reward Yamada et al., 2004; Joshua et al., 2008; Deffains et al., 2010). An important feature of the responses of TANs is that the responses are usually transferred from the reward to the preceding condi- tioned stimulus during learning (Apicella et al., 1997; Ravel et al., 2003), and TANs’ modulations are more pronounced in response to unpredictable, highly rewarding stimuli. It has also been re- ported that the activity of TANs can be modulated, under some conditions, by movement in both nonhuman primates and ro- dents (Lee et al., 2006; Benhamou et al., 2014). between 0 and 20% of the maximal force, defined experimentally at 900 g based on the capabilities of the animals, during a 1 s preparatory period. After this period, two visual stimuli, a green one and a red one, each being either a filled circle or filled square, were presented vertically in the center of the screen. The shape of the green stimulus indicated the amount of force the animals had to develop on the lever; that of the red stimulus indicated the amount of the upcoming reward. A green filled circle indi- cated that the animals had to develop a force between 20 and 55% of the maximal force [180 to 495 g; “low force” (f)], and a green filled square a force between 55 and 90% of the maximal force [495 to 810 g; “high force” (F)]. Introduction In each recording session, the four different combinations of visual stimuli were presented pseudorandomly, from trial to trial. The first trial of a session was ran- domly chosen from a list of trials where each condition was present in the same proportion. If trials were performed correctly, the same visual stim- uli were not presented more than times sequentially. Surgery Surgery Initial anesthesia was administered by an intramuscular injection of ket- amine (10 mg/kg) and xylazine (0.5 mg/kg), followed by deep anesthesia induced by isoflurane. A polyether ether ketone recording chamber (19 mm inner diameter) was implanted over the left hemisphere. Recording chambers in both monkeys were positioned with a 20° angle laterally in the coronal plane. The targeted stereotaxic coordinates, relative to ear bars, were as follows: Monkey M, anterior, 18 mm; lateral, 16 mm; Mon- key Y, anterior, 14 mm; lateral, 16 mm [based on the atlas of Saleem and Logothetis (2007)]. During the same surgery, two titanium cylinders were embedded in the orthopedic cement (Palacos with gentamicin) and fixed to the skull with titanium orthopedic bone screws for subsequent head restraint during neuronal recordings. Following surgery, monkeys were given antibiotics (Marbocyl, 2 mg/kg) and analgesics (Tolfedine, 4 mg/kg) on the day of the surgery and for the following 4 d. The recording chamber was filled with an antibiotic solution (Marbocyl, 2 mg/ml) and sealed with a removable cap. Materials and Methods Animal and apparatus pp Two male rhesus monkeys (Macaca mulatta) weighing 8 and 7 kg at the beginning of the experiments (Monkeys M and Y, respectively) were trained to exert and maintain a force on a lever in response to visual stimuli to receive a liquid reward. All experimental procedures were in compliance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, the French laws on animal experimenta- tion, and the European directive on the protection of animals used for scientific purposes. Behavioral procedures Behavioral procedures p The monkeys were seated in a Plexiglas primate chair and faced a panel supporting a 17 inch screen on which visual stimuli could be presented. The screen was positioned 18 cm from the monkey; a lever equipped with strain gauges in the lower part of the panel was positioned at waist level. A sliding door at the front of the primate chair could be opened to allow the animal to position his hand on the lever. The liquid reward (water) was delivered via a metal spout positioned directly in front of the mon- key’s mouth. The liquid was delivered through a solenoid valve located outside the recording room. The trial structure is illustrated in Figure 1A. At the beginning of each trial, the monkey had to develop a basal pressing force on the lever, 15216 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward Preparatory period Cue-threshold period Holding period Post-reward period Beginning of the trial Occurence of the visual stimuli Force development Reward A AC + 2 AC + 3 AC +4 AC +5 AC +6 AC -1 AC AC + 1 Monkey Y B Monkey M Associative neurons Sensorimotor neurons AC -2 C D AC -1 AC AC +1 AC +2 AC +3 re1. Taskdesignandreconstructionofrecordinglocations.A,Taskdesign.Apairofvisualstimuliappearedafterthemaintenanceofabasalpressingforcebytheanimalfor1s.Inrespo esestimuli,themonkeyincreasedhispressingforce,reachedtherequiredforcerange,andheldthisforcefor1stoobtainthereward.Fourpossiblecombinationsofvisualstimuliindicate nimaltheforcehehadtodevelopandthesizeoftheupcomingreward.Thetopstimulusrepresentedtheforceandthebottomonethereward.Afilledcirclerepresentedasmallamount, ed square a large one. B–D, Reconstruction of recording locations of TANs. All electrode recording sites were obtained from MR images. Electrode artifacts are visible in MR images of b keys,andthelocationofeachrecordedneuronwasextrapolatedfromthetipoftheseelectrodes.B,RecordingsitesforMonkeyMfromAC1toAC6(onemoreneuron,recordedinAC t represented on the figure). Each dot represents the location of one neuron recorded during the task. The white ones represent the locations of associative neurons, and the black ones ons of sensorimotor neurons. C, Recording sites for Monkey Y from AC 2 to AC 3 (same representation as in B). D, Three-dimensional reconstruction of the anterior part of the stria sparent gray) of Monkey Y, AC 4 in the background. 6 • J. Behavioral procedures Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs Modulation by Force and Rew Preparatory period Cue-threshold period Holding period Post-reward period Beginning of the trial Occurence of the visual stimuli Force development Reward A A Reward Post-reward period Force development Holding period Cue-threshold period Preparatory period AC + 2 AC + 3 AC +4 AC +5 AC +6 AC -1 AC AC + 1 Monkey Y B Monkey M Associative neurons Sensorimotor neurons AC -2 C D AC -1 AC AC +1 AC +2 AC +3 Monkey M B Associative neurons Sensorimotor neurons D Associative neurons Sensorimotor neurons Monkey Y C D AC +3 AC +1 Figure1. Taskdesignandreconstructionofrecordinglocations.A,Taskdesign.Apairofvisualstimuliappearedafterthemaintenanceofabasalpressingforcebytheanimalfor1s.Inresponse tothesestimuli,themonkeyincreasedhispressingforce,reachedtherequiredforcerange,andheldthisforcefor1stoobtainthereward.Fourpossiblecombinationsofvisualstimuliindicatedto theanimaltheforcehehadtodevelopandthesizeoftheupcomingreward.Thetopstimulusrepresentedtheforceandthebottomonethereward.Afilledcirclerepresentedasmallamount,and a filled square a large one. B–D, Reconstruction of recording locations of TANs. All electrode recording sites were obtained from MR images. Electrode artifacts are visible in MR images of both monkeys,andthelocationofeachrecordedneuronwasextrapolatedfromthetipoftheseelectrodes.B,RecordingsitesforMonkeyMfromAC1toAC6(onemoreneuron,recordedinAC3 is not represented on the figure). Each dot represents the location of one neuron recorded during the task. The white ones represent the locations of associative neurons, and the black ones the locations of sensorimotor neurons. C, Recording sites for Monkey Y from AC 2 to AC 3 (same representation as in B). D, Three-dimensional reconstruction of the anterior part of the striatum (transparent gray) of Monkey Y, AC 4 in the background. Data analyses Data analyses All data analyses were performed using conventional statistical proce- dures with the R statistical computing environment (R Development Core Team, 2011). Behavioral analyses. Behavioral analyses were performed on data col- lected during the recordings of 169 TANs (81 from Monkey M; average number of trials, 92; range, 52–131; 88 from Monkey Y, average number of trials, 95; range, 55–117). Force and reward selectivity. To examine the force and reward sensi- tivities of TANs, we performed a two-way ANOVA on the spike count of each of the four determined response periods. We used the amount of force and the amount of reward as the two factors. We considered a TAN as modulated by the amount of force during a period if the p value “force” of the ANOVA was 0.05 in this period. In the same way, a TAN was considered as modulated by the amount of reward in a period if the p value “reward” of the ANOVA was 0.05 in this period. Finally, a TAN was considered as modulated by an interaction between the amount of force and the amount of reward in a period if the p value “interaction” of the ANOVA was 0.05 in this period. This analysis was performed on the entire population of TANs, not only those categorized as responsive, to avoid omission of nonrespon- sive TANs that could nevertheless be sensitive to the amount of a force and/or reward. RTs, which were the times between the onset of the visual stimuli and the time at which the monkey started to increase his pressing force on the lever, were measured only for correct trials. The time at which monkeys started to increase their pressing force on the lever to reach the required force was determined manually by moving a cursor on the force graph trial by trial using LabVIEW. An automatic detection procedure, using the force derivate compared to a threshold, failed to give reliable values of RTs since monkeys were sometimes slightly releasing the lever (and de- creasing their force) before pressing to reach the required force. Thus, manual detection was required. Since, on trials in which RTs could be automatically detected, values were identical to those that we could de- termined manually, we estimated that it was more reliable to analyze all trials with the same method, and did it manually. Data analyses RTs were changed into z-scores for normalization purposes, and a two-way ANOVA was per- formed with required force and expected reward as the two factors. Error rates (ERs; i.e., the total number of errors performed in a condition divided by the total number of trials, both correct and error trials, per- formed in this condition) were calculated and compared with a Pearson’s  2 test. Each p value was corrected by Bonferroni correction and differ- ences considered as significant when p  0.0083 (0.05/6, six possible comparisons). In each condition, the proportion of omission and execu- tion errors was determined by dividing the number of one type of error (execution or omission) by the total number of errors in the condition. For each neuron and each period, we estimated a force selectivity index (FSI) and a reward selectivity index (RSI) to determine the weight of these modulations based on the firing rate for pairs of conditions with a common factor (same reward or same force; Peck et al., 2013). For each selectivity index (SI), we computed SI  (1  2)/[(SS1  SS2)/(df1  df2)], where x is the firing rate during a given period, SSx is the sum of squares, and dfx is the degree of freedom (number of trials minus 1) for each pair of conditions de- scribed below (Peck et al., 2013). To calculate the FSI, we compared the neuronal activity during trials in the high force conditions (Fr and FR) to the neuronal activity during trials in the low force conditions (fr and fR). In the same way, to calculate the RSI, we compared, in the same periods, the neuronal activity during trials in the large reward conditions (fR and FR) with the neuronal activity during trials in the small reward conditions (fr and Fr). An index superior to zero indi- cated, for the force, a stronger modulation in the high force condi- tions, and for the reward, a stronger modulation in the large reward conditions. Similarly, an index inferior to zero indicated, for the force, a stronger modulation in the low force conditions, and for the reward, a stronger modulation in the small reward conditions. Electrophysiological analyses. Electrophysiological data were analyzed only for correct trials performed during the recording sessions. Determination of response periods. Data analyses TAN responses to rewarding stimuli have been described previously as being mainly multiphasic, with a pause followed by a rebound (Aosaki et al., 1995; Apicella et al., 1997, Ravel et al., 1999). This biphasic response could rarely be preceded by a short initial excitation. Thus, we will focus on the classically described pause and rebound (when it follows the pause) or elevation of discharge rate in the following analyses. To evaluate statistically the responses to the oc- currence of the cues and the reward, we set unique test windows for all recorded TANs, and for each TAN, we counted the number of spikes within these windows for each neuron. The test windows were deter- mined on the basis of the population histogram aligned on cues and reward onsets averaged across all recorded TANs, using the following procedure [based on the procedure of Shimo and Hikosaka, (2001)]. A sliding time window with a duration of 100 ms was moved in 10 ms steps starting at the onset of the cue, for the cue responses, and starting at the occurrence of the reward, for reward responses. We established four test windows, two for the decrease of TAN activity after cue and reward occurrence and two for the increase. The sliding window was moved until the averaged firing rate within the window was significantly different from the baseline firing rate (within a 900 ms window before the occur- rence of the cues) for five consecutive steps (two-tailed t test, p  0.001). For detection of a decrease in the firing rate, these significant differences had to be combined with a negative t value, and for detection of an Relation to the time of onset of the change in force. Based on visual inspection of the rasters, the activity of some neurons seemed to be more aligned on the onset of the change in force (OCF) than on the occurrence of the cues. To investigate the influence of the OCF on the neuronal responses and determine whether those responses were trig- gered by the movement or by sensory information (cues), we per- formed the same series of analysis as described previously with the neuronal activity aligned on the OCF of the animals. Different meth- ods described in the literature have been successfully used to investi- gate this question in other brain structures (DiCarlo and Maunsell, 2005; Lee et al., 2010). Electrophysiological recordings hil h k f i The recording electrode was lowered to isolate neurons while the mon- key performed the task. We isolated single neurons by continuously monitoring the waveform of the recorded neuronal impulses on an os- cilloscope. Tonic striatal neurons were distinguished on the basis of their spontaneous firing rate and the duration of their spikes (Kimura et al. 1984; Apicella et al. 1997). The activity of the first well-isolated neuron in a trajectory was recorded for at least ten trials per condition. After re- cording from a TAN, the electrode was moved forward until another TAN was encountered. Data from all TANs recorded were included in analyses. p y g g While the monkeys were performing the task, with head immobilization, extracellular activity of single neurons was recorded with custom-made glass-insulated tungsten microelectrodes based on the technique of Mer- rill and Ainsworth (1972). To record from the basal ganglia structures, a stainless steel guide tube (diameter, 0.6 mm) was lowered below the surface of the dura, and the microelectrode was passed inside the guide and advanced using a manual hydrolic microdrive (M096, Na- rishige). The microelectrode was connected to a preamplifier located in close proximity to the microdrive. The neuronal signal was then amplified 5000 times and filtered at 0.3–1.5 kHz, and spikes were detected on-line by threshold crossing using a window discriminator (Neurolog, Digitimer). The presentation of visual stimuli, the force developed by the animal, the delivery of the reward, and digital pulses from neuronal activity were controlled/collected by a computer using custom-designed software written in LabVIEW (National Instru- ment). The signals from the strain gauge allowing the measure of the force were digitized at 500 Hz. Localization of recordings To assess the localization of our recordings, we obtained a high- resolution magnetic resonance imaging (MRI) scan for each monkey with electrodes positioned (five for Monkey M, six for Monkey Y) in trajectories on which we recorded TANs. Based on the localization of the tip of these electrodes, we extrapolated the inferior/superior, anterior/ posterior, and medial/lateral position of each recorded neuron to gener- J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15217 Nougaret and Ravel • TANs’ Modulation by Force and Reward ate a three-dimensional reconstruction using Brainsight software (Rogue Research; Fig. 1B). One hundred and sixty-nine neurons were localized to the striatum and identified as TANs. Electrophysiological recordings hil h k f i In the anteroposterior plane from the anterior commissure (AC), neurons were recorded from AC 3 to AC 6 and AC 2 to AC 3, for Monkey M and Monkey Y, respectively. For Monkey M, only one neuron was recorded in AC 3 and none in AC 2; these two anteroposterior planes are not illustrated in Figure 1B. Neurons from AC 1 and posterior locations located in the putamen were considered as located in the sensorimotor part of the striatum. Neurons from AC 1 and posterior locations located in the caudate nucleus and all neurons anterior to AC 1 were considered as located in the associative part of the striatum (Parent and Hazrati, 1995). increase in the firing rate, with a positive t value. The onset of the test windows were taken to be the beginning of the window that was the earliest among the five consecutive steps. The 100 ms window was further moved until the averaged firing rate was not significantly different from the baseline firing rate or the t value was no longer of the same sign for five consecutive steps. The offset of the test window was taken to be the beginning of the window that was the earliest among the five consecutive steps. Presence or absence of cues responses. We then determined whether each TAN showed a response. For each trial, we calculated the response firing rate in the four test windows (converted from the spike count within the window) and the baseline firing rate in the control window (900 ms period before onset of the cues). If the difference between the response and the baseline firing rate was statistically significant (Wilcoxon signed rank test, p  0.05), it was judged that the TAN showed a response during this window. For the two windows targeting the decrease of TAN activity, it was verified that significant responses corresponded to decreases of the firing rate. For the two windows targeting the increases of TAN activity, it was also verified that significant responses were increases of the firing rate. Data analyses C, G, Acceptance lev oftheanimalsinthefourconditionsofthetask(fR,lowforce/largereward;FR,highforce/largereward;fr,lowforce/smallreward;Fr,highforce/smallreward).D,H,Meanoftheforcedevelop bytheanimalsinthefourconditionsofthetask.Darkgraylines,Largereward;lightgraylines,smallreward;thicklines,highforce;thinlines,lowforce.Asterisksindicateforthereactiontimest influenceofeachfactorontheanimal’sbehavior(two-wayANOVA.**p0.05;***p0.001)andfortheerrorratesthedifferencesamongconditions(Pearson’s 2test;***p0.001,corrected fR FR fr Fr Percentage of acceptance 100 95 90 85 80 l l l l e e C -1 0 1 2 3 1000 800 600 400 200 0 4 FR Fr fR fr Mean force developped on the lever (grams) D 0 10 20 30 40 50 Percentage of error rates l l *** *** *** *** r R Low Force High Force B 270 280 290 300 310 Average reaction time (ms) l l r R p.reward *** p.force > 0.05 A fR FR fr Fr Percentage of acceptance 100 95 90 85 80 l l l l -1 0 1 2 3 1000 800 600 400 200 0 4 FR Fr fR fr e Mean force developped on the lever (grams) C D C B D Average reaction time (ms) Percentage of error rates Percentage of acceptance r R 270 280 290 300 310 Average reaction time (ms) l l r R p.reward > 0.05 p.force > 0.05 E 0 10 20 30 40 50 Percentage of error rates l l r R ** *** *** r R F fR FR fr Fr Percentage of acceptance 100 95 90 85 80 l l l l Time -1 0 1 2 3 1000 800 600 400 200 0 4 Mean force developped on the lever (grams) G H Time E F G Percentage of error rates Percentage of acceptance Figure 2. A–H, Behavioral results for Monkey M (A–D) and Monkey Y (E–H). A, E, Reaction times of the animals in the four conditions of the task. R, Small reward; R, large reward; solid black line, high force; dashed gray line, low force. The error bars represent the SEM. B, F, Error rates of the animals in the four conditions of the task (same conventions as in A, E). C, G, Acceptance level oftheanimalsinthefourconditionsofthetask(fR,lowforce/largereward;FR,highforce/largereward;fr,lowforce/smallreward;Fr,highforce/smallreward).D,H,Meanoftheforcedeveloped bytheanimalsinthefourconditionsofthetask.Darkgraylines,Largereward;lightgraylines,smallreward;thicklines,highforce;thinlines,lowforce.Asterisksindicateforthereactiontimesthe influenceofeachfactorontheanimal’sbehavior(two-wayANOVA.**p0.05;***p0.001)andfortheerrorratesthedifferencesamongconditions(Pearson’s 2test;***p0.001,corrected). trials only (7480 from Monkey M, 8468 from Monkey Y). RTs were significantly shorter in the large reward conditions than in the small reward ones in Monkey M (two-way ANOVA on RT z-score, preward  7.97.10 15, F  60.59), and there was no sig- nificant difference among the RTs in the low or high force con- ditions for this monkey (Fig. 2A). There was no significant difference among the RTs of Monkey Y (Fig. 2E). Error rates A complementary analysis was performed, in situations in which a response was detected when the neuronal activity was aligned on cue occurrence and on the OCF, to specify which of these two events was most likely to modulate the response. A Wilcoxon rank sum test was performed to determine whether the detected pauses, aligned on the cues and on the OCF, were significantly different (in terms of magnitude) and which one was more pronounced. The same tests were performed for the elevation of the discharge rate. ERs were computed from the total number of trials performed by the animals (9115 from Monkey M, 10221 from Monkey Y), including correct and error trials. The ERs were significantly higher in the small reward than large reward conditions for the same force developed (low force, p  3.27.10 14 and 1.44.10 17,  2  57.57 and 72.79 for Monkeys M and Y, respectively; high force, p  5.43.10 43 and 1.49.10 31,  2  188.94 and 136.57 for Monkeys M and Y, respectively; Fig. 2B,F). Moreover, for the same expected reward, the ERs were significantly higher in the high than low force conditions (small reward, p  8.05.10 53 and 0.00058,  2  233.99 and 11.85 for Monkeys M and Y, respec- tively; large reward, p  9.28.10 21,  2  87.31 for Monkey M, nonsignificant for Monkey Y). Data analyses However, the electrophysiological properties of TANs made those methods unsuitable. Thus, we used an alternative method in which we successively defined periods of decrease and increase of activity around the OCF, determined whether each TAN Nougaret and Ravel • TANs’ Modulation by Force and Reward 15218 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 270 280 290 300 310 Average reaction time (ms) l l r R 0 10 20 30 40 50 Percentage of error rates l l 270 280 290 300 310 Average reaction time (ms) l l r R 0 10 20 30 40 50 Percentage of error rates l l r R p.reward *** p.force > 0.05 p.reward > 0.05 p.force > 0.05 *** *** *** *** ** *** *** fR FR fr Fr Percentage of acceptance 100 95 90 85 80 r R l l l l fR FR fr Fr Percentage of acceptance 100 95 90 85 80 l l l l -1 0 1 2 3 1000 800 600 400 200 0 4 Time FR Fr fR fr Low Force High Force Mean force developped on the lever (grams) -1 0 1 2 3 1000 800 600 400 200 0 4 Mean force developped on the lever (grams) A B C D E F G H Time Figure 2. A–H, Behavioral results for Monkey M (A–D) and Monkey Y (E–H). A, E, Reaction times of the animals in the four conditions of the task. R, Small reward; R, large reward; solid bla line, high force; dashed gray line, low force. The error bars represent the SEM. B, F, Error rates of the animals in the four conditions of the task (same conventions as in A, E). Data analyses showed a pause or an elevation of discharge rate, and examined the force and reward sensitivities of each TAN when its activity was aligned on the time of the OCF. To compare the amplitude of the response period of the whole population of TANs aligned on the occurrence of the visual stimuli versus that aligned on the OCF, a two-tailed t test was performed between the average firing rates of each significant bin defining the test window aligned on the cues versus each significant bin defining the test window aligned on the OCF. Behavioral results: modulation of the behavioral responses by the force required and the expected reward size Reaction times Average RTs to start increasing the force on the lever after the occurrence of visual stimuli were computed from the correct J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15219 Nougaret and Ravel • TANs’ Modulation by Force and Reward 0 4.5 5.0 5.5 6.0 6.5 7.0 200 400 600 800 -30 -20 -10 0 10 20 Spike/s t-value Time from cues (ms) 4.5 5.0 5.5 6.0 6.5 7.0 -30 -20 -10 0 10 20 t-value 0 200 400 600 800 Cues Reward Spike/s A B f R F R f r F r Time from reward (ms) −1000 0 2000 3000 0 10 0 10 0 10 0 10 Spk density Spk density Spk density Spk density C 1000 −1000 0 2000 3000 0 10 0 10 0 10 0 10 f R F R f r F r D 1000 0 10 0 10 0 10 0 10 −1000 0 1000 Time from reward (ms) Time from reward (ms) E F f R F R f r F r −1000 0 1000 0 15 0 15 0 15 0 15 f R F R f r F r Time from cues (ms) Time from cues (ms) ure3. Periodsofanalysisaftertheoccurrenceofcuesandreward.A,Populationactivityalignedoncueoccurrenceandcorrespondingtvaluesofthecomparisonbetweenthebaselineactiv theactivityduringa100msslidingwindowbeginningaftertheoccurrenceofthecues.Thetwograyrectanglesillustratethetwoperiodsofanalysisbasedonthestatisticaldifferencefoundw baseline. The pause occurred between 60 and 280 ms, and the elevation of the discharge rate between 300 and 530 ms after cue occurrence. B, Population histogram aligned on rewa rrence (same conventions as in A). The pause occurred between 20 and 280 ms, and the elevation of the discharge rate between 450 and 610 ms after reward occurrence. C–F, Raster plots examplesofTANs.Eachlinerepresentsatrial,andeachpointrepresentsaspike;thesmoothedlinesontherasterrepresentthespikedensity(50).Trialsarerankedaccordingtothefo itionsofthetask.C,Neuronshowingapositiveforceeffectduringtheelevationofthedischargerateaftertheoccurrenceofthevisualstimuli.D,Neuronshowinganegativerewardeffectduri elevation of the discharge rate after the occurrence of the cues E Neuron showing an interaction effect during the pause after reward occurrence Its activity decreased critically in the garet and Ravel TANs Modulation by Force and Reward J. Behavioral results: modulation of the behavioral responses by the force required and the expected reward size Neurosci., November 11, 2015 35(45):15214 15226 1521 0 4.5 5.0 5.5 6.0 6.5 7.0 200 400 600 800 -30 -20 -10 0 10 20 Spike/s t-value Time from cues (ms) Cues A 4.5 5.0 5.5 6.0 6.5 7.0 -30 -20 -10 0 10 20 t-value 0 200 400 600 800 Reward Spike/s B Time from reward (ms) B Reward A Time from cues (ms) f R F R f r F r −1000 0 2000 3000 0 10 0 10 0 10 0 10 Spk density Spk density Spk density Spk density C 1000 −1000 0 0 10 0 10 0 10 0 10 D 1000 f R F R f r F r Time from cues (ms) Time from cues (ms) Time from reward (ms) 2000 3000 f R F R f r F r 0 10 0 10 0 10 0 10 −1000 0 1000 Time from reward (ms) Time from reward (ms) E F −1000 0 1000 0 15 0 15 0 15 0 15 f R F R f r F r E C ewar F m cue D Time from reward (ms) Time from reward (ms) Figure3. Periodsofanalysisaftertheoccurrenceofcuesandreward.A,Populationactivityalignedoncueoccurrenceandcorrespondingtvaluesofthecomparisonbetweenthebaselineactivity andtheactivityduringa100msslidingwindowbeginningaftertheoccurrenceofthecues.Thetwograyrectanglesillustratethetwoperiodsofanalysisbasedonthestatisticaldifferencefoundwith the baseline. The pause occurred between 60 and 280 ms, and the elevation of the discharge rate between 300 and 530 ms after cue occurrence. B, Population histogram aligned on reward occurrence (same conventions as in A). The pause occurred between 20 and 280 ms, and the elevation of the discharge rate between 450 and 610 ms after reward occurrence. C–F, Raster plots of fourexamplesofTANs.Eachlinerepresentsatrial,andeachpointrepresentsaspike;thesmoothedlinesontherasterrepresentthespikedensity(50).Trialsarerankedaccordingtothefour conditionsofthetask.C,Neuronshowingapositiveforceeffectduringtheelevationofthedischargerateaftertheoccurrenceofthevisualstimuli.D,Neuronshowinganegativerewardeffectduring the elevation of the discharge rate after the occurrence of the cues. E, Neuron showing an interaction effect during the pause after reward occurrence. Its activity decreased critically in the FR condition. F, Neuron showing a negative reward effect during the elevation of the discharge rate after the reward occurrence. Table 1. Responses of TANs in both monkeys, after cue and reward occurrence Responsive Cues Reward Cues and reward Pause 105/169 (62.1%) 57/169 (33.7%) 67/169 (39.6%) 19/169 (11.2%) Elevation 87/169 (51.5%) 58/169 (34.4%) 47/169 (27.8%) 18/169 (10.7%) Pause and elevation 36/169 (21.3%) 23/169 (13.6%) 16/169 (9.5%) 3/169 (1.8%) At least one 136/169 (80.5%) 92/169 (54.4%) 98/169 (58.0%) 54/169 (32.0%) Therowheadingsindicatethetypeofmodulation(apause,anelevationofthedischargerate,both,oratleastone of the two). The columns indicate the period of modulation (after the cues and/or the reward, only after the cues, only after the reward, or after cues and reward). Behavioral results: modulation of the behavioral responses by the force required and the expected reward size Table 1. Responses of TANs in both monkeys, after cue and reward occurrence reward (fR); high force/large reward (FR); low force/small reward (fr); high force/small reward (Fr) (Fig. 2C,G). In both monkeys, the amount of reward seemed to be more important than the level of effort in the decision of animals to perform the task. In the fR condition, monkeys performed the action in 99.3% (Monkey M) and 97.6% (Monkey Y) of trials presented to them. On the contrary, in the Fr conditions, monkeys per- formed the action in 82.4% (Monkey M) and 83.7% (Monkey Y) of trials. For both monkeys, “intermediate” conditions (FR and fr) were ranked following the same order: they accepted FR trials more frequently (97% for Monkey M, 95.3% for Monkey Y) than fr trials (92% for Monkey M, 94.7% for Mon- key Y). The relatively low level of acceptance in the Fr condi- tion suggests that an higher effort and smaller amount of reward are perceived by the animals as being associated with a negative motivational value. Electrophysiological results l f h d f g One-hundred and sixty-nine neurons were localized to the striatum and identi- fied as TANs. All recorded neurons were located in the associative (138 neurons, 81.7%) and the sensorimotor (31 neurons, 18.3%) parts of the striatum. We did not investi- gate the ventral striatum. No obvious dif- ference between each population of neurons during the defined periods of analysis in the encoding of the force and the reward was found. Neurons with pos- itive and negative FSI (indicating a stron- ger variation in activity in high force conditions if positive, and in low force conditions if negative) and RSI (indicat- ing a stronger variation in activity in large reward conditions if positive, and in small reward conditions if negative) were inter- mixed throughout the regions of the stria- tum from which we recorded. −1.5 1.5 −1.5 1.5 C Reward Selectivity Index −1.5 1.5 −1.5 1.5 Elevation of discharge rate following cues occurrence Elevation of discharge rate following reward occurrence D C Reward Selectivity Index −1.5 1.5 −1.5 1.5 Elevation of discharge rate following cues occurrence Elevation of discharge rate following cues occurrence Force Selecti it Inde −1.5 1.5 −1.5 1.5 Elevation of discharge rate following reward occurrence D C D Elevation of discharge rate following reward occurrence Response of TANs to motivational relevant stimuli and to reward We recorded from 169 TANs in two mon- keys. The firing rate of TANs was 6.01  1.1 (mean  SD; n  169), ranging from 2.7 to 9.1 spikes  s 1. Force Selectivity Index 0 3000 4000 1 -1 Spk density 4 8 7 6 5 4 Fr fr FR fR E 0 Spk density 8 7 6 5 1 Time from cues (s) -1 Time from cues (s) Force effect only (n = 29) Reward effect only (n = 20) Force Selectivity Index F Figure 4. A–D, Force and reward selectivity indices (FSI and RSI) of TANs during the pause (A, B) and the elevation of the discharge rate (C, D) after cue (A, C) and reward occurrence (B, D). Scatter plots of force versus reward selectivity indices for each individual neuron in both periods. Electrophysiological results l f h d FSIs of 0 indicate higher firing rate in the high force conditions, and indices of 0 indicate Force Selectivity Index Force Selectivity Index Force Selectivity Index 0 3000 4000 1 -1 Spk density 4 8 7 6 5 4 Fr fr FR fR E 0 Spk density 8 7 6 5 1 Time from cues (s) -1 Time from cues (s) Force effect only (n = 29) Reward effect only (n = 20) Force Selectivity Index F F E Data from the literature have shown that TANs respond to unpredictable stim- uli, associated with reward (Aosaki et al., 1995; Apicella et al., 1997). In line with previous reports, we found that most TANs responded to motivational relevant visual stimuli (called “cues” in the follow- Spk density higher firing rates in the low force conditions. RSIs of 0 in- dicate higher firing rates in the large reward conditions, and indices of 0 indicate higher firing rates in the small reward conditions. Symbols indicate the significance of the modula- tion for each neuron when we performed a two-way ANOVA (squares, force; filled circles, reward; crosses, interaction; smallfilledcircles,neuronswithoutsignificantmodulation).E, Average spike density (  80) of the neurons modulated only by the amount of force (n  29) during the elevation of the discharge rate after the occurrence of the cues. The hori- zontaldashedlinerepresentsthebaselineactivityofthispop- ulation of neurons. The four shades represent, from the black to the lightest gray, the high force/small reward, low force/ small reward, high force/large reward, and small force/large reward conditions, respectively. F, Average spike density (  80) of the neurons modulated only by the amount of reward (n  20) during the elevation of the discharge rate after the occurrence of the cues (same conventions as in E). higher firing rates in the low force conditions. RSIs of 0 in- dicate higher firing rates in the large reward conditions, and indices of 0 indicate higher firing rates in the small reward conditions. Symbols indicate the significance of the modula- tion for each neuron when we performed a two-way ANOVA (squares, force; filled circles, reward; crosses, interaction; smallfilledcircles,neuronswithoutsignificantmodulation).E, Average spike density (  80) of the neurons modulated only by the amount of force (n  29) during the elevation of the discharge rate after the occurrence of the cues. The hori- zontaldashedlinerepresentsthebaselineactivityofthispop- ulation of neurons. Acceptance level Acceptance level p For each monkey, we ranked the level of motivation in each of the four conditions by computing an acceptance level from the num- ber of omission errors. For both monkeys, the four conditions could be ranked in the same order of preference: low force/large 15220 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward Force Selectivity Index −1.5 1.5 −1.5 1.5 Force Reward Interaction C ensity 8 7 6 E ensity 8 7 6 Force effect only (n = 29) Reward effect only (n = 20) N. S. Reward Selectivity Index −1.5 1.5 −1.5 1.5 −1.5 1.5 −1.5 1.5 Force Selectivity Index Reward Selectivity Index −1.5 1.5 −1.5 1.5 Pause following cues occurrence Pause following reward occurrence Elevation of discharge rate following cues occurrence Elevation of discharge rate following reward occurrence D F A B Figure 2, D and H, represents the aver- age force developed by each monkey in the four conditions and shows that the monkeys did not modulate their force on the lever as a function of the expected reward. Force Reward Interaction N. S. Reward Selectivity Index −1.5 1.5 −1.5 1.5 −1.5 1.5 −1.5 1.5 Pause following cues occurrence Pause following reward occurrence A B Force Selectivity Index −1.5 1.5 −1.5 1.5 Force Reward Interaction C 0 3000 4000 1 -1 Spk density 4 8 7 6 5 4 Fr fr FR fR E 0 Spk density 8 7 6 5 1 Time from cues (s) -1 Time from cues (s) Force effect only (n = 29) Reward effect only (n = 20) N. S. Reward Selectivity Index −1.5 1.5 −1.5 1.5 −1.5 1.5 −1.5 1.5 Force Selectivity Index Reward Selectivity Index −1.5 1.5 −1.5 1.5 Pause following cues occurrence Pause following reward occurrence Elevation of discharge rate following cues occurrence Elevation of discharge rate following reward occurrence D F A B Figure 4. A–D, Force and reward selectivity indices (FSI and RSI) of TANs during the pause (A, B) and the elevation of the discharge rate (C, D) after cue (A, C) and reward occurrence (B, D). Scatter plots of force versus reward selectivity indices for each individual neuron in both periods. FSIs of 0 indicate higher firing rate in the high force conditions, and indices of 0 indicate 5 Pause following reward occurrence B A Electrophysiological results l f h d The four shades represent, from the black to the lightest gray, the high force/small reward, low force/ small reward, high force/large reward, and small force/large reward conditions, respectively. F, Average spike density (  80) of the neurons modulated only by the amount of reward (n  20) during the elevation of the discharge rate after the occurrence of the cues (same conventions as in E). Figure 4. A–D, Force and reward selectivity indices (FSI and RSI) of TANs during the pause (A, B) and the elevation of the discharge rate (C, D) after cue (A, C) and reward occurrence (B, D). Scatter plots of force versus reward selectivity indices for each individual neuron in both periods. FSIs of 0 indicate higher firing rate in the high force conditions, and indices of 0 indicate J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15221 Nougaret and Ravel • TANs’ Modulation by Force and Reward responded to the cues, to the reward, or to both. Fifty-seven neurons responded by a pause only to the cues, 67 neurons re- sponded only to the reward, and 19 neurons showed a pause to both the cues and the reward. Similarly, 58 neurons responded by an elevation of the discharge rate only to the cues, whereas 47 only showed this elevation after the reward. Eighteen neurons pre- sented an elevation of the discharge rate to both the cues and the reward. The number of neurons showing modulations to both events (N  19 and 18, respectively, for pause and elevation of the discharge rate) is not significantly different from the number we could have observed by chance. We calculated the percentile re- flecting the chance to find this number of neurons that would respond to both events, replicated 20,000 times. We got an equiv- alent p value of 0.025, which means that the population of neurons showing a response to the cues is independent of the one showing a response to the reward for both pause and elevation of the discharge rate. ing text) while the monkeys performed the instrumental task. Based on the population histogram aligned on the cues onset (Fig. 3A), a significant decrease (called “pause” in the following text) in TAN activity after the occurrence of the cues was found between 60 and 280 ms. Electrophysiological results l f h d In summary, in our task, TANs did not respond preferentially to the cues or the reward, and responses to each event were inde- pendent; i.e., a response to one event did not preclude nor predict a response to the other, unlike what has been shown in previous reports using different behavioral tasks, in which neurons tend to lose their responses to the reward during learning of the associa- tion of reward to a conditioned stimulus. After the occurrence of the cues, we observed an equivalent number of neurons showing a pause or an elevation of the discharge rate, whereas after the reward occurrence, the predominant response was a pause. Modulation of TAN activity by the amounts of force and reward Responses of TANs have been shown to be modulated by the motivational significance of stimuli (Ravel et al., 1999, 2003; Blazquez et al., 2002; Joshua et al., 2008). In our task, the visual cues carried different motivational significance depending on the required force and the expected reward. We examined whether this motivational significance, translated behaviorally as shown by the acceptance levels and error rates of the animals and which should be computed from the information on force and reward, would also modulate the activity of TANs, qualitatively or quan- titatively. A two-way ANOVA on the spike count within each of the four determined response periods was performed. For better reading, a modulation of the neuronal activity by the amount of force will be called a “force effect,” and a modulation by the amount of reward a “reward effect.” All neurons, not only those categorized as responsive, were taken into account to include neurons that would respond in only two conditions and show a modulation by the amount of force or reward but might not be considered as responsive if all conditions were combined. Results described in the following paragraphs are displayed in Table 2. After the occurrence of the cues, only 12% (21 of 169) of TANs showed a modulation during the pause: 4% (7 of 169) showed a force effect, 3% (6 of 169) a reward effect, and 5% (8 of 169) an interaction effect. Conversely, during the elevation of the dis- charge rate after the occurrence of the cue, we observed a strong modulation of the neuronal activity, with 37% (62 of 169) of TANs modulated. Electrophysiological results l f h d Surprisingly, we observed a similar proportion of TANs showing a reward effect (29 of 169; 17%) and a force effect (34 of 169; 20%). TANs showed an interaction effect (10 of 169; 6%) usually associated with a force or reward main effect (7 of 169; 4%). We noticed that very few neurons showed both force and reward main effects (4 of 169; 1%), suggesting that these types of information were encoded by independent groups of neurons. We calculated the percentile reflecting chance to find the number of neurons that would encode both force and reward g p ) TANs show responses to unpredictable rewards and usually lose their responses to reward when it is associated with a condi- tioned stimulus (Apicella et al., 1997; Ravel et al., 2003). Here, the majority of TANs (98 of 169; 58%) showed a modulation of their activity in response to the reward, even though it was preceded by visual cues and predictable. The population of TANs showing a pause (67 of 169; 40%) was larger than that showing an elevation of the discharge rate (47 of 169; 28%;  2  4.78, p  0.029). Nine percent (16 of 169) showed a pause followed by a rebound. There was no significant difference between the number of responses to the cues and to the reward ( 2  0.30, p  0.58), for both pauses ( 2  1.03, p  0.31) and elevations of the discharge rate ( 2  1.38, p  0.24). As for the cues, we checked, for modulations after the reward, that the neurons showing a pause or an elevation in discharge rate were belonging to comparable populations. We found no difference in the average firing rate nor in the average ISI of neurons showing a pause or a combination of a pause followed by a rebound (N  138; average firing rate, 6  1.2; average ISI, 159.13  34.38) and of neurons showing only an elevation of discharge rate (N  31; average firing rate, 6.07  0.77; average ISI, 156.77  19.26; t test, p  0.05). It has been shown that TAN responses transfer from the re- ward to the preceding conditioned stimulus during learning (Apicella et al., 1997). However, in our task, we found a large number of neurons presenting modulations after cues and also after reward occurrence. Electrophysiological results l f h d A significant increase (called “rebound” if preceded by a pause or “elevation of discharge rate” in the following text) occurred between 300 and 530 ms. Following the same procedure for the reward occurrence (Fig. 3B), the pause in TAN activity was found between 20 and 280 ms, and the elevation of the discharge rate occurred between 450 and 610 ms. In re- sponse to cues and reward, no significant difference in the num- ber of neurons showing a pause ( 2  1.85, p  0.17 for cues,  2  0, p  1 for reward) or in the number of neurons showing an elevation of the discharge rate ( 2  3.42, p  0.06 for cues,  2  0.49, p  0.49 for reward) was found between the two monkeys. Consequently, data from Monkey M and Monkey Y were pooled for further analyses of cues and reward responses. Results described below are summarized in Table 1. The ma- jority of TANs (92 of 169; 54%) showed a modulation of their activity in response to the cues: 33% (57 of 169) showed a pause, 34% (58 of 169) an elevation of the discharge rate, and 14% (23 of 169) a pause followed by a rebound. Previous reports on TAN responses to appetitive stimuli have mainly described decreases in activity (pauses; Aosaki et al., 1995; Apicella et al., 1997; Ravel et al., 1999; Morris et al., 2004). Given the large number of eleva- tions in the discharge rate observed in our data, we checked that neurons recorded were not belonging to different populations by comparing the average firing rates and interspike intervals (ISIs). There was no difference in the average firing rate nor in the aver- age ISI of neurons showing a pause or a combination of a pause followed by a rebound (N  134; average firing rate, 6.02  1.1; average ISI, 158.17  31.86) or of neurons showing only an ele- vation of discharge rate (N  35; average firing rate, 5.99  1.21; average ISI, 160.72  33.43; t test, p  0.05). Electrophysiological results l f h d “All”correspondstoneuronsshowingaforce,areward,and/oraninteractioneffect.Thecolumnsindicateperiods(cuesandreward)andtypesofmodulation(pauseorelevationindischargerate).Ontheright,Venndiagramsreprensent the distribution of neurons showing a force (top) or a reward (bottom) effect during the elevations of the discharge rate after the cues, the reward, or both. tions (Fig. 4A,B). During the elevation of the discharge rate fol- lowing the occurrence of the cues, more force-modulated neurons exhibited a positive FSI than a negative one (27 of 34, 79% vs 7 of 34, 21%; mean, 0.36; binomial test, p  0.001; Fig. 4C). Additionally, during the same period, more reward- modulated neurons exhibited a negative RSI than a positive one (22 of 29, 76% vs 7 of 29, 24%; mean, 0.38; binomial test, p  0.01; Fig. 4C). None of the neurons was part of the two groups of neurons (positive FSI and negative RSI). We calculated the per- centile reflecting the chance to find no neuron that would encode both a positive FSI and negative RSI (replicated 20,000 times). We got an equivalent p value of 0.025, meaning that the group of neurons showing a positive FSI is different from the one show- ing a negative RSI. Thus, the encoding of a positive FSI excludes the encoding of a negative RSI, and vice versa. This result sup- ports the idea that positive force and negative reward information were integrated by two populations of neurons during the eleva- tion of the discharge rate in neuronal activity after the cues (Fig. 4E,F). One population of neurons encoded the force informa- tion carried by the cues when the force to exert on the lever, the cost of the action, was high, whereas another one encoded the reward information carried by the cues when the expected re- ward, the benefit of the action, was small (Fig. 4C). information (replicated 20,000 times). We got an equivalent p value of 0.025, meaning that the group of neurons showing a force effect is independent of the one showing a reward effect. Thus, the encoding of force was neither predictive nor preclusive to the encoding of reward and vice versa. Figure 3 shows two examples of neurons illustrating the force effect (Fig. 3C) and the reward effect (D) during this period. Electrophysiological results l f h d The neuron in Figure 3C has more pronounced pauses and elevations of the discharge rate in the high force conditions (FR and Fr) compared to the low force conditions (fR and fr), whereas the neuron in Figure 3D shows a stronger elevation of the discharge rate in the small reward con- ditions (Fr and fr) than in the large reward ones (fR and FR). After the reward occurrence, 24% (40 of 169) of TANs showed a modulation during the pause: 7% (12 of 169) showed a force effect, 11% (18 of 169) a reward effect, and 9% (15 of 169) an interaction effect. Only 10% (4 of 40) of the modulated neurons showed a combination of two of the three effects (force, reward, and/or interaction). Figure 3E shows an example of neuron with an interaction effect during the pause following the reward oc- currence. This neuron shows a stronger response in the FR con- dition compared to the fR condition and does not respond in fr and Fr conditions. During the elevation of the discharge rate after the reward occurrence, 25% (43 of 169) showed a modulation. A larger proportion of neurons showed a reward effect (26 of 169; 15%) than a force effect (12 of 169; 7%;  2  5.01; p  0.025) or an interaction effect (9 of 169;  2  8.16; p  0.004). Only 9% (4 of 43) of the modulated neurons showed a combination of two of the three effects (force, reward, and/or interaction). Figure 3F shows an example of neuron with a reward effect during the elevation of the discharge rate following the reward occurrence, with a stronger elevation in the small reward conditions (fr/Fr) compared to the large ones (fR/FR). During the pause following the reward occurrence, 83% (10 of 12) of TANs modulated by the amount of force exhibited a neg- ative FSI, against only 17% (2 of 12) exhibiting a positive FSI (mean, 0.45; binomial test, p  0.05). During the same period, similar proportions of neurons modulated by the amount of re- ward exhibited a negative RSI (8 of 18; 44%) and a positive one (10 of 18; 56%). Only two neurons were included in both popu- lations. Electrophysiological results l f h d We thus examined whether the same population of neurons responded to the cues, the reward, or both or whether there were distinct populations. We found that neu- rons showing significant pause or elevation in their firing rate 15222 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward Table 2. The row headings indicate the type of modulation (all, force effect, positive FSI, negative FSI, reward effect, positive RSI, and negative RSI) or an interaction effect e c r o f a g ni w o h s s n o r u e n f o n oit u birtsi D d r a w e R s e u C or a reward effect during the elevations of discharge rate Pause Elevation Pause Elevation % 7 . 6 3 ( 9 6 1 / 2 6 ) % 4 . 2 1 ( 9 6 1 / 1 2 ll A ) 40/169 (23.7%) 43/169 (25.4%) Force effect Force effect 7/21 (33.3%) 34/62 (54.8%) 12/40 (30%) 12/43 (27.9%) FSI+ 4/7 (57.1%) 27/34 (79.4%) 2/12 (16.7%) 5/12 (41.7%) ) % 3 . 8 5 ( 2 1 / 7 ) % 3 . 3 8 ( 2 1 / 0 1 ) % 6 . 0 2 ( 4 3 / 7 ) % 9 . 2 4 ( 7 / 3 -I S F Reward effect 6/21 (28.6%) 29/62 (46.8%) 18/40 (45%) 26/43 (60.5%) Reward effect RSI+ 5/6 (83.3%) 7/29 (24.1%) 10/18 (55.6%) 6/26 (23.1%) ) % 9 . 5 7 ( 9 2 / 2 2 ) % 7 . 6 1 ( 6 / 1 -I S R 8/18 (44.4%) 20/26 (76.9%) Interaction effect 8/21 (38.1%) 10/62 (16.1%) 15/40 (37.5%) 9/43 (20.1%) 33 1 11 d r a w e R s e u C 18 11 15 Cues Reward “All”correspondstoneuronsshowingaforce,areward,and/oraninteractioneffect.Thecolumnsindicateperiods(cuesandreward)andtypesofmodulation(pauseorelevationindischargerate).Ontheright,Venndiagramsreprensent the distribution of neurons showing a force (top) or a reward (bottom) effect during the elevations of the discharge rate after the cues, the reward, or both. Table 2. The row headings indicate the type of modulation (all, force effect, positive FSI, negative FSI, reward effect, positive RSI, and negative RSI) or an interaction effect 15 “All”correspondstoneuronsshowingaforce,areward,and/oraninteractioneffect.Thecolumnsindicateperiods(cuesandreward)andtypesofmodulation(pauseorelevationindischargerate).Ontheright,Venndiagramsreprensent the distribution of neurons showing a force (top) or a reward (bottom) effect during the elevations of the discharge rate after the cues, the reward, or both. Electrophysiological results l f h d During the elevation of the discharge rate following the reward occurrence, a similar proportion of neurons modulated by the amount of force exhibited a positive FSI (5 of 12; 42%) and a negative FSI (7 of 12; 58%), whereas most of the neurons mod- ulated by the amount of reward exhibited a negative RSI (20 of 26; 77%), and few a positive one (6 of 26; 23%; mean, 0.28; bino- mial test, p  0.01; Fig. 4D). The encoding of the amount of force during the elevation of the discharge rate following the cues and the encoding of the amount of force during the elevation of the Weight and direction of activity modulation by force and reward after the occurrence of the cues and the reward An FSI and an RSI were estimated for each neuron in each period (see Materials and Methods). Results are detailed in the following section and are summarized in Table 2. During the pause follow- ing the occurrence of the cues, the small number of significant neurons did not allow us to test the direction of these modula- Weight and direction of activity modulation by force and reward after the occurrence of the cues and the reward An FSI and an RSI were estimated for each neuron in each period (see Materials and Methods). Results are detailed in the following section and are summarized in Table 2. During the pause follow- ing the occurrence of the cues, the small number of significant neurons did not allow us to test the direction of these modula- J. Electrophysiological results l f h d Neurosci., November 11, 2015 • 35(45):15214–15226 • 15223 Nougaret and Ravel • TANs’ Modulation by Force and Reward 2000 3000 −1 0 1 Time from OCF (s) Aligned on the OCF B 0.7 t-value 20 10 0 -0.5 -0.3 -0.1 0.1 0.3 0.5 0.9 Spike/s 4.5 5 5.5 6 6.5 7 -10 -20 -30 Time from OCF (s) Aligned on the OCF B B A Time from cues (s) Time from cues (s) Time from OCF (s) C Cues OCF Pause 9 48 48 10 9 Cues > OCF OCF > Cues Cues Cues > OCF 29 Cues = OCF Cues Cues > OCF Cues = OCF Reward effect (N = 45) Force effect (N = 50) N = 16 N = 6 N = 16 N = 12 N = 12 N = 7 N = 16 N = 10 D Reward effect (N = 45) N = 12 N = 7 N = N = cues (s) cues (s) C Cues OCF Pause 9 48 48 10 9 Cues > OCF OCF > Cues 29 Cues = OCF Cues = OCF D Force effect (N = 50) N = 16 N = 6 N = 16 N = 12 D C Force effect (N = 50) Cues Cues > OCF Cues Cues > OCF Reward effect (N = 45) N = 12 N = 7 N = 16 N = 10 Reward effect (N = 45) N = 12 N = 7 N = N = Reward effect (N = 45) N = 12 N = 7 N = 16 N = 10 Reward effect (N = 45) N = 12 N = 7 N = N = Figure5. Influenceoftheonsetofthechangeinforceonthepauseandtheelevationofthedischargerateaftercueoccurrence.A,ExampleoftheactivityofaTAN(allconditionsmingled).Left, Activityisalignedonthecueoccurrence.Center,ActivityisalignedoncueoccurrenceandsortedbythetimeoftheOCFoftheanimal(down,trialwiththeshortestRT;up,trialwiththelongestRT). Right, Activity aligned on the OCF. This neuron exhibited a more pronounced pause and a more pronounced elevation of the discharge rate. B, Population histogram aligned on the OCF (same conventionsasinFig.3A).Thepauseoccurredbetween190and10ms,andtheelevationofthedischargeratebetween0and250msaroundtheOCF.Theperiodofpauseisnarrowerandmore pronounced when aligned on the OCF. C, Venn diagrams reprensent the number of neurons showing a pause (top) or an elevation of the discharge rate (bottom) when the activity was aligned on cue occurrence (left), the OCF (right), and both. Electrophysiological results l f h d Neurosci., November 11, 2015 • 35(45):15214–15226 • 15223 3000 −1 0 2000 1 −1 0 2000 3000 1 −1 0 1 10 0 2 4 6 8 Spk density Time from cues (s) Time from cues (s) Time from OCF (s) B 0.7 t-value 20 10 0 -0.5 -0.3 -0.1 0.1 0.3 0.5 0.9 Spike/s 4.5 5 5.5 6 6.5 7 -10 -20 -30 Time from OCF (s) A Aligned on cues occurence Aligned on cues occurence Sorted by the OCF Aligned on the OCF C Reward effect (N = 45) Force effect (N = 50) N = 16 N = 6 N = 16 N = 12 N = 12 N = 7 N = 16 N = 10 D Cues OCF Pause 9 48 48 10 9 Cues > OCF OCF > Cues Cues Cues > OCF 29 Cues = OCF Reward effect (N = 45) N = 12 N = 7 N = N = Cues Cues > OCF Cues = OCF Figure5. Influenceoftheonsetofthechangeinforceonthepauseandtheelevationofthedischargerateaftercueoccurrence.A,ExampleoftheactivityofaTAN(allconditionsmingled).Left, Activityisalignedonthecueoccurrence.Center,ActivityisalignedoncueoccurrenceandsortedbythetimeoftheOCFoftheanimal(down,trialwiththeshortestRT;up,trialwiththelongestRT). Right, Activity aligned on the OCF. This neuron exhibited a more pronounced pause and a more pronounced elevation of the discharge rate. B, Population histogram aligned on the OCF (same conventionsasinFig.3A).Thepauseoccurredbetween190and10ms,andtheelevationofthedischargeratebetween0and250msaroundtheOCF.Theperiodofpauseisnarrowerandmore pronounced when aligned on the OCF. C, Venn diagrams reprensent the number of neurons showing a pause (top) or an elevation of the discharge rate (bottom) when the activity was aligned on cue occurrence (left), the OCF (right), and both. For this latter case, the numbers of neurons showing a statistically significant or nonsignificant pause or elevation in discharge rate with one or the otheralignmentareindicatedatthebottomofeachdiagram.D,Piechartsillustratethenumberofneuronsshowingaforce(top)orareward(bottom)effectduringtheelevationofthedischarge ratewhentheactivitywasalignedonthecueoccurrence(black)orontheOCF(gray).Thehatchedblackarearepresentsthenumberofneuronswithastrongereffectwhentheactivitywasaligned on the cue occurrence, and the hatched gray area represents the number of neurons with a stronger effect when the activity was aligned on the OCF. Nougaret and Ravel • TANs’ Modulation by Force and Reward J. Electrophysiological results l f h d For this latter case, the numbers of neurons showing a statistically significant or nonsignificant pause or elevation in discharge rate with one or the otheralignmentareindicatedatthebottomofeachdiagram.D,Piechartsillustratethenumberofneuronsshowingaforce(top)orareward(bottom)effectduringtheelevationofthedischarge ratewhentheactivitywasalignedonthecueoccurrence(black)orontheOCF(gray).Thehatchedblackarearepresentsthenumberofneuronswithastrongereffectwhentheactivitywasaligned on the cue occurrence, and the hatched gray area represents the number of neurons with a stronger effect when the activity was aligned on the OCF. pattern of activity of TANs when the neuronal activity was aligned on the OCF. We observed an influence of the perfor- mance of the animal on the cue responses of TANs (Fig. 5A). Aligned on the time of OCF of the monkeys, the significant de- crease of TAN activity was found between 190 and 10 ms. This pause had a shorter duration than that found when the activity was aligned on the cues; it was also more pronounced when the activity was aligned on the time of OCF (mean, 5.14 Hz; minimal frequency, 4.52 Hz) than on cue occurrence (mean, 5.42 Hz; minimal frequency, 5.07 Hz; t  2.23; p  0.036; Fig. 5B). When aligned on the time of OCF, the significant increase of TAN activity was found between 0 and 250 ms. The size of the elevation of the discharge rate was found to be similar if aligned on the time of OCF of the animals (mean, 6.64 Hz; maximal frequency, 7.16 Hz) or aligned on the occurrence of the cues (mean, 6.60 Hz; discharge rate following the reward were found in separate neu- ronal populations. Only one neuron was included in both groups. In contrast, of the 26 neurons showing a reward effect during the elevation of the discharge rate following the reward occurrence, 11 (42%) showed this modulation following the occurrence of the cues as well (Table 2). discharge rate following the reward were found in separate neu- ronal populations. Only one neuron was included in both groups. In contrast, of the 26 neurons showing a reward effect during the elevation of the discharge rate following the reward occurrence, 11 (42%) showed this modulation following the occurrence of the cues as well (Table 2). Influence of the OCF on the activity of TANs Influence of the OCF on the activity of TANs f f y f Response period time locked on the onset of the change in force. Previous reports have mentioned TAN responses occurring at the time of movement onset (Lee et al., 2006; Benhamou et al., 2014). In their tasks, the movement is either the only event leading to a reward or plays a crucial role in succeeding in a trial. Even though it was not the case in our task, the control of the required force was an important parameter in performing a correct trial. Thus, despite the fact that it has rarely been reported, we studied the 15224 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward maximal frequency, 6.87 Hz; t  0.54; p  0.59; Fig. 5B). How- ever we observed that when the neuronal activity was time locked to the OCF, the beginning of the elevation of the discharge rate corresponded exactly to the beginning of the movement of the animals. animal. Another group of TANs showed significant or larger modulations when aligned to the time of OCF. Seventeen out of 29 neurons (59%) were modulated by the amount of reward for both alignments. The activity of 16 addi- tional neurons was modulated only when their activity was aligned to the time of OCF. Of the 17 neurons modulated by the amount of reward in both cases, 10 neurons showed a higher RSI with their activity time locked to the OCF, versus a higher RSI in 7 neurons when time locked to cue occurrence (Fig. 5D). There was no difference in the distribution of the RSIs between these two populations. In both cases, the majority of TANs were more modulated in the small reward conditions (time locked to cues, mean, 0.38; time locked to OCF, mean, 0.46). As before, one population of TANs seemed to encode the information related to the amount of reward independently of the movement initiated by the animal, whereas another one was very sensitive to the OCF. Number of responsive neurons time locked to the onset of the change in force. The analysis of the activity of TANs time locked to the OCF revealed that 57% of the recorded neurons (96 of 169) showed a significant pause in their activity in relation to this event. TANs respond to both cues and reward TANs respond to both cues and reward TANs are known to respond to temporally unpredictable reward or conditioned stimulus associated with reward and to lose their response to reward with learning (Aosaki et al., 1994, 1995; Api- cella et al., 1997). Compared to these findings, our results have shown that a relatively small number of TANs were responsive to the temporally unpredictable visual cues associated with reward. Conversely, a large number of TANs was found to be responsive to the reward itself, even though it was predictable in time and quantity. It could be concluded that TANs held their response until the reward delivery because the animals did not know the significance of the cues; however, the behavioral results clearly demonstrate that they understood it and modulated their behav- ior accordingly (ERs and acceptance levels). Finding neurons re- sponding to cues and to reward in the TAN population suggests that when there is an added complexity in the task, these neurons will encode more than the temporal predictability of a rewarding stimulus. Neurons responsive to the cues could encode the stim- ulus–reward association, as described previously, neurons re- sponsive to the reward could encode the reward value. In the present task, the reward value can change from trial to trial. One hypothesis is that there is an estimation of this value by the ani- mals at the cue’s occurrence, and that its accuracy is updated by the neurons responding to the reward. Influence of the OCF on the force and reward selectivity indices. The distributions of the FSIs and the RSIs were compared when the elevation of the discharge rate of TANs was aligned on cue occurrence or time locked to the OCF. Eighteen of 34 neurons (53%) were modulated by the amount of force for both align- ments. The activity of 16 additional neurons was modulated only when their activity was aligned on the time of OCF. On the 18 neurons modulated by the amount of force in both cases, 12 (67%) neurons showed a higher FSI with their activity time locked to the OCF, and 6 neurons when their activity was time locked to cue occurrence (Fig. 5D). There were no differences in the distribution of the FSIs between these two populations. TANs respond to both cues and reward In both cases, the majority of TANs were more modulated in the high force conditions (time locked to cues, mean, 0.36; time locked to OCF, mean, 0.43). Consequently, one population of TANs seemed to encode the information related to the effort to develop force independently of the movement initiated by the Influence of the OCF on the activity of TANs This proportion of neurons was higher than that observed when the activity was aligned on the occurrence of the cues (96 of 169 vs 57 of 169,  2  17.24, p  3.29.10 5). Among these neurons, half of them (48 of 96; 50%) showed a pause in their activity only if the electrophysiological signal was time locked to the OCF. In contrast, among the neurons showing a pause when their activity was aligned on the cues, 16% (9 of 57) showed a pause only if their activity was time locked to the cues. Conse- quently, in 48 neurons, the pause was detected in both cases. Among these neurons, the pause was more pronounced when the activity was time locked to the OCF in 9 neurons (19%; Wilcoxon rank sum text, p  0.05) and to cue occurrence in 10 neurons (21%; Wilcoxon rank sum text, p  0.05; Fig. 5C). Since the pause occurred before the initiation of the movement, it could not be concluded that this response was triggered by the force- generating movement. However, considering the higher number of responses detected and the higher magnitude of the responses when activities ware aligned on the time of OCF, we can hypoth- esize that the characteristics of the pause of TANs were dependent on the OCF. For example, beside the variation of magnitudes observed, the end of the pause was time locked on the OCF. Discussion The present study revealed new features of TAN responses in an instrumental task modulating motivation. First, we found a large number of TANs responding to cues and/or reward occurrence by a pause or an elevation of the discharge rate in their activity. Second, this elevation, but not the pause, was modulated by the force required and expected reward in each trial, and was most sensitive to high force or small reward conditions. Finally, TAN responses appeared to be better aligned on the time of OCF than on cue occurrence (triggered by the latter, but time locked on the former). These data suggest that the striatal TAN population is not as homogeneous as previously reported, and that TANs can be involved in encoding either force or reward parameters. p When the activity was time locked to the OCF, 39% (66 of 169) of the recorded neurons showed an elevation of the dis- charge rate in their activity, versus 33% (58 of 169) when the activity was aligned on the cues ( 2  0.62, p  0.43). Among these neurons, only 13 of 66 (20%) showed an elevation of the discharge rate in their activity only if it was time locked to the OCF, and 9% (5 of 58) only if it was aligned on the occurrence of the cues. Consequently, in 53 neurons, the elevation of the dis- charge rate was detected in both cases. Among these neurons, the elevation of the discharge rate was greater when the activity was time locked to the OCF in 18 neurons (34%; Wilcoxon rank sum text, p  0.05) and to cue occurrence in 26 neurons (49%; Wil- coxon rank sum text, p  0.05; Fig. 5C). Consequently, compared to the pause, the elevation of the discharge rate seemed to be less influenced by the OCF. TAN responses are triggered by the cues but largely time locked to the OCF Pauses in TAN activity triggered by the cues were more pro- nounced when aligned on the movement onset, and elevations of the discharge rate were better time locked to the OCF. This sug- gests that TAN activity could be modulated by the movement of the animals, a feature rarely reported. One hypothesis to explain this new property could be that, in the present task, the behavioral response required to succeed in the trial relies on movement modulation. In monkeys, Lee et al. (2006) described modulations around the time of the movement linked to the fact that the movement could be self-initiated in some conditions in their task. In rats, Benhamou et al. (2014) reported an excitation dur- ing movement and hypothesized that the absence of such modu- lation in monkeys could rely on the differences in the protocols used, and thus on the behavioral context. In the present study, the modulation of the movement and the condition in which it is performed, as in the study by Lee et al. (2006), are prominent features of succeeding in the task, and might explain the influence of the OCF on TAN activity. Functionally, the pauses time locked to the OCF could serve to potentiate the movement, when it is a key feature of a task, by influencing striatal projection neuron excitability and allowing the expression of thalamic and cortical afferents. After the reward, the pause was equally modulated by force, reward, or a force–reward interaction, suggesting that the task parameters are integrated by the pause of the TANs at the time of the outcome. As discussed above, TANs showing a pause after the reward could allow a check in the accuracy of the reward value prediction and an update of this value if necessary, via the plas- ticity window created by the decrease in ACh release. It is also in line with the properties of TANs to encode reward prediction error reported previously (Apicella et al., 2011). In our data, the elevation of the discharge rate after reward occurrence was bigger for a smaller reward. It has been suggested that this increase in activity is involved in maintaining the behavior in response to external stimuli (Yarom and Cohen, 2011; Schulz and Reynolds, 2013). References dl b Adler A, Katabi S, Finkes I, Israel Z, Prut Y, Bergman H (2012) Temporal convergence of dynamic cell assemblies in the striato-pallidal network. J Neurosci 32:2473–2484. CrossRef Medline Akins PT, Surmeier DJ, Kitai ST (1990) Muscarinic modulation of a tran- sient K conductance in rat neostriatal neurons. Nature 344:240–242. CrossRef Medline Pauses and elevations of the discharge rate in TAN activity encode different features of the task Biphasic properties of TAN responses have been observed in re- sponse to unpredictable rewards or conditioned stimuli with mo- tivational significance (Aosaki et al., 1994; Ravel et al., 2003; Yamada et al., 2004; Joshua et al., 2008). The neurons recorded here also showed cue- and reward-dependent pauses and eleva- Nougaret and Ravel • TANs’ Modulation by Force and Reward J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15225 tions of the discharge rate. However, in contrast to prior findings, only a small proportion of TANs responded by a full biphasic combination of a pause followed by a rebound (Aosaki et al., 1995; Apicella et al., 1997; Ravel et al., 1999). These results build upon prior findings that TAN activity is largely influenced by context (Yamada et al., 2004; Apicella et al., 2009, 2011). In the context of our more complex cognitive task, TANs present less homogeneous responses. TANs can influence the surrounding striatal PANs by a pause or an elevation of the discharge rate in their activity. Concerning the latter, TANs were found to inde- pendently encode force and reward selectively. Since TANs are considered to be cholinergic interneurons, their tonic activity should correspond to a continuous acetylcholine (ACh) release that would stabilize PANs in an “up” (depolarized) or “down” (hyperpolarized) excitability state (Wilson et al., 1983; Calabresi et al., 1990a,b, 2000). A pause corresponds to ACh decrease, which creates a plasticity window for a change in the excitability state of PANs triggered by other afferents. In contrast, an eleva- tion of the discharge rate should correspond to increased ACh release, serving to stabilize this new excitability state (Akins et al., 1990; Aosaki et al., 1995; Stern et al., 1998). Schulz and Reynolds (2013) suggest that the pause is relatively independent of stimu- lus significance, whereas the elevation of the discharge rate is more likely to be influenced by the context and involved in the mediation of action in response to external stimuli. This concep- tualization is in line with the present results showing that the cue-dependent pause showed little modulation by force or re- ward. Conversely, both factors were encoded during the elevation of the discharge rate, with very little interaction and by indepen- dent neuronal populations. The pause after the cues might thus signal the presence of salient stimuli, triggering trial perfor- mance, as described in tasks with cues carrying single informa- tion. Pauses and elevations of the discharge rate in TAN activity encode different features of the task The elevation of the discharge rate might carry more specific information regarding the motivational level of the animals, given the force required and the expected reward. alternative explanation. It could be that, in both the task in the study by Apicella et al. (2011) and the current task, TANs encode the high cost–small benefit condition. In the present task, the elevation of the discharge rate is most modulated by the least attractive cues, either indicating a high effort or a small reward, in two different groups of TANs. In the task used by Apicella et al. (2011), the elevation in discharge rate modulation could reflect the fact that an animal could have to work up to four times more to receive the same reward in the lowest probability condition. In both studies, the modulation occurred after the event carrying information about effort and reward. In the present study, this modulation was after the cues, whereas in the study by Apicella et al. (2011) it was after the reward when information was com- puted by the animal. TAN responses are triggered by the cues but largely time locked to the OCF Thus, in the present results, it could be a signal to PANs to reinforce the movement despite the small amount of reward re- ceived, while the pause modulation could allow a differential influence of striatal afferents onto PANs such that a reward re- ceived can have a distinct value depending on its size and the effort required to obtain it. The present results demonstrate new features in TAN re- sponse properties, particularly in poorly attractive conditions, but also their potential involvement in movement modulation, in a task demanding different efforts to get various rewards. If TAN responses can differentiate the animals’ preference for perform- ing high cost and low benefit ratio trials, it would be interesting to test whether TAN response properties at the time of the cue pre- dict the decision of an animal to perform any given trial, based on its motivation. The elevation in discharge rate is mainly modulated after least attractive cues and small reward Aosaki T, Tsubokawa H, Ishida A, Watanabe K, Graybiel AM, Kimura M (1994) Responses of tonically active neurons in the primate’s striatum undergo systematic changes during behavioral sensorimotor condition- ing. J Neurosci 14:3969–3984. Medline Modulation of the elevation of the discharge rate in TAN activity after cue occurrence was higher in the high force and small re- ward conditions. Apicella et al. (2011) described similar modu- lations of TANs’ elevation in discharge rate after the reward when its probability was lowest, suggesting that TANs could encode a positive reward prediction error. 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Physical and Mechanical Properties Analysis of Wood-waste Composite Panels
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Physical and Mechanical Properties Analysis of Wood-waste Composite Panels IMAD ANTYPAS*, ALEXEY DYACHENKO Don State Technical University. Gagarin square 1, Rostov-on-Don, 344000, Russian Federation Abstract: The timber industry takes an important place in our modern life, and there has been an increased interest in it for a long time, especially in industrially developed countries. The concerns of the environmental organization regarding the plant wealth preservation have caused scientific trends to search for sources or industries that can achieve acceptable alternatives to natural wood. The materials in this article are related to industrial timber manufacturing of greenhouse wood waste, low density polyethylene and non-standard joinery waste. Moreover, panel samples made from the above materials were briefly subjected to tests as well as tested by fresh and salt water absorption.The density calculation results for all samples have shown the possibility of obtaining a wide range of wooden planks from the lightest to the heaviest ones, depending on the type of filler used. Additionally, the conducted research has shown the possibility of producing artificial wood panels made from the studied materials with good quality and fully satisfactory physical and mechanical properties. MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 *email: imad.antypas@mail.ru MATERIALE PLASTICE https://revmaterialeplastice.ro p p https://doi.org/10.37358/Mat.Plast.1964 Panels were manufactured by adding watermelon skin waste after drying, mixing and precision separation into recycled low density polyethylene, after which its microstructure, absorption, moisture, tensile modulus, elastic modulus, strength modulus, internal adhesion and impact strength were determined. Research objective: industrial timber manufacturing of greenhouse wood waste, low density polyethylene and non-standard joinery waste. 1.Introduction The timber industry takes an important place in modern life, and there has been an increased interest in it for a long time, especially in developed industrialized countries. Timber consumption volume in the world is constantly increasing; therefore, resource-efficient technologies developing is important for ensuring the forest resources protection by exploiting the recycling of raw materials left over from timber processing [1-4]. Scientific studies [5-7] show the use of thermoplastic waste or mixtures of recycled plastics with new plastics in the timber processing industry. The study [8] demonstrates the possibility of using RLDPE waste in the industrial timber panel production; moreover, these panels were pressure formed, with an increase in the percentage of low density polyethylene waste (30-50%) at temperatures of 140°C and 180°C, forming pressure of 30-40 kg/cm2 with a pressure time of 3-7 min. This article also investigates the mechanical properties and stability of these wood-plastic materials panels affected by various conditions, in addition to determining their microstructure. Chow Poo and his colleagues [9] developed thermoplastic composite panels using corn fiber mesh as the main material. Corn stalk fibers were cropped from the western part of the United States of America and the panels were formed in various ways. Fatih Mengel and his colleague [10] studied the thermal behavior of recycled high-density polyethylene (RHDPE) composites as base material, to which eucalyptus timber waste was added. Nwigho Solomon and his colleague [11] studied the evaluation of industrial wood panels mechanical properties made from recycled LDPE as base material stuffed with ‘Iroko’ sawdust as a filler. The RLDPE was accordingly mixed with sawdust by applying molding at a pressure of 150 MPa and a temperature of 160°C. The tests have shown a tensile strength decrease and impact resistance as well as an increase in hardness with a sawdust increased amount; the researchers concluded that it was possible to use sawdust to increase the RLDPE/timber composites hardness and impact resistance. Idris and his colleagues [12] investigated the possibility of watermelon skin waste to produce timber- plastic mixtures. *email: imad.antypas@mail.ru https://doi.org/10.37358/MP.22.2.5585 Mater. Plast., 59 (2), 2022, 61-72 61 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 2.1. Plastic matrix Chopped plastic greenhouse crumbles, made from low density polyethylene (RLDPE) and shown in Figure 1, were used as the base matrix material: Figure 1. Low Density Polyethylene (RLDPE) Figure 1. Low Density Polyethylene (RLDPE) Figure 1. Low Density Polyethylene (RLDPE) 2.2. Joinery waste, resulted from the processes of creating wood products in the form of organic material having different particle sizes (powder, medium particles, large particles, long particles), is shown in Figure 2: Figure 2. Wood waste: a) powder, b) large particles, c) medium particles, d) long particles Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic substance were used as a matrix: - a wood powder set; Figure 2. Wood waste: a) powder, b) large particles, c) medium particles, d) long particles Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic substance were used as a matrix: - a wood powder set; Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic substance were used as a matrix: - a wood powder set; Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic substance were used as a matrix: - a wood powder set; Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 62 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 - a group of medium particle sizes; - a group of long particle sizes; - a random set of particles group. Each of these groups contained five samples with the following proportions by weight - 20% organic filler + 80% RLDPE; - 30% organic filler + 70% RLDPE; - 40% organic filler + 60% RLDPE; - 50% organic filler + 50% RLDPE; - 60% organic filler + 40% RLDPE. These samples were manufactured using a semi-hydraulic molding press according to the following system: - mold temperature T = 160°C; - initial molding pressure P = 4 kg/cm2; - initial pressure time t = 5 min; - final molding pressure P = 70 kg/cm2; - final pressure time t = 10 min. At the end of the molding cycle, the mold was cooled at a cooling rate of V = 10°C / min, while the final pressure remained unchanged until the temperature, at which the final product could be removed without deformation, was reached. Figure 3. 2.1. Plastic matrix an artificial wood sample made from: a) 60% wood powder + 40% RLDPE, b) 20% large particles + 80% RLDPE, c) 50% medium particles + 50% RLDPE, d) 30% long particles + 70% RLDPE Figure 3. an artificial wood sample made from: a) 60% wood powder + 40% RLDPE, b) 20% large particles + 80% RLDPE, c) 50% medium particles + 50% RLDPE, d) 30% long particles + 70% RLDPE 3. Results and discussions The samples density measurement results in this study have shown the different values, that can be explained by a clear difference in the density of the fillers used. As it is known (3), the timber density is about 1.5 g / cm3, while the density of other different sizes types fillers (large, medium and long) is the same as density of wood obtained therefrom, which is generally in 0.4-0.8 g/cm3 range. If it is known that only RLDPE samples density was 0.898 g/cm3, the obvious difference in the density of the matrix and fillers used showed a different behavior of the density curves compared to the samples with an organic filler. Figure 4 shows the density curve of chopped LDPE samples with different percentages of wood powder waste. Figure 4-1 shows that the density increases with an increase in the percentage of an organic filler from 0.898 g/cm3 at 0% to 1.066 g/cm3 at 60%. This result can be divided due to the difference in density values between an organic filler and RLDPE, showing that the compounds density enlarges with an increasing proportion of a filler. The obtained results have revealed that all compounds in Figure 4-1 belong to high density hardwoods according to the classification in [13]. The density measurement results, made on the samples consisting of chopped LDPE and medium- sized wood particles, have shown that the differences in density values relative to the matrix density are small, as shown in Figure 4-2, where a slight increase in the sample density of 0.898 g/cm3 can be observed at a zero level particle, then the density with a composition of 40% medium wood particles becomes 0.908 g/cm3, further it decreases and at 60% it becomes equal to 0.811 g/cm3. The results presented in Figure 4-2 show that these compounds are very close in density to industrial hardwoods according to [13], and the addition of medium wood waste up to 60% leads to a slight decrease in density values and it is possible to produce a wide range of mixtures approximately equal to the density of the base material. Figure 4-3 shows the density development curve of chopped RLDPE matrix composites with various percentages of fillers, consisting of long wood particles and at 60% filler the density value is 0.42 g/cm3. 2.3.2. Short-time absorption The test was made with samples prepared within a short period of time (2 h, 24 h and 48 h). The test samples were cut from 2.2 cm plates. The test samples weight was measured before the immersion into water to find the percentage absorption that occurred after the immersion for the specified time. The absorption percentage was calculated by the ratio: 𝑊= 𝑊2 −𝑊1 𝑊1 . 100 𝑊= 𝑊2 −𝑊1 𝑊1 . 100 𝑊1 where: W1 - sample weight before immersion (g); W1 - sample weight before immersion (g); W2 - sample weight after immersion (g); W - absorption percentage (%). MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 p p https://doi.org/10.37358/Mat.Plast.1964 Charpy Test: the samples for tests were prepared in accordance with the standard specifications (ASTM D256-93) and were 10 mm in length and 10.5 mm2 in cross section. 2.3. Experimental procedure 2.3.1. The density was calculated by subtracting samples from the prepared panels to determine whether the formed panels are light or heavy and whether their density is close to that of natural wood or not. Density was calculated using the following formula: , /v m P = gr/cm3 - m - sample mass, gr; - v - sample size, cm3. Mater. Plast., 59 (2), 2022, 61-72 63 63 https://doi.org/10.37358/MP.22.2.5585 Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 63 MATERIALE PLASTICE 3. Results and discussions The results shown in Figure 4-3 show a wide range of variable density panels production: from high density panels up to 30% to low density panels, comparable to that of insulation panels [14]. Alternatively, this result presents the compositions, which are similar to those of certain light woods, such as spruce, that is the main purpose of our study - developing of industrial mixtures with properties as close as possible to those of wood. The search for specific particle sizes of an organic filler increases the cost of the production process due to the production stages lack, and since the main purpose was to develop waste wood and plastic Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 64 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 products as an organic filler, subsequent research entailed the use of various random shapes and sizes waste without sorting or categorizing. The experimental work has shown that it is advisable to use such random organic waste and to produce products whose properties can be considered good to obtain the required consistency of the studied samples after immersion them into water for more than 40 days. This percentage amounted to 60% for all types and shapes of organic fillers, because using higher ratios, which is the main purpose of our study, it was impossible to obtain cohesive samples with acceptable properties under the same conditions. Since experimental objective of this study was to develop products at the lowest cost, an increase in the proportion of an organic filler to a value greater than the declared value requires preliminary procedures to develop compounds before they are formed, increasing the economic cost. Figure 4-4 shows the density evolution curve of the low density polyethylene chopped with a random set of wood particles samples. In addition, the density values are almost constant up to 30% of the composition, and a gradual decrease in the density values is due to an increase in the filling percentage from 30 to 60%. Figure 4. The RLDPE composites density development curve containing different proportions of a 1) wood powder, 2) medium-sized wood, 3) long wood, 4) random set of wood particles as an organic filler Figure 4. 3. Results and discussions The RLDPE composites density development curve containing different proportions of a 1) wood powder, 2) medium-sized wood, 3) long wood, 4) random set of wood particles as an organic filler The results given in Figure 3-4 show that the use of random waste gave approximately the same results for a long particle filler, and the density measurements of all previous samples mentioned in Figures 3 have demonstrated the possibility of a wide range of industrial wood panels producing comparable to light wood of (0.40 - 0.45) g/cm3, used as insulation panels, as well as high-density heavy wood of 0.9 g/cm3, close in density to polymer - based materials. 3.1. Impact tests It can be noted that when using a medium size organic filler, the impact resistance of the studied compositions improves and it is clear that the impact stress values have decreased from 20 kJ / m2 to 20% to 11.2 kJ / m2 at 60%, that is 2 times less. This result confirms a marked improvement in impact resistance over the impact strength values of compositions containing wood powder, and is due to an improvement in the distribution of the base material due to a reduction in the surface area of an organic filler. Figure 5-2 also shows that when using medium size fillers, the impact strength values remain almost constant until the percentage of organic fillers reaches 30%, followed by a slight decrease in impact resistance at a value of 18.2 kJ/m2. This result is explained by the ability of the base material for better wetting and encapsulation until this percentage is reached, then there is a significant decrease in impact strength values to 11.21 kJ/m2 at 60%. These results are considered important from the point of view of application, as they show that the optimal percentage of the filling process for this type of filler is up to 40% with keeping the good values of impact resistance, and that an increase in an organic filler volume improves the impact resistance up to 40%: when using wood powder of 10.12 kJ/m2 and when using medium-sized particles, it reaches up to 18.2 kJ/m2, hence, there is a result importance, that showed an improvement in impact resistance using medium-sized particles, therefore, the effect of larger particles on impact resistance has been investigated. g Figure 5-3 gives the impact resistance evolution curve of mixtures containing long wood particles, and it is clear that the general behavior of the impact resistance curve is identical to the previous curves, where it decreases with an increase in the percentage of organic filler, and the value of this stress at 20% is 31.26 kJ/m2, and at 40% it becomes 18.24 kJ/m2, i.e., it is more than the impact resistance at the same ratio in the case of using wood powder, or when using medium-sized particles, and this result confirms the improvement of the penetrating power of the base material used due to the reduction of the surface area of the organic filler particles. 3.1. Impact tests The impact strength values of plastic products give an adequate representation of the final product resistance to dynamic loads, since it is known [15] that most objects are destroyed under the influence of dynamic loads, such as hit or regular loads, while the effect of static loads, such as creep, remains very insignificant and lasts for a long time compared to the product life expectancy under the influence of dynamic loads. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 65 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 Determining the RLDPE impact resistance in terms of organic loads percentage is an important point for the future study of the resistance of products made from these materials to the dynamic loads to which they may be subjected during the tests. Figure 5-1 shows that the impact strength values of chopped RLDPE compositions with wood powder as a filler decrease with increasing percentage of an organic filler. This result complies with many reference studies [15] and is predictable since, as it is known, additives generally cause a decrease in the impact strength values, regardless of their percentage value. According to Griffith's theory [14], fillers, regardless of their composition in general, are as stress concentrators in the final product, that in its turn causes different types of loads (static - dynamic) concentration in certain parts of the structure. Figure 5-1 shows the development curve of the impact RLDPE resistance as a function of the percentage of an organic filler (wood powder). A high percentage of wood powder results in a decrease in wetting and penetration of the base material, that, in addition to an increase in agglomeration, makes it difficult to develop the batch-to-batch uniformity final structure. It should be noted that in order to reduce the size difference between the chopped RLDPE and the wood powder a separation process was performed using a set of sieves, with the approximately equal particle sizes of wood powder to achieve uniformity of the components of the mixture. The curve in Figure 5-1 shows a decrease in impact resistance from 20 kJ/m2 at 20% wood powder to 7.91 kJ/m2 at 60%, i.e., about threefold. Figure 5-2 shows that as the particle size of an organic filler increases, the impact resistance of the tested compositions improves. 3.1. Impact tests The replacement of the polymeric material with low cost and high performance polymeric materials aims at reducing the economic cost of the final product with obtaining satisfactory physical and mechanical properties. A simple comparison of the impact strength values at 60% shows that the values are generally close to each other and that the best of them are associated with the use of compounds containing organic fillers with large or medium particles, compared to a wood powder filler. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 66 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 https://revmaterialeplastice.ro https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 The effect of randomly mixed (short - medium - long) particle sizes material on the impact resistance was studied to demonstrate the clear effect of the filler particle size and its uniformity. Figure 5-4 shows the development curve of impact resistance due to the percentage of organic fillers with any particles set. The results have shown that at 40% composition of this type of fillers the stress value was 24.75 kJ/m2, while at 60% it was 14.54 kJ/m2 and compared to previous results these values were higher than when using fillers with long and medium particles. Figure 5. The LDPE impact resistance development curve depending on the percentage of organic fillers 1) wood powder, 2) medium-sized wood, 3) long wood, 4) random particles Figure 5. The LDPE impact resistance development curve depending on the percentage of organic fillers 1) wood powder, 2) medium-sized wood, 3) long wood, 4) random particles According to Charpy, the results of impact tests show that if there is a need for impact-resistant products, the effective range to be investigated is in the range of 20% organic fillers, regardless of the difference in shape of these particles, and that the intensification of the research should not depend on the particle size of this filler and should be aimed to improve the adhesion conditions by preliminary treating of the wood particles with matrix, they provide the group "polymer - filler" with the functional basis, which is necessary to ensure the adhesion of the elements of this group together. Short Term absorption study p y The water absorption study over a short period of time focuses on the rapid assessment of the water resistance of the samples produced. This test is considered to be one of the rapid tests performed on industrial timber and it has been approved by the international standards as a resistance test as one of the physical tests for this type of material [13]. These tests usually involve measurements of swelling or thickening of the materials to determine any irreversible changes that may occur due to the size of the wood particles. zing the results obtained, presented in the Figures 6-13, the following can be noted: p By analyzing the results obtained, presented in the Figures 6-13, the following can 1. The high resistance of cellulose compounds to water is explained by an increase of the absorption percentage with an increase in the percentage of a filler and a time increase in water. 1. The high resistance of cellulose compounds to water is explained by an increase of the absorption percentage with an increase in the percentage of a filler and a time increase in water. 2. A decrease in the coating and wetting capacity of the base material with an increase in the filler percentage is explained by an increase in the absorbency with an increase in the organic filler percentage. 3. There a low absorption capacity of salt water compared to fresh water due to the existence of high molecular weight compounds such as sodium chloride in salt water. 2. A decrease in the coating and wetting capacity of the base material with an increase in the filler percentage is explained by an increase in the absorbency with an increase in the organic filler percentage. p g p y y g p g 3. There a low absorption capacity of salt water compared to fresh water due to the existence of high molecular weight compounds such as sodium chloride in salt water. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 67 MATERIALE PLASTICE https://revmaterialeplastice.ro p p https://doi.org/10.37358/Mat.Plast.1964 Figure 6. The fresh water absorption by RLDPE samples depending on the percentage of wood powder Figure 7. The salt water absorption by RLDPE samples depending on the percentage of wood powder Figure 6. The fresh water absorption by RLDPE samples depending on the percentage of wood powder Figure 6. Short Term absorption study The fresh water absorption by RLDPE samples depending on the percentage of wood powder Figure 7. The salt water absorption by RLDPE samples depending on the percentage of wood powder Figure 7. The salt water absorption by RLDPE samples depending on the percentage of wood powder The density measurements results have shown a clear discrepancy of the density values of compounds with different particle sizes of an organic filler, as shown in Figures 8, 9. The results of measuring the absorption rate of fresh and salt water by medium particle compounds showed that the values reach high levels at a filler composition of 60%, with the absorption exceeding approximately at 40% after immersion in water for 24 h and 48 h. This result shows that the RLDPE base coating capacity is low and does not guarantee the necessary of an organic filler all particles cohesion. g y g p This result conforms with the impact tests, which have shown that the lowest impact resistance values were for mixtures with a high composition of an organic filler. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 https://doi.org/10.37358/MP.22.2.5585 68 MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro p p https://doi.org/10.37358/Mat.Plast.1964 Figure 8. The fresh water absorption by RLDPE samples due to the percentage of organic fillers - medium-sized particles Figure 9. The salt water absorption by RLDPE samples depending on the percentage of an organic filler – medium-sized particles Figure 8. The fresh water absorption by RLDPE samples due to the percentage of organic fillers - medium-sized particles Figure 8. The fresh water absorption by RLDPE samples due to the percentage of organic fillers - medium-sized particles Figure 9. The salt water absorption by RLDPE samples depending on the percentage of an organic filler – medium-sized particles Figure 9. The salt water absorption by RLDPE samples g p y p depending on the percentage of an organic filler – medium-sized particles The rapid density measurements were made on the samples containing large wood particles precisely the same way. As can be seen from two Figures 10,11, the average absorption in fresh and salt water after 48 h is approximately 16% for a 60% filler, and Figures 12, 13 show a change in the density value as a percentage of an organic filler represented by wood large-sized particles, differing in a random way. Short Term absorption study p g g p y g p , g y As can be seen from Figures 12, 13 the highest absorption values are achieved when the percentage of a filler is 60%, with the average absorption value to be 31% in fresh and 28% in salt water after 24 h of immersion. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 https://doi.org/10.37358/MP.22.2.5585 69 MATERIALE PLASTICE https://revmaterialeplastice.ro p p https://doi.org/10.37358/Mat.Plast.1964 Mat.Plast.1964 Figure 10. The fresh water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 11. The salt water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 10. The fresh water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 11. The salt water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 10. The fresh water absorption by RLDPE samples as the percentage of an organic filler - long particles 2022 61 72 70 https://doi org/1 Figure 10. The fresh water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 11. The salt water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 12. Fresh water absorption by RLDPE samples as the percentage of organic fillers – random particles Figure 10. The fresh water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 11. The salt water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 11. The salt water absorption by RLDPE samples as the percentage of an organic filler - long particles Figure 12. Fresh water absorption by RLDPE samples as the percentage of organic fillers – random particles Figure 12. Fresh water absorption by RLDPE samples as the percentage of organic fillers – random particles Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 70 https://doi.org/10.37358/Mat.Plast.1964 Figure 13. The salt water absorption by RLDPE samples as the percentage of organic fillers - random particles Figure 13. The salt water absorption by RLDPE samples as the percentage of organic fillers - random particles 4. Conclusions The density calculation results for all samples show a wide spectral range of panels that can be made from these compounds, from the lightest with the density of 0.40-0.45 g/cm3 to the heaviest with the density of 1.066-0.89 g/cm3. The impact energy decreases with an organic filler percentage increase and declines with a particles size and volume increase up to a certain percentage due to the distribution improvement of the material base according to a filler area decrease. The fresh and salt water absorption coefficient increases with increasing immersion in water time, an organic filler percentage and its size. This means that these panels can be used in a wet environment. That it depends on the nature of the wood and on the initial moisture. p The test results have shown that it is possible to produce good quality artificial wood panels without preliminary procedures for the materials used in the study. The given studies have shown that it is possible to use wood waste of different sizes and that any filler size allows developing a product with its own final properties. Manuscript received: 3.01.2022 Reference A., IGBOANUGO A.C., Potential of Using Recycled Low Density Polyethylene in Wood Composite Board – Tribolgy in industry, Vol 5 (5), 2011, 389-396. 9.POO CHOW, CHARLIE T. BOWERS, JOHN. A., YOUNGQUIST, JIM H. MUEHI, ANDREZEJ M. KRZYSIK, Fasteners Resistance of Non-Woven and Melt-Blended Composite Made from Cornstalk Fibers and Recycled Plastic - International Wood and Natural Fiber Composites Symposium April 10- 11, in Kassel/Germany, 2002, 1-9. 7.YONG LEI, QINHLIN WU, FEI YAO, YANJUN XU, Preparation and properties of recycled HDPE/natural fiber composites, 38, 2007, 1664-1667.DOI 10.1016/j.compositesa.2007.02.001 8 ATUANYA C U IBHADODE A O A IGBOANUGO A C Potential of Using Recycled Low 7.YONG LEI, QINHLIN WU, FEI YAO, YANJUN XU, Preparation and properties of recycled HDPE/natural fiber composites, 38, 2007, 1664-1667.DOI 10.1016/j.compositesa.2007.02.001 8.ATUANYA C. U., IBHADODE. A. O. A., IGBOANUGO A.C., Potential of Using Recycled Low Density Polyethylene in Wood Composite Board – Tribolgy in industry, Vol 5 (5), 2011, 389-396. 9 POO CHOW CHARLIE T BOWERS JOHN A YOUNGQUIST JIM H MUEHI ANDREZEJ M HDPE/natural fiber composites, 38, 2007, 1664-1667.DOI 10.1016/j.compositesa.2007.02.001 8.ATUANYA C. U., IBHADODE. A. O. A., IGBOANUGO A.C., Potential of Using Recycled Low Density Polyethylene in Wood Composite Board – Tribolgy in industry, Vol 5 (5), 2011, 389-396. 9.POO CHOW, CHARLIE T. BOWERS, JOHN. A., YOUNGQUIST, JIM H. MUEHI, ANDREZEJ M. KRZYSIK, Fasteners Resistance of Non-Woven and Melt-Blended Composite Made from Cornstalk Fibers and Recycled Plastic - International Wood and Natural Fiber Composites Symposium April 10- 11, in Kassel/Germany, 2002, 1-9. 10. MENGEL OGLU FAITH, KARAKUS KADIR - Some Properties of Eucalyptus Wood Flour Filled Recycled High Density Polyethylene Polymer -Composites - Turk J Agric, 32, 2008, 537-546 11.NWIGBO SOLOMON, C. U. ATUANYA - Evaluation of the Mechanical Properties of Recycled Polyethylene - Iroko wood Saw Dust Particulate Composite - J. Basic. Appl. Sci. Res, 1(12), 2011, 2806- 2810. 12.IDRISU. D, AIGBODION. V. S, ATUANYA. C. U, ABDULL AHI. J- Eco-Friendly Water Melon Peels Alternative to Wood - Based Particleboard Composite - Tribology in Industry, Vol. 33(4), 2011, 173-181. 13.ABDOLLAH NAJAFI, HABIBOLLAH KHADEMI-ESLAM. Lignocelluloses Filler / Recycled HDPE Composites Filler Type on Physical and Flexural Properties. BioResources, 2011, 6(3), 2411- 2424. 13.ABDOLLAH NAJAFI, HABIBOLLAH KHADEMI-ESLAM. Lignocelluloses Filler / Recycled HDPE Composites Filler Type on Physical and Flexural Properties. BioResources, 2011, 6(3), 2411- 2424. 14.SUCHSLAND. O, WOODSON. G. E., Fiberboard Manufacturing in the United State, Agric- Handbook 1987, 640. 14.SUCHSLAND. O, WOODSON. G. Reference Reference 1.ANTYPAS IMAD RIZAKALLA, DYACHENKO ALEXEY G., Effect of wood particulate filler content on durability of composite materials, Herald of Dagestan State Technical University, Tom 17, 1(88), 2017, 67-74.DOI: 10.23947/1992-5980-2017-17-1-67-74 2.SAMSON AYELE BEKALO, HANS WOLF REINHARDT, Fiber of Coffee and Husk and Hulls for Production of Particleboard - Materials and Structure, 43, 2010, 1049-1060. DOI 10.1617/s11527-009-9565-0 3.ANTYPAS, I.R., The influence of polyethylene processing on the plastic containers blowing. Journal of Physics: Conference Series, 1515 (4), 2020, 1-7. doi: 10.1088/1742-6596/1515/4/042042 4.MADI, N.K. Thermal and mechanical properties of injection molded recycled high Telekom fivdensity polyethylene blends with virgin isotactic polypropylene. Materials and Design, 46, 2013, 435-441. doi: 10.1016/j.matdes.2012.10.004 5 AZADE KIAEIFAR MOHSEN SAFFARI BEHZAD KORD C ti I ti ti Th ( ) 2.SAMSON AYELE BEKALO, HANS WOLF REINHARDT, Fiber of Coffee and Husk and Hulls for Production of Particleboard - Materials and Structure, 43, 2010, 1049-1060. DOI 10.1617/s11527-009-9565-0 3.ANTYPAS, I.R., The influence of polyethylene processing on the plastic containers blowing. Journal of Physics: Conference Series, 1515 (4), 2020, 1-7. doi: 10.1088/1742-6596/1515/4/042042 4.MADI, N.K. Thermal and mechanical properties of injection molded recycled high Telekom fivdensity polyethylene blends with virgin isotactic polypropylene. Materials and Design, 46, 2013, 435-441. doi: 10.1016/j.matdes.2012.10.004 3.ANTYPAS, I.R., The influence of polyethylene processing on the plastic containers blowing. Journal of Physics: Conference Series, 1515 (4), 2020, 1-7. doi: 10.1088/1742-6596/1515/4/042042 4.MADI, N.K. Thermal and mechanical properties of injection molded recycled high Telekom fivdensity polyethylene blends with virgin isotactic polypropylene. Materials and Design, 46, 2013, 435-441. doi: 10.1016/j.matdes.2012.10.004 5.AZADE KIAEIFAR, MOHSEN SAFFARI, BEHZAD KORD, Comparative Investigation on The Mechanical Properties of Wood Plastic Composites Made of Virgin and Recycled Plastics – World Applied Sciences Journal, 14(5), 2011, 735-738. 5.AZADE KIAEIFAR, MOHSEN SAFFARI, BEHZAD KORD, Comparative Investigation on The Mechanical Properties of Wood Plastic Composites Made of Virgin and Recycled Plastics – World Applied Sciences Journal, 14(5), 2011, 735-738. https://doi.org/10.37358/MP.22.2.5585 Mater. Plast., 59 (2), 2022, 61-72 71 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 6.BLEZKI A. K., REIHMANE S, J. GASSAN, Thermoplastics reinforced with wood fillers - A literature review, Polymer-Plastics Technology and Engineering, 37, 2006:451-468. https://doi.org/10.1080/03602559808001373 6.BLEZKI A. K., REIHMANE S, J. GASSAN, Thermoplastics reinforced with wood fillers - A literature review, Polymer-Plastics Technology and Engineering, 37, 2006:451-468. https://doi.org/10.1080/03602559808001373 https://doi.org/10.1080/03602559808001373 7.YONG LEI, QINHLIN WU, FEI YAO, YANJUN XU, Preparation and properties of recycled HDPE/natural fiber composites, 38, 2007, 1664-1667.DOI 10.1016/j.compositesa.2007.02.001 8.ATUANYA C. U., IBHADODE. A. O. Reference E., Fiberboard Manufacturing in the United State, Agric- Handbook 1987, 640. 15.V. P. SOLOMKO - FILLED CRYSTALLINE POLMERS - Kiev - NAUKOVA DUMKA - 1980,264 16.G. KAUSHR - POLYMER DESTRUCTION - Publishing House of the World - Moscow, 1981,440 Manuscript received: 3.01.2022 Mater. Plast., 59 (2), 2022, 61-72 72 https://doi.org/10.37358/MP.22.2.5585 https://doi.org/10.37358/MP.22.2.5585
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https://projecteuclid.org/journals/communications-in-mathematical-physics/volume-172/issue-3/Vertex-IRF-correspondence-and-factorized-L-operators-for-an-elliptic/cmp/1104274318.pdf
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Vertex-IRF correspondence and factorizedL-operators for an ellipticR-operator
Communications in mathematical physics/Communications in Mathematical Physics
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Received: 16 September 1994/in revised form: 27 March 1995 Abstract: As for an elliptic /^-operator which satisfies the Yang-Baxter equation, the incoming and outgoing intertwining vectors are constructed, and the vertex-IRF correspondence for the elliptic i^-operator is obtained. The Boltzmann weights of the corresponding IRF model satisfy the star-triangle relation. By means of these inter- twining vectors, the factorized L-operators for the elliptic /^-operator are also con- structed. The vertex-IRF correspondence and the factorized L-operators for Belavin's /^-matrix are reproduced from those of the elliptic /^-operator. Commun. Math. Phys. 172, 661 - 677 (1995) Commun. Math. Phys. 172, 661 - 677 (1995) Communications IΠ Mathematical Physics © Springer-Verlag 1995 Vertex-IRF Correspondence and Factorized L-operators for an Elliptic /?-operator Youichi Shibukawa Department of Mathematics, Hokkaido University, Sapporo 060, Japan. Email: shibu@math.hokudai.ac.jp Youichi Shibukawa Department of Mathematics, Hokkaido University, Sapporo 060, Japan. Email: shibu@math.hokudai.ac.jp Youichi Shibukawa R(ξn)(Vk(ξι) ® Vk(ξ2 + μ)) C Vk(ξ2) 0 Vk(ξλ + μ) , where ξn := ζ\ — ζi. Then we define outgoing intertwining vectors φ\{ξ) κ λ(z) G Vk(ξ+ |A|k), which are the duals of Φχ\vk(ξ+\λ\ky Making use of the properties of the incoming and outgoing intertwining vectors, we can easily construct factorized L-operators. p This paper is organized as follows. In Sect. 1, we review the properties of the elliptic ^-operator R(ξ) proved in [12,13,14,4]. In Sect. 2, we shall define in- coming intertwining vectors φλ and Boltzmann weights W λ ξ V of an IRF K model. Then we have the vertex-IRF correspondence for the elliptic ^-operator (Theorem 2.1). Theorem 0.1 (Vertex-IRF Correspondence). For Λ,κ,v G Λ, φλ ® φκR(ξ) = κ'eΛ λ ξ v K Φλ Because the elliptic i?-operator satisfies the Yang-Baxter equation, we can show that these Boltzmann weights satisfy the star-triangle relation. This IRF model can be regarded as the limiting case n —> oo of the A^\ face model. In Sect. 3, making use of the results obtained by Felder and Pasquier [4], we shall construct outgoing intertwining vectors in the same way as [6,7,10]. We can consequently define factorized L-operators L^(ξ) (Theorem 3.4). heorem 0.2 (Factorized L-operator). For ξuξ2 $Έ + Zτ, Theorem 0.2 (Factorized L-operator). For ξuξ2 $Έ + Zτ, Theorem 0.2 (Factorized L-operator). For ξuξ2 $Έ + Zτ, In the last section, after stating the results obtained by Felder and Pasquier [4] more precisely, we show that the vertex-IRF correspondence and the factorized L-operators for the elliptic i?-operator imply those for Belavin's /^-matrix. 0. Introduction In [12,13,14] we have introduced an infinite-dimensional /^-matrix. It is a new solution of the Yang-Baxter equation. By means of the Fourier transformation of the i?-matrix, we defined an /^-operator acting on some function space. This ^-operator also satisfies the Yang-Baxter equation. Since this operator is deeply linked to an- alytic properties of an elliptic theta function, we call it the elliptic ί?-operator. We have shown some properties satisfied by the elliptic i?-operator, for example, first in- version relation, fusion procedure, etc. For the trigonometric degenerate case of the elliptic /^-operator, we proved that the finite-dimensional, trigonometric i^-matrices are constructed from the ^-operator through restricting the domain of the ^-operator to some finite-dimensional subspaces. Recently Felder and Pasquier [4] showed that Belavin's i?-matrix [3,11] can be obtained through restricting the domain of a modified version of the elliptic ^-operator to a suitable finite-dimensional subspace. In [1], Baxter has introduced the intertwining vectors for the eight-vertex model. Jimbo, Miwa and Okado [8] constructed the outgoing intertwining vectors between Belavin's vertex model and the A^\ face model. We call this relation the vertex- IRF correspondence for Belavin's i?-matrix. Hasegawa [6,7], Quano and Fujii [10] defined the incoming intertwining vectors which are the dual vectors of the outgoing intertwining vectors. Then they constructed the factorized L-operators for Belavin's i^-matrix. The vertex-IRF correspondence plays a central role in their methods. 662 Y. Shibukawa Y. Shibukawa The aim of this paper is to extend the result above to the elliptic i?-oρerator. p p p Our strategy to construct factorized Z-operators for the elliptic ^-operator is as follows. At first we define incoming intertwining vectors φλ of the elliptic ^-operator R(ξ) and establish a vertex-IRF correspondence. The vertex-IRF correspondence plays the most important role in this paper. Next we find finite-dimensional sub- spaces with the following property (cf. Theorem 1.3); 1. Review of the Properties of an Elliptic /^-operator In this section, we review the construction and the properties of an elliptic R- operator [4,12,13,14]. We fix τ G (C such that Im τ > 0 and define an open subset D c C b y Imτ\ = jz G C; D= lz G <C;|Imz| 2 J ' Vertex-IRF Correspondence for an Elliptic /^-operator Vertex-IRF Correspondence for an Elliptic /^-operator Vertex-IRF Correspondence for an Elliptic /^-operator 663 Let Y be a space of all functions / holomoφhic on D and such that Similarly let property be a space of all functions / holomorphic on D x D with the Similarly let property be a space of all functions / holomorphic on D x D with the /(zi + l,z2) = /(Z!,z2 + 1) = /(zi,z 2) Vz1?z2 G D . Now we define an elliptic /^-operator R(ξ) on Y®Ϋ". Let μ be a complex number such that μ $Z + Έτ and let #i(z) = i?i(z,τ) be an elliptic theta function = £exp Lemma 1.1. There is a unique function F holomorphic on D x D and such that F(zuz2) = (R(ξ)f)(zuz2) for (zuz2) e D x D\X. The elliptic theta function ϋ\(z) satisfies the following properties. The elliptic theta function ϋ\(z) satisfies the following properties. (1) ϋ\(z) is entire, (1) ϋ\(z) is entire, (3) ΰλ(z + τ) = - exp(-2π λ/ =Tz - π v ^ λ λ (4) ϋ\{z) has simple zeros at z G Z + Zτ, (5) i?i(z) satisfies the three term equation (cf. [15] p. 461); (5) i?i(z) satisfies the three term equation (cf. [15] p. 461); - y) - z) + ΰι(x + z)ϋi(x - z)i?, + ϋi(x + w)ΰι(x - w)ΰ (6) M-z) = -i?!(z). Definition 1.1 (Elliptic Λ-operator). For f e , we define z2i : = z 2 — zi, ^ί(O) = ^-(z,τ)| z =o called a spectral parameter. G C. The complex number ξ is We set X = {(zi,z2) e D x D; z2\ £%}. By the property (4) of the elliptic theta function ι?i(z), the function R(ξ)f has the singularities at the points (zi,z2) G X. The lemma below tells us that all singularities are removable. Lemma 1.1. There is a unique function F holomorphic on D x D and such that F(zuz2) = (R(ξ)f)(zuz2) for (zuz2) e D x D\X. 664 Y. Shibukawa Proof. For (zuz2)eD x D\X and m G Z Proof. For (zuz2)eD x D\X and m G Z, (R(ξ)f)(zuz2) (ξ)f)(zuz2) ( Q ) / ( ) ϋ{-μ-m)-ϋx{-μ) ^ z2 - zλ - m z2-zλ-m ϋx{z2X-m) — z\—m V\(z2\—m) -ξ-m)- 1?,(-< z2-z\-m f(zuz2 -/w)-. z2-zx-m Thus there is a function F continuous o n D x D and such that F(z\,z2) = (R(ξ)f) (z\,z2) for (zi,z2) G D x Z)\X. In fact, we define Thus there is a function F continuous o n D x D and such that F(z\,z2) = (R(ξ)f) (z\,z2) for (zi,z2) G D x Z)\X. In fact, we define F(zuz2) F(zuz2) ,^2), otherwise. otherwise. otherwise. Making use of the Riemann removable singularity theorem (cf. [5]), this function F is holomorphic on D x D. D We also denote by R(ζ)f this holomorphic function F. It is easy to see that Ui) = for (zi,z2) £DxD. Hence Λ(ξ)/ ^ ^ Θ ^ for / G 'T&'T, and ^ ( 0 is an operator on i^(§i^ as a result. (§ Let Y&'V&'V be a space of all functions / on D x D x D and such that /(zi + l,Z2,z3) = /(zi,z2 + l,z3) = /(zi,z2?z3 + 1) = /(zi,z2,z3) V zi,z2,z3 eD. We have ^ I 2 ) ( ^ ~ ( ^ i ) ® F-(ξ 2 + μ)) C V-(ξ2) 0 F - ( 6 + μ) . ^ I 2 ) ( ^ ~ ( ^ i ) ® F-(ξ 2 + μ)) C V-(ξ2) 0 F - ( 6 + μ) . A basis of V~(ξ) is as follows. A basis of V~(ξ) is as follows. A basis of V~(ξ) is as follows. jez/nz Remark 1.2. Let J b e a space of the meromorphic functions on (C 2. Then we note that the elliptic /^-operator R(ξ) can be regarded as an operator on Jt and satisfies the Yang-Baxter equation (1.1). The elliptic theta function ϋ\(z) satisfies the following properties. Let Y&'V&'V be a space of all functions / on D x D x D and such that Let Y& V& V be a space of all functions / on D x D x D and such that /(zi + l,Z2,z3) = /(zi,z2 + l,z3) = /(zi,z2?z3 + 1) = /(zi,z2,z3) V zi,z2,z3 eD. (zi + l,Z2,z3) = /(zi,z2 + l,z3) = /(zi,z2?z3 + 1) = /(zi,z2,z3) V zi,z2,z3 eD. By the three term equation of τ?i(z) (the property (5)), we get the following theo- rem. Theorem 1.2 ([12,13,14]). R(ξ) satisfies the Yang-Baxter equation on Theorem 1.2 ([12,13,14]). R(ξ) satisfies the Yang-Baxter equation on orem 1.2 ([12,13,14]). R(ξ) satisfies the Yang-Baxter equation on (1 ®Λ(ξi2))(Λ(£13)® 1)(1 ®Λ(ξ23)) - (Λ(ξ23)Θ 1)(1 (8>Λ(ξi3))(Λ(ίi2)®l), (1.1) h ξij ξ ξj (1 ®Λ(ξi2))(Λ(£13)® 1)(1 ®Λ(ξ23)) - (Λ(ξ23)Θ 1)(1 (8>Λ(ξi3))(Λ(ίi2)®l), (1.1) where ξij = ξt - ξj. (1 ®Λ(ξi2))(Λ(£13)® 1)(1 ®Λ(ξ23)) - (Λ(ξ23)Θ 1)(1 (8>Λ(ξi3))(Λ(ίi2)®l), (1.1) where ξij = ξt - ξj. where ξij = ξt - ξj. For <J G C and « = 1,2,..., let Kw(ξ) be a space of all functions / holomorphic on <C and such that / ( z + l ) = /(z), /(z + τ) = (-l)"exp(2π>/=ϊ(£ - nz))/(z). /(z + τ) = (-l)"exp(2π>/=ϊ(£ - nz))/(z). Vertex-IRF Correspondence for an Elliptic /^-operator 665 Vertex-IRF Correspondence for an Elliptic /^-operator 665 665 It is well known that Vn(ξ) has dimension n. We easily see that It is well known that Vn(ξ) has dimension n. We easily see that (1.2) Z/nZ (1.2) is a basis of Vn(ξ). Here ϋ \ a (z,τ) is a theta function with rational characteristics; ϋ I 11 (z, τ) = £ expfπV^Tίm + α) 2τ -f 2πv /ΓT(m + a)(z + b)]. 1°} mez In [4] Felder and Pasquier show the following. In [4] Felder and Pasquier show the following. Theorem 1.3 ([4]). R{ξn){Vn(ξx) ® Vn(ξ2 + μ)) C FΛ(&) ® Vn{ξx + μ). Theorem 1.3 ([4]). R{ξn){Vn(ξx) ® Vn(ξ2 + μ)) C FΛ(&) ® Vn{ξx + μ). Remark 1.1. Let Ί ^ ~ be a space of all functions / holomorphic on D and suc that Theorem 1.3 ([4]). R{ξn){Vn(ξx) ® Vn(ξ2 + μ)) C FΛ(&) ® Vn{ξx + μ). Remark 1 1 Let Ί ^ be a space of all functions / holomorphic on D a Remark 1.1. The elliptic theta function ϋ\(z) satisfies the following properties. Let Ί ^ ~ be a space of all functions / holomorphic on D and such that We set Ψ"~®Ϋ"~ and V~(&i r~&i r~ in the same way as TΓ. Then we can define the elliptic i?-operator ^((^) on i r~®i r~, which is the same as in Definition 1.1. It is easy to see that R(ξ) on Ί^~<8)^~ satisfies the Yang-Baxter equation (1.1). It is easy to see that R(ξ) on Ί 8) satisfies the Yang Baxter equation (1.1). We denote V~(ξ) as a space of all functions / holomorphic on (C and such that We have 2. Incoming Intertwining Vectors and Vertex-IRF Correspondence In what follows μ e 1R\Z, and let A be a set of sequences λ — (λt) (i G TL) such that λij := λi ~ λj$ Z + Zμ V/φy € Z . λij := λi ~ λj$ Z + Zμ V/φy € Z . We take r e 1R such that r^ Q + Qμ, and set ηi:=ir ( i G l ) . We take r e 1R such that r^ Q + Qμ, and set ηi:=ir ( i G l ) . ηi:=ir ( i G l ) . Y. Shibukawa 666 Y. Shibukawa Then η = (ηt) G A. Hence, for any μ, the set A is not empty. For i G Έ, we define the sequences ε, = (βij) (j G Έ), and for λ G A, let λ -f μεz denote the sequence We note that λ 4- με, G A for all / G Έ by the definition of A Definition 2.1 (Boltzmann Weight of the IRF Model). For λ, K, K', V G Λ, Boltzmann „ r *' i weights W \λ ξ v \ e <C of an interaction-round-a-face (IRF) ratfίfe/ are g/i eH L K: J as follows (cf. [1,6,7,8,10]). For /I G Λ, we put Definition 2.1 (Boltzmann Weight of the IRF Model). For λ, K, K', V G Λ, Boltzmann „ r *' i weights W \λ ξ v \ e <C of an interaction-round-a-face (IRF) ratfίfe/ are g/i eH L K: J as follows (cf. [1,6,7,8,10]). For /I G Λ, we put λ 4- με. W λ 4- 2μεi 4- λ 4- με7- 4- με7- 4- otherwise we set W λ ξ K : = 0 . Next we define incoming intertwining vectors of the elliptic i?-operator. Next we define incoming intertwining vectors of the elliptic i?-operator. Definition 2.2 (Incoming Intertwining Vector). For λ,κ G Λ, define an incoming intertwining vector φλ G 'V* as follows: 3ί G TL s.t. κ = otherwise. μεi The incoming intertwining vectors are the Dirac delta functions essentially. By Definition 1.1 we can get a vertex-IRF correspondence for the elliptic ^-operator. Theorem 2.1 (Vertex-IRF Correspondence). For λ, κ9 v G Λ, φ K λ®φ V κR(ξ) = κ'eΛ ξ v K φ K λ (2.1) (2.1) C where both sides are the operators o i j. Since satisfies the Yang-Baxter equation (1.1), we can show j Since satisfies the Yang-Baxter equation (1.1), we can show Proposition 2.2. The Boltzmann weights of the IRF model satisfy the star-triangle relation', Σ w w K λ ξ2i K a. λ ξn K' K W K' i β ξv. V W Γ β y w A y ξ X 12 K' β (2.2) (2.2) for λ, K, v, oc,β,ye A. for λ, K, v, oc,β,ye A. Proof Unless there exist i9j,k G Έ such that K — λ + μεt, v = λ -f μ(ε, + εy) and y = λ + μ(εz + εy + ε^), both sides of Eq. (2.2) are zero. Then we assume that κ = λ + μεi9 v = λ + μ(ε, + ε, ), y = A H- μ(ε, εk) κ = λ + μεi9 v = λ + μ(ε, + ε, ), y = A H- μ(ε, εk) Moreover both sides of Eq. (2.2) are zero unless α = λ + με/? A + μεy or A + Moreover both sides of Eq. (2.2) are zero unless and jS = A 4- μ(ε, + εy ), A + μ(ε/ + ε^) or A jS = A 4- μ(ε, + εy ), A + μ(ε/ + ε^) or A so it suffices to show Eq. (2.2) in each case. Since R(ξ) satisfies the Yang-Baxter equation (1.1), ?2, Z 3 ) . C where both sides are the operators It is to be noted that, by Definition 2.1 and 2.2, both sides of Eq. (2.1) are zero unless there exist ij G Έ such that K = λ 4- με/, v = λ 4- μ(εz 4- εy). The other cases Vertex-IRF Correspondence for an Elliptic /^-operator 667 Vertex-IRF Correspondence for an Elliptic /^-operator Vertex-IRF Correspondence for an Elliptic /^-operator 667 are as follows: R(ξ) = Φx ΦΪ με' for i+j. for i+j. ?2, Z Putting z\ — λf, Z2 = Ay + μ(5^y and z-^ — λΛ- μ(βik Λ- δjk) in the coefficient of /(zi,Z2?z3)? we obtain Eq. (2.2) in the case α = A + με^ and β = A + μ(ε; + εy). We can prove the other cases in the similar way, so we omit the proof. D Remark 2.1. We define an incoming intertwining vector φ} G (Ϋ"~)* in the same way as Definition 2.2; for / G i^~, κ (f(λi\ λf•= i Λ L 0, otherwise. Y. Shibukawa Y. Shibukawa 668 In this case, we also get a vertex-IRF correspondence; for λ,κ,v e A, K' λ ξ V K -K -V Φλ ® Φκ f ' 3. Outgoing Intertwining Vectors and Factorized L-operators To begin with, we define outgoing intertwining vectors of the elliptic i?-operator (cf. [6,7,10]). Let k\ and k2 be integers such that k\ ^ Aτ2, and we set k:~ (k\,k2) and k = k2 - k\ + 1. For λ, K e A and kx ^ j ^ k2, we define φk(ξ)f e C by )? := Φ'. where |λ|k = Efi* 1^ Proposition 3.1. For λ £ Λ and ξ tfϊ Έ + Έτ, the k — by — k matrix i is invertible. Proof. Since λ +μει' J )k{ £ijzk2 = d i aS ( e xP πy/^Tkλkι, , exp πλ/ it suffices to prove + 0. The Weyl-Kac denominator formula for Ak\ (cf. [9,7]) yields det [ϋ 2 k k i=\ det [ϋ 2 k k i=\ Here η(τ) is Dedekind's ^-function Here η(τ) is Dedekind's ^-function Here η(τ) is Dedekind's ^-function πyfΛτ η(τ) = exp- 12 fi (1 ~~ w=l Vertex-IRF Correspondence for an Elliptic ^-operator 669 Vertex-IRF Correspondence for an Elliptic ^-operator Vertex-IRF Correspondence for an Elliptic ^-operator Vertex-IRF Correspondence for an Elliptic ^-operator Vertex-IRF Correspondence for an Elliptic ^-operator 669 669 Then we obtain l j-h det ( 7? \λ\k-kλi9kτ) 1 _ L 2 k k 2 thereby completing the proof. D thereby completing the proof. D The proposition above says that for λ, K G Λ, k\ S j S ^2 and ξ £ Z + Zτ, there exist φk(O;, ^ ^ which are characterized by the following duality relations; (3.1) (A:i ^ Vz ^ A:2) we set (3.1) and for K Φ λ + μεz (A:i ^ Vz ^ A:2) we set Definition 3.1 (Outgoing Intertwining Vector). For A, K G A and ξ £ ΊL + Zτ, «w outgoing intertwining vector φ^{ξ) κ λ{z) e Vk(ξ + |A|k) o///ze elliptic R-operator is defined as follows (cf (1.2)): *ω := Σ μ|k - fe, Equation (3.1) is equivalent to Equation (3.1) is equivalent to Equation (3.1) is equivalent to ; + μ ε / = id on Vk(ξ (3.2) (3.2) The outgoing intertwining vectors satisfy the following: The outgoing intertwining vectors satisfy the following: The outgoing intertwining vectors satisfy the following: Proposition 3.2. For λ,κ,v G A and ξ\,ξι i TL + Zτ, Proposition 3.2. For λ,κ,v G A and ξ\,ξι i TL + Zτ, Proposition 3.2. For λ,κ,v G A and ξ\,ξι i TL + Zτ, K λ ξU V κ f K λ ξU V κ f Proof By Definition 2.1 and 3.1, it suffices to show λ + μ λ ξu λ + μ Y. Shibukawa 670 for any λ G A and kλ <. Vι, j ^ k2. With the aid of Theorem 2.1 and Eq. (3.2), we obtain for k\ ^ V#, b ^ k2, /I + με, ζn /I + με, ζn Then Σ Σ h = Σ w t+εj) λ + μεa λ ξl2 λ + μεi 3/) /+μ(εα+ε6) λ ζ\2 h = Σ t+εj) w λ + μεa λ ξl2 λ + μεi 3/) /+μ(εα+ε6) L /I + By virtue of Definition 3.1 and Theorem 1.3 we deduce \λ\k + μ) By virtue of Definition 3.1 and Theorem 1.3 we deduce \λ\k + μ) From Eq. (3.2), we are led to the desired result. D From Eq. (3.2), we are led to the desired result. D From Eq. (3.2), we are led to the desired result. D For λ, K G A and ξ £ Έ -f Zτ, we define an operator ^ by From Eq. (3.2), we are led to the desired result. D For λ, K G A and ξ £ Έ -f Zτ, we define an operator ^ by From Eq. (3.2), we are led to the desired result. D For λ, K G A and ξ £ Έ -f Zτ, we define an operator (4(£)5/)( ) (ξf ( ) κ f (/ f) ^ by For λ, K G A and ξ £ Έ -f Zτ, we define an operator ^ by (4(£)5/)(z) := φk(ξfλ(z)φ κ λf (/ef). Theorem 2.1 and Proposition 3.2 say Theorem 2.1 and Proposition 3.2 say Lemma 3.3. i w A, v e A and ξu ξ2 £ TL + Zτ, Lemma 3.3. i w A, v e A and ξu ξ2 £ TL + Zτ, Vertex-IRP Correspondence for an Elliptic ^-operator Vertex-IRP Correspondence for an Elliptic ^-operator Vertex-IRP Correspondence for an Elliptic ^-operator 671 Proof. For / G T Proof. For / G T Σ κ,κ'eΛ λ ξn v (Φλ®Φκ)f We have thus proved the lemma. D We have thus proved the lemma. D We have thus proved the lemma. D We have thus proved the lemma. D Now we are in the position to construct factorized I-operators for the elliptic i?-operator. Let if be a space of all (C-valued functions on A, and let 'V&'Ψ (resp. i^&i^) be a space of all functions g : D x Λ —• (C (resp. Λ x D —> (C) such that G f (resp. g(λ, ) G f^) for any λ £ A. We define a factorized L-operator 1T01Γ as follows [2,6,7,10]. For g e Ϋ~®ir zxAξ^Έ + TLτ, (Lk(ξ)g)(λ,z) := E (Z k(O;^( , *))(*) . (3.3) (3.3) For λ G Λ we set <5 ;v G ^ as follows: δ\κ) = δ λ ί C. We note that TT = Y[κeA^δ κ (cf. [6]). Then, for / G TΓ, We note that TT = Y[κeA^δ κ (cf. [6]). Then, for / G TΓ, We note that TT = Y[κeA^δ κ (cf. [6]). Then, for / G TΓ, and Eq. (3.3) is hence equivalent to We define 'V&'V&IV (resp. I^&I^&Ψ*) by a space of all functions g : D x D x y l - ^ C (resp. / I X D X D ^ C ) such that g( , ,/ί) G Ί ^ Θ ^ (resp. g(λ, , ) G i^®i^) for any λ e Λ. By means of Lemma 3.3, we immediately obtain the following theorem. Theorem 3.4 (Factorized L-operator). For ζ\9ξ2 ί- % + % τ> are ίήe operators 'V& Remark 3.1. In the same way as this section, we can construct factorized L-operators for R(ξ) on T T - ^ T T " by using FΛ"(O instead of Fn(ξ) (cf. Remark 1.1 and 2.1). 672 Y. Shibukawa Y. Shibukawa Y. Shibukawa In this case, outgoing intertwining vectors are characterized by the following duality relation: In this case, outgoing intertwining vectors are characterized by the following duality relation: Here, for λ9κ e Λ, h <± j <L k2, we define φk(ξ)f e € as follows (c Remark 1.1): Here, for λ9κ e Λ, h <± j <L k2, we define φk(ξ)f e € as follows (c Remark 1.1): _.. / Γ 1 j-h Ί \ / ZT(^+ l)z) . 4. Vertex-IRF Correspondence and Factorized L-operators for Belavin's /^-matrix In this section, we apply Theorem 2.1 to the /^-matrix obtained through restricting the domain of the elliptic i?-operator to some finite-dimensional subspace. Then we will show that the vertex-IRJF correspondence for Belavin's /^-matrix proved by Baxter [1], Jimbo, Miwa and Okado [8] is obtained from Theorem 2.1. Moreover we will construct the factorized L-operators for Belavin's i?-matrix obtained by Hasegawa [6], Quano and Fujii [10]. First let us state the results proved by Felder and Pasquier [4] more precisely. q p y For k — 1,2,..., let Vk(ζ) be a space of entire functions / of one variable such that f(z + τ) = (-l/exp(-2πΛ/^T(^ - ξ + We note that Vk(ξ) c ^ if k is even and that Vk(ξ) c V~ if k is odd. In the same fashion as Theorem 1.3 and Remark 1.1, we obtain + μ)) C Vk(ξi) The space Vk(ζ) is of A: dimensions and a basis is given by {ej(ξ)(z) := ΰ \j 7 * ] (ξ - kz,kτ)}jeΣ/kπ . For k — 1,2,..., define a translation operator Tk(ξ) on the space of all holomoφhic functions on C [4] by (Tk(ξ)f)(z) ;= Tk(ξ) maps isomorphically V^ := Vk(0) onto ^t(ξ). We modify the elliptic ^-operator as Vertex-IRF Correspondence for an Elliptic i?-operator Vertex-IRF Correspondence for an Elliptic i?-operator 673 Vertex-IRF Correspondence for an Elliptic i?-operator 67 673 We note that ^(£12) is determined by the difference ξn In fact, We note that ^(£12) is determined by the difference ξn In fact, + 7> zl ~ 7 Felder and Pasquier prove Theorem 4.1 ([4]). Rk(ζ) preserves Vk 0 Vk and obeys the Yang-Baxter equation (1.1). Theorem 4.1 ([4]). Rk(ζ) preserves Vk 0 Vk and obeys the Yang-Baxter equation (1.1). Let {e j}Jez/kπ C Vk be the dual basis of {ej := e;(0)} C Vk; e'iej) = δtj . Now we define an operator Rk(ζ)* on Vk (8) Vk , the transpose of Rk(ζ) on F^ 0 j ^ . ® ea). Proposition 4.2 (cf. [4]). The R-matrix Rk(ξ)* is Belavin's R-matrix up to constant. Proof. Let A and B be operators on the space of all holomorphic functions on C as (Bf)(z) = -exp (iπV^Λ (z + ^ ) ) / (z + | ) . (Bf)(z) = -exp (iπV^Λ (z + ^ ) ) / (z + | ) . The space F^ is invariant under the actions of A and B. 4. Vertex-IRF Correspondence and Factorized L-operators for Belavin's /^-matrix In fact, ^4 and B are expressed on Vk as - βj exp Bej = eJ+ι . Bej = eJ+ι . We define operators A* and B* on F^ to be the transposes of A and B on F^ respectively; To prove this proposition, it is enough to show the following [3,6,7]. (1) Rk(ζ)* is an entire End(F/ (g> F/)-valued function in ξ. (1) Rk(ζ)* is an entire End(F/ (g> F/)-valued function in ξ. (2)&k(ξ) χ®x=x®x&k(ξ)* x=A*,B*. k k (3) Rk{ξ + 1)* = (1 ®A*y 1Rk(ξ)*(A* ® 1) x (-1). (4) Rk(ξ + τ)* = (1 ® B*)-'Rk(ξ)*{B* ® 1) x (-exp2π^/=T(ξ + | - £))-». (4) Rk(ξ + τ)* = (1 ® B*)-'Rk(ξ)*{B* ® 1) x (-exp2π^/=T(ξ + | - £))-». 674 u/'t Y. Shibukawa i. oiiiuuivαwα Y. Shibukawa i. oiiiuuivαwα Y. Shibukawa i. oiiiuuivαwα The operator Rk(ξ) on Vk 0 Vk has the properties below, which imply the properties (2), (3), (4), and (5) above, respectively. The operator Rk(ξ) on Vk 0 Vk has the properties below, which imply the properties (2), (3), (4), and (5) above, respectively. (2) Rk(ξ)x ® x = x ® xRk(ξ) x=A9B. (3) Rk(ξ + 1) = (1 ®A)Rk(ξ)(A 0 I)" 1 x (-1). (Λ) Rk(ξ + τ) = (1 ® B)Rk(ξ)(B ® iyι x (- exp2πv /zT(ξ + f - f))" 1. (5) 4 ( 0 ) = t?ί(0)id. The proof is quite straightforward, so we omit it. The proof is quite straightforward, so we omit it. To prove (1), it suffices to show that Rk(ξ) is an entire End(^ function in ξ. Let us introduce another basis of Vk (cf. [4]); ^)-valued In the same way as [4], we can calculate the matrix coefficients of Rk(ζ) on Vk 0 Vk with respect to the basis {ey 0 ej} and can check that all matrix coefficients are entire in ξ. This completes the proof. D In the same way as [4], we can calculate the matrix coefficients of Rk(ζ) on Vk 0 Vk with respect to the basis {ey 0 ej} and can check that all matrix coefficients are entire in ξ. This completes the proof. 4. Vertex-IRF Correspondence and Factorized L-operators for Belavin's /^-matrix D For λ,ιc £ A, we put φ(ξ)« := φ λ κ oTk(ξ + |A|k - kμ)\Ϋk Since For λ,ιc £ A, we put φ(ξ)« := φ λ κ oTk(ξ + |A|k - kμ)\Ϋk Since 0, 1 _ L 2 k k 2 (ξ - kλhkτ) ^ 3ί ^ otherwise , otherwise , otherwise , otherwise , we get we get k-\ _λ 7=0 ί To \ u 'I _ I . 2 - kμ)(ej)e> ^ 3/ ^ otherwise. ί To \ u 'I _ I . 2 ^ 3/ ^ otherwise. otherwise. otherwise. Hence the vector φ{ζ) κ λ is nothing but the outgoing intertwining vector of Belavin's 7?-matrix [6,7], which was first discovered by Baxter [1], Jimbo, Miwa and Okado [8]. Hence the vector φ{ζ) κ λ is nothing but the outgoing intertwining vector of Belavin's 7?-matrix [6,7], which was first discovered by Baxter [1], Jimbo, Miwa and Okado [8]. [ ] On the other hand, we put On the other hand, we put W λ ξ v := W V ξ λ K and then Theorem 2.1 and Remark 2.1 lead us to and then Theorem 2.1 and Remark 2.1 lead us to Vertex-IRF Correspondence for an Elliptic i?-operator Vertex-IRF Correspondence for an Elliptic i?-operator 675 Theorem 4.3 (Vertex-IRF Correspondence for Belavin's ^-matrix [1,8]). For λ,κ, v G Λ, K 1 λ ξn v . Next we construct the factorized Z-operators for Belavin's 7?-matrix proved by Hasegawa [6], Quano and Fujii [10]. To begin with, we introduce outgoing inter- twining vectors in Vk(ζ) in the same fashion as Definition 3.1. In the sequel, we fix k\,k2 G % such that k = k2 — k\ + 1 and assume that λ,κ,v e Λ and the ξ,ξ\,ξ2 £ Έ + Έτ. For k\ S j S k2, we define φk(ξ)f € <C by For k\ S j S k2, we define φk(ξ)f € <C by For k\ S j S k2, we define φk(ξ)f € <C by nd also define ψk(ζ)fj £ & by the following duality relations (cf. Proposition 3.1): ί l-ki λj ( ζ ζ l i=kι Pv\ζ)χ For κή=λ + μsi (k\ ^ Vz ^ i 2) we set l i=kι Pv\ζ)χ For κή=λ + μsi (k\ ^ Vz ^ i 2) we set Φk(ί)ϊy := 0 . 4. Vertex-IRF Correspondence and Factorized L-operators for Belavin's /^-matrix Outgoing intertwining vectors ψk{ξ) κ λ{z) G Vk(ξ + |/l|k) of the elliptic i?-operator are defined as ,(ξ + μ| kχ z). Then we define the operators Ly{ζ)\ as follows: Then we define the operators Ly{ζ)\ as follows: L){z) := φk(ξ)t(z)φ K λf where / G y if k is even and / G i r~ if k is odd. In the same way as Sect. 3, these operators satisfy (cf. Lemma 3.3) We put Z k(O" := Tk{ξ + |κ| k - kμ)- ιLk (ξ - kμ) λ κTk(ξ + |ιc|k - kμ)\^ , and denote its transpose as Lγ(ξ) κ λ : Vk —> Vk . Thus, for Belavin's i^-matrix Rk(ξ)*, M l l = Σ Ki&l ® 1*^)1 Rk(ξn)* • KEΛ Z k(O" := Tk{ξ + |κ| k - kμ)- ιLk (ξ - kμ) λ κTk(ξ + |ιc|k - kμ)\^ , and denote its transpose as Lγ(ξ) κ λ : Vk —> Vk . Thus, for Belavin's i^-matrix Rk(ξ)*, M l l = Σ Ki&l ® 1*^)1 Rk(ξn)* • KEΛ Z k(O" := Tk{ξ + |κ| k - kμ)- ιLk (ξ - kμ) λ κTk(ξ + |ιc|k - kμ)\^ , note its transpose as Lγ(ξ) κ λ : Vk —> Vk . Thus, for Belavin's i^-matrix Rk(ξ)*, M l l = Σ Ki&l ® 1*^)1 Rk(ξn)* • KEΛ Y. Shibukawa Y. Shibukawa 676 We define an operator l£(ξ) : Ϋl 0 iV -» IT 0 V* by The theorem below tells us that the operator Lk(ξ) is the factorized Z-operator for Belavin's ^-matrix, which were first constructed by Hasegawa [6], Quano and Fujii [10]. Theorem 4.4 (Factorized L-operator for Belavin's i?-matrix). For ζ\,ζι £ Z + Έ ^ 2 ) * ) ( I k ( i i ) ® 1)(1 ®Ll(ξ2)) = (tk(ξ2) ® 1)(1 £ & ^ 2 ) * ) ( I k ( i i ) ® 1)(1 ®Ll(ξ2)) = (tk(ξ2) ® 1)(1 £ & ^ 2 ) * ) ( I k ( i i ) ® 1)(1 ®Ll(ξ2)) = (tk(ξ2) ® 1)(1 £ & 57'ίfos 1 are ί/*e operators Ϋζ 0 ί^* 0 #^ —> #" Acknowledgement. The author would like to express his deep gratitude to Professor Kimio Ueno for useful advice, to Professor Takayuki Hibi for constant encouragement, and to Professor Michio Jimbo for pointing out to him another proof of Proposition 2.2 which is used in this paper. 4. Vertex-IRF Correspondence and Factorized L-operators for Belavin's /^-matrix Note added in proof. We have two remarks about the incoming and outgoing intertwining vectors. Note added in proof. We have two remarks about the incoming and outgoing intertwining vectors. (1) We canaddonemore parameter to the incoming intertwining vector φ in Definition 2 2 Note added in proof. We have two remarks about the incoming and outgoing intertwining vectors. (1) We can add one more parameter to the incoming intertwining vector φλ in Definition 2.2. (1) We can add one more parameter to the incoming intertwining vector φλ in Definition 2. For α € 1R, we set ί f( λi + α)> 3i e % s t. κ = λ + μεi9 Φλ(*)f := 1 , . ^ 0 otherwise , . otherwise 0, otherwise . These incoming intertwining vectors also satisfy the vertex-IRF correspondence (Theorem 2.1). Making use of the incoming intertwining vectors φλ(a) instead of φλ, we can construct the factorized L-operators (Theorem 3.4). p ( ) (2) By means of the Weyl-Kac denominator formula (cf. Proposition 3.1), we obtain the explicit form of the outgoing intertwining vector in Definition 3.1. For k\ S i S h, References 1. Baxter, R.: Eight-vertex model in lattice statistics and one-dimensional anisotropic Heisenbe chain. II. Equivalence to a generalized ice-type lattice model. Ann. Phys. 76, 25-47 (1973 1. Baxter, R.: Eight-vertex model in lattice statistics and one-dimensional anisotropic Heisenberg chain. II. Equivalence to a generalized ice-type lattice model. Ann. Phys. 76, 25-47 (1973) q g yp y ( 2. Bazhanov, V.V., Kashaev, R.M., Mangazeev, V.V., Stroganov, Yu. G.: (ZNx) n~ ι generaliz tion of the chiral Potts model. Commun. Math. Phys. 138, 393-408 (1991) y , ( ) 3. Belavin, A.A.: Dynamical symmetry of integrable quantum systems. Nucl. Phys. B180 [FS2], 189-200 (1981) ( ) 4. Felder, G., Pasquier, V.: A simple construction of elliptic i?-matrices. Lett. Math. Phys. 32, 167-171 (1994) ( ) 5. Gunning, R.C., Rossi, H.: Analytic functions of several complex variables. Englewood Cliffs, NJ: Prentice-Hall, 1965 6. Hasegawa, K.: Crossing symmetry in elliptic solutions of the Yang-Baxter equation and a new I-operator for Belavin's solution. J. Phys. A. Math. Gen. 26, 3211-3228 (1993) p y 7. Hasegawa, K.: L-operator for Belavin's i?-matrix acting on the space of theta functions. J. Math. Phys. 35 (11), 6158-6171 (1994) 8. Jimbo, M., Miwa, T., Okado, M.: Local state probabilities of solvable lattice models: An A^lx family. Nucl. Phys. B300 [FS22], 74-108 (1988) Vertex-IRF Correspondence for an Elliptic /^-operator 677 9. Kac, V.G.: Infinite dimensional Lie algebras. (3 rded.) Cambridge: Cambridge University Press, 1990 10. Quano, Y., Fujii, A.: Yang-Baxter equation for broken Z®"" 1 models. Mod. Phys. Lett. A8, 1585-1597 (1993) ( ) 11. Richey, M.P., Tracy, C.A.: TLn Baxter model: Symmetries and the Belavin parametrization. Stat. Phys. 42 3/4, 311-348 (1986) 11. Richey, M.P., Tracy, C.A.: TLn Baxter model: Symmetries and the Belavin parametrization. J. Stat. Phys. 42 3/4, 311-348 (1986) 12 Shib k Y U K C l l Έ i R i L M h Ph 25 239 248 y ( ) 12. Shibukawa, Y., Ueno, K.: Completely Έ symmetric R matrix. Lett. Math. Phys. 25, 239-24 (1992) 13. Shibukawa, Y., Ueno, K.: A new solution of the Yang-Baxter equation with complete TL symmetry. Int. J. Mod. Phys. A (Proc. Suppl.) 3A. Yang, C.N., Ge, M.L., Zhou, X.W. (eds.) Differential geometric methods in theoretical physics. Proceedings, Tianjin 1992, Singapore: World Scientific, 1993, pp. 309-312 14. Shibukawa, Y., Ueno, K.: Infinite-dimensional R matrix with complete TL symmetry. In: Ge, M.L., de Vega, H.J. References (eds.) Quantum groups, integrable statistical models and knot theory. Singapore: World Scientific, 1993, pp. 302-318 g p , , pp 15. Whittaker, E.T., Watson, G.N.: A course of modern analysis. Fourth edition. Cambridge: Cambridge University Press, 1927 Communicated by M. Jimbo
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CORDIAL LABELING FOR CYCLE OF COMPLETE BIPARTITE GRAPHS AND CYCLE OF WHEELS
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PA ijpam.eu PA ijpam.eu ijpam.eu AMS Subject Classification: 05C78 Key Words: cordial labeling, cycle of complete bipartite graphs and cycle of wheels V.J. Kaneria1, Meera Meghpara2 §, H.M. Makadia3 1Department of Mathematics Saurashtra University Rajkot, 360005, 2Om Engineering College Junagadh, 362001, 3Govt. Engineering College RAJKOT, 360005, Abstract: In this paper we have obtained cordial labeling for cycle of complete bipartite graphs and cycle of wheels. AMS Subject Classification: 05C78 CORDIAL LABELING FOR CYCLE OF COMPLETE BIPARTITE GRAPHS AND CYCLE OF WHEELS V.J. Kaneria1, Meera Meghpara2 §, H.M. Makadia3 1. Introduction The concept of cordial labeling was introduced by Cahit [2] in 1987 as a weaker version of graceful and harmonious labelings. Many researchers have studied cordiality of graphs. Ho et al. [3] proved that unicyclic graph is cordial unless it is C4k+2. Kaneria et al. [6] introduced a graph known as cycle of graphs. In [7] Kaneria et al. proved that cycle of a cycle is cordial. c⃝2015 Academic Publications, Ltd. url: www.acadpubl.eu Received: September 4, 2014 §Correspondence author 2 V.J. Kaneria, M. Meghpara, H.M. Makadia The recent survey on graph labeling can be found in Gallian [4], which pro- vide vast amount of literature on graph lableling. Labelled graph have variety of applications in coding theory. A detailed study about applications of graph labeling is carried out in Bloom and Golomb [1]. For all terminology and no- tations we follws Harary [5]. First of all we shall recall some definitions, which are used in this paper. Definition 1.1. A function f : V (G) −→{0, 1} is called binary vertex labeling of a graph G and f(v) is called label of the vertex v of G under f. For an edge e = (u, v), the induced function f ∗: E(G) −→{0, 1} defined as f ∗(e) = |f(u) −f(v)|. Let vf(0), vf(1) be number of vertices of G having labels 0 and 1 respectively under f and let ef(0), ef(1) be number of edges of G having labels 0 and 1 respectively under f ∗. A binary vertex labeling f of a graph G is called cordial labeling if |vf(0) −vf(1)| ≤1 and |ef(0) −ef(1)| ≤1. A graph which admits cordial labeling is called cordial graph. A graph which admits cordial labeling is called cordial graph. Definition 1.2. For a cycle Cn, each vertices of Cn is replace by connected graphs G1, G2, . . . , Gn is known as cycle of graphs and we shall denote it by C(G1, G2, . . . , Gn). If we replace each vertices by a graph G i.e. G1 = G, G2 = G, . . ., Gn = G, such cycle of a graph G, we shall denote it by C(n · G). 2. Main Results Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. , n, 1 when t ≡0 1 (mod 4) and k = n + 1 n + 2 n or f(v1,k) = ( 0, ∀k = 1, 2, . . . , n 2 , 1, ∀k = n 2 + 1, n 2 + 2, . . . , n; f(ut,j) =            0, when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m 2 or t ≡3 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m, 1, when t ≡0, 1, 2 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m or t ≡3 (mod 4) and j = 1, 2, . . . , m 2 ; f(vt,k) =            0, when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n 2 or t ≡2, 3 (mod 4) and k = n 2 + 1, n 2 + 2, . . . , n, 1, when t ≡0, 1 (mod 4) and k = n 2 + 1, n 2 + 2, . . . , n or t ≡2, 3 (mod 4) and k = 1, 2, . . . , n 2 . f(vt,k) =            0, when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n 2 or t ≡2, 3 (mod 4) and k = n 2 + 1, n 2 + 2, . . . , n, 1, when t ≡0, 1 (mod 4) and k = n 2 + 1, n 2 + 2, . . . , n or t ≡2, 3 (mod 4) and k = 1, 2, . . . , n 2 . Case II. W.l.o.g. we assume that m is even and n is odd. Therefore: Case II. W.l.o.g. we assume that m is even and n is odd. Therefore: f(u1,j) = ( 0, ∀j = 1, 2, . . . , m 2 , 1, ∀j = m 2 + 1, m 2 + 2, . . . Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. Proof. Let G be a cycle of t copies of the complete bipartite graph Km,n. Let ui,j (1 ≤j ≤m) and vi,k (1 ≤k ≤n) be vertices of ith copy of Km,n, ∀ i = 1, 2, . . . , t. We shall join ui,m vertex of ith copy of Km,n with vi+1,1 vertex of (i + 1)th copy of Km,n by an edge, ∀i = 1, 2, . . . , t −1. We also join ut,m with v1,1 by an edge to form the cycle graph C(t · Km,n). , , To define the labeling function f : V (C(t · Km,n)) −→{0, 1} we shall con- sider following three cases. Case I. m and n are even. Then: f(u1,j) = ( 0, ∀j = 1, 2, . . . , m 2 , 1, ∀j = m 2 + 1, m 2 + 2, . . . , m; CORDIAL LABELING FOR CYCLE OF COMPLETE... 3 3 f(v1,k) = ( 0, ∀k = 1, 2, . . . , n 2 , 1, ∀k = n 2 + 1, n 2 + 2, . . . , n; f(ui,j) = ( f(u1,j), when i ≡0, 1 (mod 4), 1 −f(u1,j), when i ≡2, 3 (mod 4); f(vi,k) = ( f(v1,k), when i ≡0, 1 (mod 4), 1 −f(v1,k), when i ≡2, 3 (mod 4), where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . , t −1. Hence f(ut,j) =            0, when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m 2 or t ≡3 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m, 1, when t ≡0, 1, 2 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m or t ≡3 (mod 4) and j = 1, 2, . . . , m 2 ; f(vt,k) =       0, when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n 2 or t ≡2, 3 (mod 4) and k = n 2 + 1, n 2 + 2, . . . Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. , m; f(v1,k) = ( 0, ∀k = 1, 2, . . . , n−1 2 , 1, ∀k = n+1 2 , n+3 2 , . . . , n; f(ui,j) = ( f(u1,j), when i ≡0, 1 (mod 4), 1 −f(u1,j), when i ≡2, 3 (mod 4); f(vi,k) = ( f(v1,k), when i ≡0, 1 (mod 4), 1 −f(v1,k), when i ≡2, 3 (mod 4), where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . , t −1. So, we have f(ut,j) =            0, when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m 2 or t ≡3 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m, 1, when t ≡0, 1, 2 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m or t ≡3 (mod 4) and j = 1, 2, . . . , m 2 ; f(ui,j) = ( f(u1,j), when i ≡0, 1 (mod 4), 1 −f(u1,j), when i ≡2, 3 (mod 4); f(ui,j) = ( f(u1,j), when i ≡0, 1 (mod 4), 1 −f(u1,j), when i ≡2, 3 (mod 4); f(vi,k) = ( f(v1,k), when i ≡0, 1 (mod 4), 1 −f(v1,k), when i ≡2, 3 (mod 4), where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . , t −1. So, we have f(ut,j) =            0, when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m 2 or t ≡3 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m, 1, when t ≡0, 1, 2 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m or t ≡3 (mod 4) and j = 1, 2, . . . , m 2 ; where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. , t −1. So, we have f(ut,j) =            0, when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m 2 or t ≡3 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m, 1, when t ≡0, 1, 2 (mod 4) and j = m 2 + 1, m 2 + 2, . . . , m or t ≡3 (mod 4) and j = 1, 2, . . . , m 2 ; 4 V.J. Kaneria, M. Meghpara, H.M. Makadia Figure 1: Cycle graph C(6 · K3,5) and its cordial labeling Figure 1: Cycle graph C(6 · K3,5) and its cordial labeling f(vt,k) =            0, when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n−1 2 or t ≡2, 3 (mod 4) and k = n+1 2 , n+3 2 , . . . , n, 1, when t ≡0, 1 (mod 4) and k = n+1 2 , n+3 2 , . . . , n or t ≡2, 3 (mod 4) and k = 1, 2, . . . , n−1 2 . Case III. m and n both are odd. Hence f(ui,j) = ( 0, ∀j = 1, 2, . . . , m−1 2 , ∀i = 1, 2, . . . , t, 1, ∀j = m+1 2 , m+3 2 , . . . , m, ∀i = 1, 2, . . . , t; f(vi,k) = ( 1, ∀k = 1, 2, . . . , n−1 2 , ∀i = 1, 2, . . . , t, 0, ∀k = n+1 2 , n+3 2 , . . . , n, ∀i = 1, 2, . . . , t. The above labeling pattern give rise cordial labeling to the given graph G, as it satisfies |vf(0) −vf(1)| ≤1 and |ef(0) −ef(1)| ≤1 in above three cases. Thus G = C(t · Km,n) is a cordial graph, ∀t, m, n ∈N −{1}. Example 2.2. C(6·K3,5) (it is related with case-III) and its cordial labeling shown in Figure 1. Theorem 2.3. C(t · Wn) is cordial, t, n ∈N −{1, 2}. Proof. Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. Let G be cycle of t copies of wheel Wn. Let vi,j (0 ≤j ≤n) be vertices of ith copy of Wn, where vi,0 is vertex of apex of the wheel Wn, ∀ CORDIAL LABELING FOR CYCLE OF COMPLETE... 5 CORDIAL LABELING FOR CYCLE OF COMPLETE... 5 i = 1, 2, . . . , t. We shall join vi,0 with vi+1,0, ∀i = 1, 2, . . . , t −1 and vt,0 with v1,0 unless t ≡2 (mod 4), otherwise join v1,0 with vt,1 to form the cycle graph C(t · Wn). ( ) To define the labeling function f : V (t · Wn) −→{0, 1, }, we have following four cases. Case I. Let t ≡0, 2 (mod 4). Then Case I. Let t ≡0, 2 (mod 4). Then Case I. Let t ≡0, 2 (mod 4). Then f(vi,0) =      0, when i ≡0, 1 (mod 4), 1, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t; f(vi,j) =      1, when i ≡0, 1 (mod 4), 0, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t, ∀j = 1, 2, . . . , n. Case II. Let t ≡1, 3 (mod 4) and n ≡0, 1, 2 (mod 4). Then Case II. Let t ≡1, 3 (mod 4) and n ≡0, 1, 2 (mod 4). Then f(vi,0) =      0, when i ≡0, 1 (mod 4), 1, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t −1; f(vi,0) =      0, when i ≡0, 1 (mod 4), 1, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t −1; f(vi,j) =      1, when i ≡0, 1 (mod 4), 0, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n; f(vt,j) =      1, when j ≡1, 2 (mod 4), 0, when j ≡0, 3 (mod 4), ∀j = 0, 1, 2, . . . , n. 3. Concluding Remarks Cordial labeling of some cycle of graphs discussed. Here we provide cordial labeling to C(t · Km,n) and C(t · Wn). V.J. Kaneria, M. Meghpara, H.M. Makadia 6 f(vt,j) =      0, when j = 1, 2 or j ≡2, 3 (mod 4), 1, when j = 0, 3, 4, 5 or j ≡0, 1 (mod 4), ∀j = 6, 7, . . . , n. Case IV. Let t ≡3 (mod 4) and n ≡3 (mod 4). Then Case IV. Let t ≡3 (mod 4) and n ≡3 (mod 4). Then f(vi,0) =      0, when i ≡0, 1 (mod 4), 1, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t −1; f(vi,j) =      1, when i ≡0, 1 (mod 4), 0, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n; f(vt,j) =      1, when j = 1, 2 or j ≡2, 3 (mod 4), 0, when j = 0, 3, 4, 5 or j ≡0, 1 (mod 4), ∀j = 6, 7, . . . , n. The above labeling pattern give rise cordial labeling to the given graph G, as it satisfies |vf(0) −vf(1)| ≤1 and |ef(0) −ef(1)| ≤1 in above four cases. Thus G = C(t · Wn) is a cordial graph, ∀t, n ∈N −{1, 2}. Example 2.4. C(5W7) and its cordial labeling (it is related with Case III) shown in Figure 2. Example 2.5. C(5W4) and its cordial labeling (it is related with Case II) shown in Figure 3. Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. f(vt,j) =      1, when j ≡1, 2 (mod 4), 0, when j ≡0, 3 (mod 4), ∀j = 0, 1, 2, . . . , n. Case III. Let t ≡1 (mod 4) and n ≡3 (mod 4). Then Case III. Let t ≡1 (mod 4) and n ≡3 (mod 4). Then f(vi,0) =      0, when i ≡0, 1 (mod 4), 1, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t −1; f(vi,j) =      1, when i ≡0, 1 (mod 4), 0, when i ≡2, 3 (mod 4), ∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n;   1, when i ≡0, 1 (mod 4),   1, when i ≡0, 1 (mod 4), f(vi,j) =  0, when i ≡2, 3 (mod 4),    ∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n;    ∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n; V.J. Kaneria, M. Meghpara, H.M. Makadia V.J. Kaneria, M. Meghpara, H.M. Makadia [7] V.J. Kaneria, H.M. Makadia, Meera Meghpara, Gracefulnes of cycle of cycles and complete bipartite graphs, I.J.M.T.T., 12, No. 1 (2014), 19-26. V.J. Kaneria, M. Meghpara, H.M. Makadia References [1] G.S. Bloom, S.W. Golomb, Application of numbered undirected graphs, Proc. of IEEE, 65, No. 4 (1977), 562-570. CORDIAL LABELING FOR CYCLE OF COMPLETE... 7 7 Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 3: Cycle graph C(5 · W4) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 3: Cycle graph C(5 · W4) and its cordial labeling Figure 3: Cycle graph C(5 · W4) and its cordial labeling [2] I. Cahit, Cordial graphs: A weaker version of graceful and harmonious graphs, Ars Combin, 23 (1987), 201-207. [3] Y.S. Ho, S.M. Lee, S.C. Shee, Cordial labeling of unicyclic graphs and generalized Petersen graphs, Congress. Numer., 68, 109-122. [4] J.A. Gallian, The Electronics Journal of Combinatorics, 19 (2013). [5] F. Harary, Graph Theory, Addition Wesley, Massachusetts, 1972. [6] V.J. Kaneria, H.M. Makadia, M.M. Jariya, Graceful labeling for cycle of graphs, Int. J. of Math. Res., 6, No. 2 (2014), 173-178. V.J. Kaneria, M. Meghpara, H.M. Makadia 8 [7] V.J. Kaneria, H.M. Makadia, Meera Meghpara, Gracefulnes of cycle of cycles and complete bipartite graphs, I.J.M.T.T., 12, No. 1 (2014), 19-26.
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Advanced Fabrication and Characterization of Hydrothermal Responsive Fabric from Microcrystalline Cellulose-Reinforced Shape Memory Polyurethane Filament
Textile & leather review
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Textile & Leather Review ISSN 2623-6281 | www.tlr-journal.com | 10.31881/TLR Advanced Fabrication and Characterization of Hydrothermal Responsive Fabric from Microcrystalline Cellulose-Reinforced Shape Memory Polyurethane Filament Direselgn Molla Semanie, Lei Zhang, Bewuket Teshome Wagaye, Buguang Zhou, Yalin Dong, Jiansheng Guo How to cite: Semanie DM, Zhang L, Wagaye BT, Zhou B, Dong Y, Guo J. Advanced Fabrication and Characterization of Hydrothermal Responsive Fabric from Microcrystalline Cellulose-Reinforced Shape Memory Polyurethane Filament. Textile & Leather Review. 2024; 7:670-687. https://doi.org/10.31881/TLR.2024.074 How to link: https://doi.org/10.31881/TLR.2024.074 Published: 8 May 2024 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 Advanced Fabrication and Characterization of Hydrothermal Responsive Fabric from Microcrystalline Cellulose-Reinforced Shape Memory Polyurethane Filament Direselgn Molla SEMANIE, Lei ZHANG, Bewuket Teshome WAGAYE, Buguang ZHOU, Yalin DONG, Jiansheng GUO* Key Laboratory of Textile Science & Technology, Ministry of Education, College of Textiles, Donghua University, Shanghai, China *jsguo@dhu.edu.cn Article https://doi.org/10.31881/TLR.2024.074 Received 2 April 2024; Accepted 30 April 2024; Published 8 May 2024 ABSTRACT Demands for smart textiles have recently increased quickly in terms of functionality and responsiveness to wearers and environmental changes. This paper explores the development of hydrothermal responsive shape memory woven fabric from Microcrystalline Cellulose Reinforced Shape Memory Polyurethane Microcomposite Filament. The concentration of microcrystalline cellulose and drawing ratio of the filaments were first optimized according to the tensile strength and shape recovery ratio and taken as 15 wt% and 2.0 respectively. Hydrothermal responsive shape memory micro-composite filaments were then produced from shape memory polyurethane (SMPU) and microcrystalline cellulose (MCC) with optimized concentration and draw ratio by wet spinning process. The physical, mechanical, thermal and shape memory performances of the filaments were studied. The optimized filament was found to have a tensile strength of 0.91 cN/dtex and elongation of 385.2% in which the strength is much more improved when compared to a pure SMPU filament of strength 0.72 cN/dtex. The shape fixity and shape recovery results of the micro-composite filament were found to be 71.2% and 93.6% respectively. A woven fabric was manufactured from pure polyester, cotton as warp and SMPU-MCC filaments as weft and its breathability and shape memory properties were investigated. The air permeability of SMPU-PE fabrics was found to be 172.9 mm/s and 155.1 mm/s in its fixed temporary shape and recovered shape respectively. The water vapour permeability of SMPU-CT fabric was found to be 612.01 g/m2.h and 540.28 g/m2.h in its fixed temporary and recovered shape respectively which shows smart breathable fabrics can be made and adopted with enhanced properties. KEYWORDS cellulose, micro-composite filaments, hydrothermal responsive fabrics, shape memory polyurethane INTRODUCTION Shape memory materials (SMMs) have gained significant attention in recent years due to their ability to change their shape in response to specific stimuli (temperature, water or moisture, magnetic field, etc.), and memorize their original shape and potential applications in various fields such as smart textiles, energy harvesting, biomedical engineering and responsive materials [1]. The two most used https://doi.org/10.31881/TLR.2024.074 670 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 types of SMMs are shape-memory alloys (SMAs) and shape-memory polymers (SMPs) [2]. SMAs exhibit their shape memory effect through the transformation of austenite to martensite, a process that has found applications in textiles in various forms such as filaments or springs [3]. On the other hand, the shape memory effect of SMPs is due to the movement of hard and soft segments within polymer chains. This allows a new temporary shape to be formed and later returned to its original shape [2,4]. SMPs offer several advantages over SMAs, including greater production versatility, low manufacturing cost, high shape recovery rate, and a broad triggering temperature range. The relatively low melting point, ability to withstand up to 800% deformation, and thermoplastic properties of SMPs also enable them to be produced as thin filament yarns, composite, and various types of textile fabrics [5]. In the textile industry, SMPs can be used for temperature regulation and maintenance applications such as breathable sportswear that can provide better comfort under variable temperature and moisture environments. Since their discovery in 1980, SMPs have been developed to respond to a variety of stimuli, including heat, moisture or water, electrical current, alternating magnetic fields and light [6]. There are various types of temperature and water-responsive SMPs, but shape memory polyurethane (SMPU) is suitable for the textile application since it can be applied as coating as well as shape memory filaments used to make fabrics with a triggering or glass transition temperature (Tg) very close to a human body temperature. However, SMPU pure filaments and yarns are not strong enough to be woven or knitted into a textile fabric. Therefore, the incorporation of reinforcements mainly cellulose and carbon fibres are used in different stages of textile manufacturing processes which leads to the development of SMPU filament composites [7,8]. Korkmaz et al. produced dual moisture and temperature-responsive shape memory nanocomposite filaments from SMPU and cellulose nanowhiskers and achieved a shape recovery ratio of up to 100% with remarkable strength of filaments [9]. It has been noted that the incorporation of cellulose has improved the strength and waterresponsive properties of the filaments. However, no prior studies have been conducted to produce fabrics from microcrystalline cellulose (MCC) reinforced shape memory polyurethane filaments and the effect of MCC on the breathability and overall properties of fabrics has not been investigated. The combination of MCC and SMPU provides a synergistic effect, where the MCC acts as a reinforcing agent, enhancing the mechanical strength and dimensional stability of the fabric, while the SMPU gives the shape memory effect [10]. This paper presents an advanced fabrication of woven hydrothermal responsive shape memory fabrics using a microcrystalline cellulose-reinforced shape memory polyurethane composite filament as weft and either polyester or cotton as warp yarn. The physical properties, permeability and shape memory properties of these fabrics are also examined. https://doi.org/10.31881/TLR.2024.074 671 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 EXPERIMENTAL Materials Pellet-type SMPU granules purchased from Sigma-Aldrich, USA were used as matrix material and Microcrystalline cellulose (MCC) (70-140μm) also purchased from Sigma-Aldrich was used as a reinforcement. In addition, N-dimethylformamide (DMF) and Polyoxyethylene sorbitan monooleate (Tween 80) both from Sigma-Aldrich were used as a polar solvent and nonionic surfactant respectively. Production of Optimized Micro-composite Filament A multivariate Response Surface Methodology (RSM) using central composite design (CCD) was used to determine the optimal concentration of microcrystalline cellulose (MCC) and drawing ratio. Through optimization, it was found that 15 wt% MCC (relative to the polymer mass) and a draw ratio of 2.0 were the ideal values. A spinning solution containing 25 wt% SMPU polymer, 15 wt% MCC in DMF and a non-ionic surfactant called Tween80 at a ratio of 1:2 w/w with MCC was prepared. This solution was prepared at 60 °C for 6 hours using an ultrasonic bath with a mechanical stirrer at a speed of 200 rpm [11]. The spinning solution was then extruded using Sazhuo wet spinning equipment (Shanghai Sazhuo New Material Technology CO., LTD, China) with a needle diameter of 1.05 mm with a constant flow rate of 0.81 ml/min into a coagulation bath of distilled water and then take-up speed of 1.9 m/min. The SMPU-MCC micro-composite filaments were then soaked in distilled water for 24 hours to remove any residual solvent (DMF) and dried for 10 hours at 40 °C. Production of Hydrothermal Fabrics Plain woven fabrics were manufactured using 250 dtex polyester and 280 dtex cotton yarns as warp yarn, while SMPU-MCC micro-composite filament in its fixed temporary shape was used as the weft. The fabrics were produced using SGA598 semi-automatic sampling loom (TONG YUAN TEXTILE MACHINERY CO.LTD, China) in which weft yarns were inserted manually using a bobbin. To make a comparison, plain-weave fabrics made entirely of 100% polyester and cotton were also produced, along with plain-weave fabric incorporating SMPU-MCC micro-composite filament. Hydrothermal responsive fabrics at their fixed temporary shape were immersed in hot water at a temperature of Tg+10 °C for ten minutes to recover to their original shape. Determination of Tensile Properties The mechanical properties of the filaments were assessed using the LLY-06E-500 Electronic Single Fiber Strength Tester (Laizhou Electronic Instruments Co., Ltd, China). This was done in compliance with ASTM D3822-07, using an extension rate of 200 mm/min and a gauge length of 20 mm. https://doi.org/10.31881/TLR.2024.074 672 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 Morphological Test of Filaments TM4000 Table Top Scanning Electron Microscope (Thermo Scientific, USA) was used to assess the distribution of MCC in the filament structure and the overall morphological setup. Determination of Functional Groups The chemical structure and functional group of SMPU-MCC filaments were studied using Fourier Transform Infrared (FTIR) spectroscopy (PerkinElmer, USA) within a spectral range of 4000 cm-1 to 400 cm-1. Thermal Analyses TGA 8000 Thermogravimetric analysis equipment (PerkinElmer Instruments, USA) was utilized to evaluate the thermal properties of micro-composite filaments. The test was conducted in a nitrogen environment, with temperatures ranging from 50 to 600 °C, and a heating rate of 10 °C/min. The decomposition temperature (Tmax), T10% and T50% of the micro-composite filaments were calculated based on the thermogravimetric data. The glass transition temperature (Tg) of filaments was examined using a Differential Scanning Calorimetry DSC 8500 (PerkinElmer Instruments, USA) according to ISO 11,357:1-7 standard. Filaments, weighing between 5 and 10 mg, were heated from -10 to 200 °C at a rate of 10 °C/min, then cooled to 20 °C at a rate of 20 °C/min and Tg was identified from the first heating. Physical Tests The linear density of both pure SMPU and SMPU-MCC filaments, in their original and fixed temporary shape, was measured in dtex as per the ASTM D2591-07 (Reapproved 2020) standard. The warp (EPC) and weft density (PPC) of hydrothermal responsive fabrics were determined using a thread counter. The warp or ends per centimetre (EPC) and picks or picks per centimetre (PPC) of the fabrics were determined using Pick glass (Nantong Hongda Laboratory Instrument Co., Ltd, China). The thickness of the fabrics was tested using YG (B) 141G fabric thickness tester (WENZHOU DARONG TEXTILE INSTRUMENT CO., LTD, China) according to GB/T 3820-1997 and ASTM D1777 − 96 (Reapproved 2019) with 28.7 ± 0.02 mm foot diameter and 4.14 ± 0.21 kPa applied pressure. Shape Memory Properties of Filaments The shape memory effect (SME) of filaments was evaluated using a mechanical-thermo-aqueous programming test. Initially, the filaments, with an initial length (Li) of 5 cm were stretched (100%) in hot water at a temperature of Tg +10 °C to a temporary length (Lt) for 10 minutes. Then, the filaments were cooled to a temperature below their glass transition (23 °C), and dried for 12 hours and a fixed https://doi.org/10.31881/TLR.2024.074 673 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 temporary length (Lf) was recorded. The shape memory effect (SME) was then triggered by reimmersing the filaments in water at a recovery temperature of Tg +10 °C for ten minutes and the samples returned to their recovered length (Lr) [5]. Finally, the shape-memory properties of the filaments were calculated using the shape-recovery ratio (Rr) and the shape-fixity ratio (Rf)(9)(12). Figure 1 shows a schematic representation of the mechanical-thermo-aqueous programming test and its parameters. Figure 1. Schematic representation of mechanical-thermo-aqueous programming test Air and Water Vapor Permeability Properties The air-permeability of fabrics was evaluated in accordance with GB/T 5453-1997, using the YG641E Air-permeability Tester (WENZHOU FANGYUAN INSTRUMENT CO. LTD, China). Five samples from each fabric with a size of 20 cm2 were used and the average permeability rate was reported as the air permeability of the fabric. The water vapour transmission rate (WVTR) of all (pure PE, pure CT, SMPU-PE, and SMPU-CT) fabrics was measured using a YG601H computer-type fabric moisture permeability testing apparatus (Ningbo Textile Instrument, China). A separate balance with 0.0001 g weighing precision was used to measure the consecutive mass of test samples. A pair of samples each with a sample size of 28.3 cm² from both https://doi.org/10.31881/TLR.2024.074 674 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 pure and SMPU woven fabrics were used. A desiccant technique was used in which the specimen was sealed to the open mouth of a test cup that contained a desiccant (Calcium chloride) and put in a controlled environment. The dishes were regularly measured to establish the rate at which water vapour passes through the specimen into the desiccant. All of the experiments were carried out according to GB/T 12704.1-2009 and ASTM E96 at 38.0 °C and 90% relative humidity with an air velocity of 0.5 m/s. The water vapour transmission rate (WVTR) and Water vapour permeability (WVP) of fabrics were calculated using the formulas stated in the above standard test methods as follows: WVTR = WVP = WVT ∆P ∆m−∆m′ A.t WVT = Pcb(R1−R2) Water Permeability = WVP × Thickness (1) (2) (3) Where WVTR is the water vapour transmission rate of fabrics in g/m2.h, ∆m is the difference between two weightings of the same test combination in g, ∆m' is the difference between two weightings of the blank sample in g, A is the effective test area in m2 (0.00283 m2 was used), t is test time in h, WVP is water vapour permeance of fabrics in g/m2.Pa.h. ∆P is the difference in water vapour pressure on both sides of the sample in pa, Pcb is saturated water vapour pressure at test temperature in pa (6625.056 Pa at 38.0 °C), R1 is the relative humidity of the test chamber during the test (90%) and R2 is relative humidity in the cup (0%). RESULTS AND DISCUSSION Mechanical Properties of Filaments The strength of shape-memory polyurethane fibres can be improved by the incorporation of reinforcing materials such as cellulose [3,10,11,13]. Figure 2 shows the stress-strain curves of pure SMPU and SMPU-MCC micro-composite filaments at their original and fixed temporary shapes. In its original shape, the SMPU-MCC micro-composite filament has 0.91 cN/dtex tenacity and 385.2% elongation. However, these values decreased to a tenacity of 0.74 cN/dtex and an elongation of 331.8% in its fixed temporary shape. Pure SMPU filaments have a tenacity of 0.72 cN/dtex and an elongation percentage of 519.3% in their original shape and 386.4% in their fixed temporary shape. SMPU-MCC micro-composite filaments have higher strength due to better load transfer due to matrixcellulose chain alignment [11]. https://doi.org/10.31881/TLR.2024.074 675 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 Table 1. Tensile properties of micro-composite filaments at their original and fixed temporary shape Original shape (OS) Filament code Fixed temporary shape (FTS) Tenacity (cN/dtex) Strain (%) Tenacity (cN/dtex) Strain (%) SMPU 0.72 519.3 0.61 386.4 SMPU-MCC 0.91 385.2 0.74 331.8 1.0 SMPU-OS SMPU-MCC-OS SMPU-FTS SMPU-MCC-FTS Tenacity (cN/dtex) 0.8 0.6 0.4 0.2 0 100 200 300 400 500 600 Strain (%) Figure 2. Tensile properties of Microcomposite filaments at their original and fixed temporary shape Morphological Analyses Figures 3 a & b for pure SMPU filaments and c & d for SMPU-MCC micro-composite filaments show the longitudinal and cross-sectional views of the two types of filaments. The figure shows that the crosssection of pure SMPU is slightly irregular and has foldings, microcracks and a distinct fibre edge [14]. This was explained by Korkmaz et al. as the lack of reinforcement materials such as cellulose, which affects viscosity and causes a low mass transfer rate difference between the coagulating solvent diffusion into the spinning solution and the solvent extraction into the coagulation bath [11]. Hence, the cross-sections of SMPU-MCC micro-composite filaments are comparatively regular and circular [15-17]. https://doi.org/10.31881/TLR.2024.074 676 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 Figure 3. Longitudinal (on the left) and Cross-sectional (on the right) SEM images of a) and b) pure SMPU-2.0; c) and d) SMPU-MCC Determination of Functional Groups The FT-IR spectra of SMPU-MCC and pure SMPU filaments are shown in Figure 4. A peak at 3271 cm-1 corresponds to the stretching vibrations of the (-OH-) and (-NH-) groups in SMPU polymers. The peaks 2850 cm-1 show the symmetric stretching of (-CH-) and 1627 cm-1 shows the bending vibrations of (C=O-) groups of SMPU. The particular peaks correspond to the stretching vibrations of the(-NH-) and (-OH-) shift for SMPU-MCC filaments from 3271 cm-1 to 3346 cm-1. This shift shows the presence of extra hydroxyl (-OH) groups from microcrystalline cellulose [15,18,19]. For SMPU-MCC, the distinctive peaks at 2949 cm-1 show the (-CH2-) and (-C-H-) stretching vibrations of the hydrocarbon chains. The peak at 1728 cm-1 corresponds to the (-C=C-) bending vibration of alkenes found in SMPU and cellulose, whereas the peak absorption at 1627 cm-1 and 1540 cm-1 shows (-C=O-) stretching vibrations [14,16,20,21]. https://doi.org/10.31881/TLR.2024.074 677 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 1206 1066 1728 Absorbance (%) 1540 3346 SMPU-MCC 16 32 4000 3500 523 2949 71 3000 28 27 50 SMPU 2500 2000 1500 1000 500 Wavenumber (cm-1) Figure 4. FTIR spectra values of pure SMPU and SMPU-MCC micro-composite filaments Thermal Analyses Thermogravimetric Analysis (TGA) test results of SMPU-MCC micro-composite filaments and pure SMPU filaments are shown in Figure 5. The TGA curve showed that the T10% temperature rose from 309.9 °C for pure SMPU filaments to 325.6 °C SMPU-MCC microcomposite filaments and the T50% temperature increased from 336.9 °C to 380.96 °C for SMPU-MCC microcomposite filaments compared to pure SMPU. This indicates that the presence of MCC improved the thermal stability of filaments. The degradation temperatures at T10% can be attributed to the removal of loosely bound volatile compounds whereas the first step degradations at around 343 °C is the most representative indicator of the decomposition of SMPU polymer hard segment linkages [11]. The DTG graph shows a significant increase in the maximum decomposition temperature (Tmax) of the filaments from 370 °C (SMPU) to 390 °C (SMPU-MCC), indicating better thermal stability due to interaction between the SMPU matrix and MCC through hydrogen bonding [22]. Gan et al. indicated that strong intermolecular bonding increases the energy needed for macromolecule chain breaking, which improves thermal stability [8,23]. In addition, the char residue for pure SMPU and SMPU-MCC micro-composite filaments were found to be 4.96 wt% and 10.97 wt% respectively. https://doi.org/10.31881/TLR.2024.074 678 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 100 SMPU SMPU-MCC 60 40 DTG (%) Mass () 80 SMPU SMPU-MCC 20 100 200 300 400 500 Temperature (°C) 0 100 200 300 400 500 600 Temperature (°C) Figure 5. TGA and DTG curves of SMPU and MCC-SMPU micro-composite filaments As indicated in Figure 6 and Table 2, the DSC test results showed that the Tg of the pure SMPU filament and SMPU-MCC micro-composite filaments were 41.65 °C and 41.02 °C respectively. The presence of MCC in the SMPU filament structure resulted in slightly lower Tg and higher heat absorption than pure SMPU micro-composite filaments [24]. This is consistent with Korkmaz et al. and Gan et al. where a reduction in Tg of composite fibres after the incorporation of cellulose was reported [8,11]. Korkmaz et al. studied that the plasticizing effect and micro-Brownian thermal movements of molecular chain segments caused by CNW particles contribute to a reduction in Tg [11]. Heat Flow Endo Up (mW) SMPU-MCC Pure SMPU 50 100 150 Temperature (°C) Figure 6. First heating DSC curves for pure SMPU and SMPU-MCC microcomposite filaments https://doi.org/10.31881/TLR.2024.074 679 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 Table 1. First heating DSC results for pure SMPU and SMPU-MCC micro-composite filaments Sample Code Tg (°C) SMPU 41.65 SMPU-MCC 41.02 Shape Memory Properties The shape memory characteristics of micro-composite filaments and pure SMPU at a triggering temperature of Tg+10 oC are shown in Figure 7. The micro-composite filament (SMPU-MCC) has a shape fixity ratio of 71.3%, which is higher than that of pure SMPU filaments (64%) and suggests that the SMPU-MCCC filaments will be able to retain its fixed temporary shape when the load is removed [14]. However, the shape recovery ratio decreases when Microcrystalline Cellulose (MCC) is added to the SMPU filaments. This is due to the reduced mobility of the soft segments as the addition of MCC increases the strength and rigidity, which is associated with an increased hard segment portion of the filaments [14]. For MCC-SMPU, the shape recovery ratio was found to be 93.6%, while the shape recovery of pure SMPU filaments was 96%. These values align with Korkmaz et al., which reported a shape recovery ratio between 91% and 100% for SMPU reinforced with cellulose nanowhiskers [11]. The moisture-responsive shape memory effect of MCC-SMPU filaments is primarily due to the incorporation of MCC, which introduces hydrophilic properties to the shape memory polyurethanes. Hydrogen bonds will be formed between water molecules and the hydroxyl groups in MCC when the filaments are exposed to hot water above Tg [11]. Shape Fixity (Rf) Shape Recovery (Rr) 100 96 93.6 80 71.2 % 64 60 40 20 0 SMPU SMPU-MCC Microcomposite Code Figure 7. Shape memory properties of pure SMPU and SMPU-MCC filaments https://doi.org/10.31881/TLR.2024.074 680 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 Physical Property of Filaments Figure 8 shows the linear densities of SMPU-MCC micro-composite filaments and pure SMPU in their original shape (OS) and fixed temporary shapes (FTS), ranging from 155.3 to 182 dtex. Incorporating MCC into the SMPU matrix influences the linear density of the microcomposite filaments. Pure SMPU filaments have a linear density of 176.6 dtex, whereas SMPU-MCC filaments exhibit a higher linear density value of 181.4 dtex. This increase in linear density indicates that the stiffness and density of MCC contribute to the overall mass of the composite filament [2,11]. The linear density of both filaments in their fixed temporary shape is smaller compared to their original shape. This reduction in linear density is related to the stretching of the shape memory filaments, which leads to a decrease in the mass per unit length. Original shape (OS) Fixed temporary shape (FTS) 180 Linear Density (dTex) 150 120 90 60 30 0 SMPU SMPU-MCC Figure 8. Linear density of filaments at their a) original shape b) Fixed temporary shape Physical Property of Fabrics Table 3 shows the warp density and thickness values for pure fabrics with SMPU micro-composite filaments in both recovered shape (RS) and fixed temporary shape (FTS). The thermodynamics of hydrothermal shape memory woven fabric is based on the variation in pore size between yarns in the fabric. The fabric had an open structure in fixed temporary shape (FTS), but a more closed structure in recovered shape (RS), due to dynamic changes in the diameter of shape memory filaments. These diameter changes affect the fabric density and thickness [3]. As a result, the warp density and thickness of the fabrics in the fixed temporary shape for both SMPU-polyester (PE) and SMPU-cotton (CT) fabrics are greater than that of pure polyester and cotton fabrics. This increase is related to the recovery of the weft SMPU filaments to their original shape after the shape memory effect was triggered by https://doi.org/10.31881/TLR.2024.074 681 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 treating the fabrics in hot water at Tg+10 oC. These results are consistent with the results reported by Bertran et al., where polyester yarns are used as the warp yarn and pure SMPU filaments as the weft yarn in different weft ratios and found that the fabric parameters including tightness and thickness increase after the load was removed from weft yarns [3]. Table 2. Physical properties of different fabrics Fabric Ends per cm (EPC) Picks per cm (PPC) Thickness (mm) Pure PE 18 46.2 0.60 Pure CT 17.6 42.6 0.69 FTS RS FTS RS FTS RS SMPU-PE 17.8 22 56 56.4 0.77 0.80 SMPU-CT 18.2 21.2 59 58.6 0.78 0.79 Figure 9. A simplified representation of the hydrothermal plain-weave fabric with weft SMPU-MCC filament Air and Water Vapor Permeability Properties Air and water vapour permeability tests were carried out to assess the breathability and shape memory properties of hydrothermal responsive fabrics. Results from pure polyester (PE) and pure cotton fabrics were used as references. As shown in Table 4, the air permeability of shape Memory PolyurethaneCotton (SMPU-CT) fabric in its fixed temporary shape (FTS) (213.2 mm/s) is higher than that of its recovered shape (RS) (187.4 mm/s). Additionally, the air permeability of pure PE fabric (214.9 mm/s) is higher than that of SMPU-PE fabrics in both shapes and the air permeability of pure cotton fabric is also higher than SMPU-CT fabric in both shapes due to the difference in fabric density. Despite having a slightly lower air permeability due to its high thread density, hydrothermal responsive fabrics still exhibit comparable air permeability to pure cotton and polyester fabrics. This indicates that the use of https://doi.org/10.31881/TLR.2024.074 682 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 shape-memory polyurethane micro-composite filaments as the weft can provide similar air circulation and comfort as cotton and polyester fabrics, while also providing shape-memory properties for body moisture and temperature regulation. Air permeability, which shows how porous the fabrics are, affects both heat and sweat flow within the fabric and is affected by filament structure and properties. This can be observed in SMPU-PE hydrothermal responsive fabrics, where the air permeability at their fixed temporary shape (172.9 mm/s) is higher than their recovered shape (155.1 mm/s). SMPU-MCC filaments enable the fabric to change its air permeability by adjusting the porosity. This helps the wearer be comfortable in various body or environmental conditions [3,10]. Table 4. Air permeability properties of fabrics Fabric Air permeability (mm/s) Pure PE 214.9 Pure CT 221.2 Fixed temporary shape (FTS) Recovered shape (RS) SMPU-PE 172.9 155.1 SMPU-CT 213.2 187.4 Figure 10 shows the water vapour transmission rate (WVTR) and water permeability (WP) of pure and hydrothermal responsive fabrics. The WVTR of SMPU-PE and SMPU-CT hydrothermal responsive fabrics is 541.7 g/m2.h and 612.01 g/m2.h respectively in their fixed temporary shape, and 505.65 g/m2.h and 540.28 g/m2.h respectively in their recovered shape. The WVTR of pure polyester fabrics is lower than that of SMPU-PE fabrics in their fixed temporary shapes. This can be attributed to the smooth fibre structure, crystallinity, hydrophobicity and the lack of -OH- groups in polyester morphology which affects the movement or passage of water molecules through it. In addition, SMPUPE hydrothermal responsive fabrics exhibit a higher WP than pure polyester fabrics, even at higher fabric density (as stated in Table 3). This is due to the incorporation of microcrystalline cellulose, which improves the interaction and absorption of fabric with water and facilitates the movement of water molecules through the fabric by creating hydrogen bonds with water molecules. This is consistent with the findings of Jahid et al. and Korkmaz et al. where the water vapour transmission largely depends on the water affinity of the functional group of polymers [10,25]. The WVTR of pure cotton fabrics (657.24 g/m2.h) is higher than hydrothermal responsive fabrics made from microcrystalline cellulose incorporated shape memory polyurethane at both fixed temporary and recovered shapes. This is due to the low water affinity and reaction property of polyurethane molecules compared to cellulose. Pure cotton fabrics can create more hydrogen bonds with water than SMPU-CT fabrics. Both hydrothermal responsive shape memory fabrics in their fixed temporary shape have higher WVTR than fabrics in their recovered shape, facilitating the passage of water vapour in and out of the body related to the https://doi.org/10.31881/TLR.2024.074 683 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 temperature and humidity of the environment. This can be attributed to the change in the pore size of the fabrics, which occurs when shape memory filaments recover to their original shape. This is consistent with the findings of Bertran et al. where fabric breathability increases with the incorporation of more shape-memory polyurethane [3]. 700 0.28 (a) 657.24 600 541.7 540.28 505.65 500 0.21 WP (g/m.Hg.h) 539.93 WVTR (g/m2h) (b) 612.01 400 300 200 0.14 0.07 100 T RS SM PU -C PU -C T FT S RS SM PU -P E FT S SM -P E re CT SM PU Pu re P Pu T -C -C T PU SM PU RS FT S RS SM PU -P E FT S SM -P E PU SM Pu re CT E re P Pu E 0.00 0 Figure 10. Water vapour transmission rate (WVTR) (a) and water permeability (WP) (b) of fabrics CONCLUSION This study investigated the production and characterization of plain woven, shape memory fabrics with responsive properties to moisture and heat. The fabrics were constructed using shape memory microcomposite filaments as weft yarns, woven with either polyester or cotton warp yarns. The presence of microcrystalline cellulose in the composite filament increased the mechanical, crystallinity and waterresponsive shape memory properties of the filaments. The shape fixity and recovery of pure SMPU and SMPU-MCC filaments were evaluated by using a mechanical-thermo-aqueous programming test in hot water at a temperature of Tg +10 °C and exhibited a shape recovery of 93.6%. The air permeability of hydrothermal responsive fabrics was 172.9 mm/s and 213.2 mm/s for SMPU-PE and SMPU-CT at their fixed temporary shape and 155.1 mm/s and 187.4 mm/s at their recovered shape respectively. In addition, the water vapour transmission rate of hydrothermal responsive fabrics is 541.7 g/m2.h and 612.01 g/m2.h for SMPU-PE and SMPU-CT at their fixed temporary shape and 505.65 g/m2.h and 540.28 g/m2.h at their recovered shape respectively. These results show that hydrothermal responsive fabrics can offer better breathability and maintain body thermodynamics indicating a potential application in smart textiles such as sportswear, waterproof and breathable clothes, socks etc. https://doi.org/10.31881/TLR.2024.074 684 SEMANIE DM et al. TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687 Author Contributions Conceptualization – Semanie DM; methodology – Semanie DM; formal analysis – Semanie DM; investigation – Semanie DM; writing-original draft preparation – Semanie DM; writing-review and editing – Zhang L, Wagaye BT, Zhou B; visualization – Dong YL; supervision – Guo JS. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Funding This research received no external funding. REFERENCES [1] Vaithylingam R, Ansari MNM, Shanks RA. Recent Advances in Polyurethane-Based Nanocomposites: A Review. Polymer - Plastics Technology and Engineering. 2017; 56(14):1528– 1541. https://doi.org/10.1080/03602559.2017.1280683 [2] González J, Ardanuy M, González M, Rodriguez R, Jovančić P. Polyurethane shape memory filament yarns: Melt spinning, carbon-based reinforcement, and characterization. Textile Research Journal. 2023; 93(3–4):957–970. https://doi.org/10.1177/0040517522111416 [3] Gonzalez Bertran J, Ardanuy M, González M, Rodriguez R, Jovančić P. Design and characterization of reversible thermodynamic SMPU-based fabrics with improved comfort properties. Journal of Industrial Textiles. 2023; 53:1–20. https://doi:10.1177/15280837231166390 [4] Andersons J, Kirpluks M, Cabulis U. Reinforcement Efficiency of Cellulose Microfibers for the Tensile Stiffness and Strength of Rigid Low-Density Polyurethane Foams. Materials. 2020; 13(12), 2725. https://doi:10.3390/ma13122725 [5] Santiago D, Fabregat-Sanjuan A, Ferrando F, Flor S. 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Auburn, Alabama: Graduate Faculty of Auburn University; 2009. 210 p. Available from: https://etd.auburn.edu/handle/10415/1934 [14] Zhang Y, Hu J, Zhao X, Xie R, Qin T, Ji F. Mechanically Robust Shape Memory Polyurethane Nanocomposites for Minimally Invasive Bone Repair. ACS Applied Bio Materials. 2019; 2(3):1056– 1065. https://doi.org/10.1021/acsabm.8b00655 [15] Shojaeiarani J, Bajwa DS, Chanda S. Cellulose nanocrystal based composites : A review. Composites Part C: Open Access. 2021; 5:100164. https://doi.org/10.1016/j.jcomc.2021.100164 [16] Gupta B, Revagade N, Anjum N, Atthoff B, Hilborn J. Preparation of poly(lactic acid) fiber by dryjet-wet-spinning. I. Influence of draw ratio on fiber properties. Journal of Applied Polymer Science. 2006; 100(2):1239–1246. https://doi.org/10.1002/app.23497 [17] Raja M, Ryu SH, Shanmugharaj AM. Thermal, mechanical and electroactive shape memory properties of polyurethane (PU)/poly (lactic acid) (PLA)/CNT nanocomposites. 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Optical and mechanical properties of nanofibrillated cellulose. Carbohydrate polymers. 2015; 126:40-46. https://doi.org/:10.1016/j.carbpol.2015.03.032 [22] Ahmed N, Kausar A, Muhammad B. Advances in Shape Memory Polyurethanes and Composites : A Review. Polymer-Plastics Technology and Engineering. 2015; 54(13):1410–1423. http://doi.org/10.1080/03602559.2015.1021490 [23] Tomisawa R, Ikaga T, Kim KH, Ohkoshi Y, Okada K, Masunaga H, Kanaya T, Masuda M, Maeda Y. Effect of draw ratio on fiber structure development of polyethylene terephthalate. Polymer. 2017; 116:357–366. http://doi.org/10.1016/j.polymer.2016.12.071 [24] Jahid MA, Hu J, Wong KH, Wu Y, Zhu Y, Luo HHS, Zhongmin D. Fabric Coated with shape memory polyurethane and its properties. Polymers. 2018; 10(6):681. http://doi.org/10.3390/polym10060681 https://doi.org/10.31881/TLR.2024.074 687
https://openalex.org/W2467090882
https://europepmc.org/articles/pmc4975768?pdf=render
English
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Dicarbonyls and glyoxalase in disease mechanisms and clinical therapeutics
Glycoconjugate journal
2,016
cc-by
11,099
Glycoconj J (2016) 33:513–525 DOI 10.1007/s10719-016-9705-z Glycoconj J (2016) 33:513–525 DOI 10.1007/s10719-016-9705-z REVIEW REVIEW REVIEW * Paul J. Thornalley P.J.Thornalley@warwick.ac.uk Dicarbonyls and glyoxalase in disease mechanisms and clinical therapeutics Naila Rabbani1 & Mingzhan Xue2 & Paul J. Thornalley1,2 Received: 28 February 2016 /Revised: 6 June 2016 /Accepted: 9 June 2016 /Published online: 12 July 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Received: 28 February 2016 /Revised: 6 June 2016 /Accepted: 9 June 2016 /Published online: 12 July 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The reactive dicarbonyl metabolite methylglyoxal (MG) is the precursor of the major quantitative advanced glycation endproducts (AGEs) in physiological systems - argi- nine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. The glyoxalase system in the cytoplasm of cells provides the primary defence against dicarbonyl glycation by catalysing the metabolism of MG and related reactive dicarbonyls. Dicarbonyl stress is the abnormal accumulation of dicarbonyl metabolites leading to increased protein and DNA modification contributing to cell and tissue dysfunction in ageing and disease. It is produced endogenously by increased formation and/or decreased metabolism of dicarbonyl metabo- lites. Dicarbonyl stress contributes to ageing, disease and activ- ity of cytotoxic chemotherapeutic agents. It contributes to age- ing through age-related decline in glyoxalase 1 (Glo-1) activity. Glo-1 has a dual role in cancer as a tumour suppressor protein prior to tumour development and mediator of multi-drug resis- tance in cancer treatment, implicating dicarbonyl glycation of DNA in carcinogenesis and dicarbonyl-driven cytotoxicity in mechanism of action of anticancer drugs. Glo-1 is a driver of cardiovascular disease, likely through dicarbonyl stress-driven dyslipidemia and vascular cell dysfunction. Dicarbonyl stress is also a contributing mediator of obesity and vascular complica- tions of diabetes. There are also emerging roles in neurological disorders. Glo-1 responds to dicarbonyl stress to enhance cytoprotection at the transcriptional level through stress- responsive increase of Glo-1 expression. Small molecule Glo- 1 inducers are in clinical development for improved metabolic, vascular and renal health and Glo-1 inhibitors in preclinical development for multidrug resistant cancer chemotherapy. Keywords Methylglyoxal . Glycation . Glyoxalase . Obesity . Diabetes, cancer . Renal failure . Cardiovascular disease . Therapeutics Keywords Methylglyoxal . Glycation . Glyoxalase . Obesity . Diabetes, cancer . Renal failure . Cardiovascular disease . Dicarbonyls and glyoxalase in disease mechanisms and clinical therapeutics Therapeutics Abbreviations ADH Aldehyde dehydrogenase AGE Advanced glycation endproduct AKR Aldoketo reductase ARE Antioxidant response element CVD Cardiovascular disease 3-DG 3-Deoxyglucosone DHAP Dihydroxyacetonephosphate ESRD End stage renal disease GA3P Glyceraldehyde-3-phosphate GdG 3-(2′-deoxyribosyl)-6,7-dihydro-6,7- dihydroxyimidazo-[2,3-b]purin-9(8)one Glo-1 Glyoxalase 1 HFD High fat diet HIF1α Hypoxia-inducible factor-1α HSP27 Heat shock protein 27 MDR Multidrug resistance MG Methylglyoxal MGdG 3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/ 7-methylimidazo-[2,3-b]purine-9(8)one MG-H1 Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine NAFLD Non-alcoholic fatty liver disease Nrf2 Nuclear factor erythroid 2–related factor 2 PBMC Peripheral blood mononuclear cells RAGE Receptor for advanced glycation endproducts * Paul J. Thornalley P.J.Thornalley@warwick.ac.uk Glycoconj J (2016) 33:513–525 514 ROS Reactive oxygen species SSAO Semicarbazide amine oxidase T2DM Type 2 diabetes mellitus g l y c e r a l d e h y d e - 3 - p h o s p h a t e ( G A 3 P ) a n d dihydroxyacetonephosphate (DHAP) - 0.05 – 0.1 % of flux. GA3P was ca. 8-fold more reactive than DHAP in degrading to MG but as the concentration ratio of DHAP/GA3P in cells in situ is ca. 9 or similar [9], both of these triosephosphates are important sources of MG formation in physiological systems in situ [10]. MG formation is a minor fate of triosephosphates: early studies with red blood cells suggested only 0.089 % glucotriose (2 x glucose consumption) was converted to MG [11] and our subsequent studies with endothelial cells and fibroblasts suggest a similar flux. The rate of total cellular formation of MG was estimated to be ca. 125 μmol/kg cell mass per day [11], which for an adult human of 70 kg body mass and 25 kg body cell mass [12] equates to a predicted whole body rate of formation of ca. 3 mmol MG per day (or ca. 3 mg/kg body weight/day). MG is also formed by the oxidation of acetone catalysed by cytochrome P450 2E1 in the catabolism of ketone bodies [13] – which is low except where ketone bodies are increased as in diabetic ketoacidosis, prolonged (>3 days) fasting or low calorie diet [13–15]. MG may also be formed from the oxidation of aminoacetone by semicarbazide amine oxidase (SSAO) in the catabolism of threonine [16]. Recent estimates of the concentration and rates of metabolism of aminoacetone in the presence and absence of SSAO inhibitor suggest this pathway has a flux of ca. 0.1 mmol MG per day in human subjects [17] or ca. Dicarbonyl stress and the glyoxalase system Dicarbonyl stress is the abnormal accumulation of dicarbonyl metabolites leading to increased modification of protein and DNA contributing to cell and tissue dysfunction in ageing and disease [1]. Highly reactive dicarbonyl metabolites often mediat- ing dicarbonyl stress in physiological systems are methylglyoxal (MG), glyoxal, 3-deoxyglucosone (3-DG) and others. The glyoxalase system is a cytoplasm enzymatic pathway, which metabolises the most highly reactive acyclic dicarbonyls – main- ly MG and glyoxal. It thereby plays a major role in suppressing dicarbonyl stress in physiological systems, keeping dicarbonyl metabolites at low, tolerable levels. Typical concentrations of glyoxal and MG are 50–150 nM in human plasma and 1– 4 μM in plant and mammalian cells [2–4]. When dicarbonyl concentrations increase beyond this there is potential for protein and cell dysfunction leading to impaired health and disease. Examples of dicarbonyl stress are the increased MG in ageing plants [2], increased MG-protein modification in ageing human lens [5], increased plasma and tissue concentration of MG in diabetes [6], and increased concentrations of MG and glyoxal in renal failure [4]. Dicarbonyl stress is caused by an imbalance of the formation and metabolism of dicarbonyl metabolites and also by increased exposure to exogenous dicarbonyls – Fig. 1a. In this review there is a particular but not exclusive focus on MG as it is a major contributor to dicarbonyl stress in physiological systems. Other recent reviews focussing mostly on dicarbonyl stress in obesity and diabetes have been given elsewhere [7, 8]. Dicarbonyls and glyoxalase in disease mechanisms and clinical therapeutics 3 % of total MG formation. Vascular adhesion protein-1 is considered the origin of SSAO activity in mammals in vivo [18]. It is found in plasma, endothelium, adipose tissue and smooth muscle and increases ca. 2-fold in congestive heart failure, diabetes and inflammatory liver diseases [19], and may relat- edly increase MG formation in these conditions. MG is also formed by the degradation of proteins glycated by glucose and the degradation of monosaccharides [20]. Under physiological conditions with low level phosphate and chelation of trace metal ion, the predicted flux of MG formation from glycated protein degradation is ca. 0.2 mmol MG per day or ca. 7 % of total MG formation. Dietary contributions to MG exposure from food are normally relatively low: sweetened soft drink, Formation of methylglyoxal In mammalian metabolism, MG is formed at relatively high flux mainly by the trace level degradation of triosephosphates, Fig. 1 Biochemistry of dicarbonyl stress. a Metabolism of MG by the glyoxalase system. b Formation of hydroimidazolone MG-H1 from arginine residues. c Formation of imidazopurinone MGdG in DNA. Adduct residue is shown with guanyl base only Fig. 1 Biochemistry of dicarbonyl stress. a Metabolism of MG by the glyoxalase system. b Formation of hydroimidazolone MG-H1 from arginine residues. c Formation of imidazopurinone MGdG in DNA. Adduct residue is shown with guanyl base only Glycoconj J (2016) 33:513–525 515 330 ml – 0.1 μmol MG [21], fruit juice, 330 ml – 0.7 μmol, bread/cakes, 100 g, 1–2 μmol and other foodstuffs [22]; that is, combined likely <0.03 mmol MG per day or <1 % MG exposure. MG in foodstuffs was also metabolised and/or reacted with proteins before absorption in the gastrointestinal tract and imposed dicarbonyl stress mainly in the gastrointes- tinal lumen [23]. The diet may contribute markedly greater to total exposure for other dicarbonyls where culinary heating is a source of formation; for example, 3-DG [24]. Sources of formation of MG and routes of metabolism are summarised in Fig. 2. abundance in human cells [29]. In principle, ADHs may also metabolise glyoxal and MG to glyoxylate and pyruvate, re- spectively. In examination of ADH-linked MG dehydroge- nase activity in human cells to date we have found very low or undetectable activity. AKRs and ADH catalyse the metab- olism of 3-DG whereas Glo-1 does not [4, 30]. Other proteins, Bglyoxalase III^ and DJ1, were proposed as glyoxalases but their low catalytic efficiency and cellular content suggests this is unlikely [31]. Basal and inducible expression of Glo-1, AKRs and ADH are under stress-responsive control by nuclear factor erythroid 2–related factor 2 (Nrf2) through regulatory antioxidant re- sponse elements (AREs) [32–36]. The cytoprotective function of Nrf2 therefore involves enhancing basal and inducible ex- pression and activities of enzymes of dicarbonyl metabolism and thereby prevention of dicarbonyl stress [32]. Other regu- latory elements in the mammalian GLO-1 gene are: metal response element, insulin response element, early gene 2 fac- tor-isoform-4, and activating enhancer binding protein-2α, as reviewed [31]. Glo-1 expression is negatively regulated by hypoxia-inducible factor-1α (HIF1α) in hypoxia [37] and also by the receptor for advanced glycation endproducts (RAGE) [31]. Formation of methylglyoxal Hypoxia may be an important physiological driver of dicarbonyl stress as it both increases MG formation by flux through anaerobic glycolysis and likely decreases Glo-1 ex- pression through activation of HIF1α. Fig. 2 Formation of methylglyoxal, metabolism and glycation of protein and DNA in vivo. Tissue levels of MG and MG-H1 adduct residues in tissues are given for mice. PBMC DNA AGEs are for human subjects and flux of MG metabolised by the glyoxalase system and urinary excretion of MG-H1 free adduct is for healthy adduct humans. Data from [2, 63, 89, 122, 137] and Masania, J, Shafie, A, Rabbani, N and Thornalley, P.J., Unpublished observations Dicarbonyl metabolism by the glyoxalase system There are also minor lysine- derived AGEs formed: Nε-carboxymethyl-lysine and Nε(1- carboxyethyl)lysine formed from glyoxal and MG, respective- ly, and others. The major source of CML formation, however, is the oxidative degradation of Nε-fructosyl-lysine residues [49]. Dicarbonyl glycation is particularly insidious as it is direct- ed to arginine – the amino acid residue with highest probabil- ity of location in functional sites of proteins, modification induces loss of charge of the side chain guanidino group and functionally important arginine residues tend to be those most reactive towards dicarbonyl glycation [50]. The extent of glycation of proteins by dicarbonyls is low, usually 1–5 %, but may increase in ageing and disease. Proteins modified by glyoxal and MG in dicarbonyl stress are recognised as mis- folded and directed to the proteasome for proteolysis. In yeast an unfocussed gene deletion analysis showed strains deleted for genes of ubiquitin-dependent protein degradation were sensitive to glyoxal and MG toxicity [51]. Examples of phys- iological dysfunction mediated by dicarbonyl glycation of ar- ginine residues of proteins are: mitochondrial protein dysfunc- tion and increased formation of reactive oxygen species (ROS) [52], inflammatory protein expression (RAGE, S100 proteins and high mobility group box-1) [53], mitochondrial pathway activated apoptosis [54] and cell detachment from the extracellular matrix and anoikis [55]. At in situ concentrations of MG, GSH and Glo-1, the for- mation and fragmentation of the hemithioacetal of MG and GSH are rapid compared to the Glo-1-catalysed step [31]. A consequence of this is that in situ activity of Glo-1 is directly proportional to GSH concentration. So cellular GSH concen- tration is an influential factor on in situ activity of Glo-1 and oxidative or non-oxidative depletion of GSH leads to in- creased glyoxal and MG [46]. The concentration of S-D- lactoylglutathione (SLG) is also maintained at low levels. This is likely so that lactoyl-transfer to protein thiol groups and related inactivation of enzymes with functional cysteinyl thiols is prevented [31]. SLG is also toxic if it leaks out of cells and is metabolised by γ-glutamyl transferase and dipeptidase with rearrangement to N-lactoylcysteine which is an inhibitor of pyrimidine synthesis [47]. A mathematical model of the glyoxalase pathway predicted very low levels of MG and SLG in cells which fitted well with experimental observation [31]. A surprising observation was increased chaperone function of αA-crystallin with very high modification by MG for re- versing dithiothreitol- and heat-induced misfolding of proteins [56]. Dicarbonyl metabolism by the glyoxalase system Glyoxal and MG are metabolised mainly by glyoxalase 1 (Glo-1) of the glutathione-dependent glyoxalase system, with minor metabolism by aldoketo reductases (AKRs) and alde- hyde dehydrogenases (ADHs). As total MG-derived glycation adducts excreted in urine of healthy human subjects was typ- ically ca. 10 μmol per day [25, 26], it can be inferred that less than 1 % MG formed endogenously modifies the proteins. Most of the MG formed (>99 %) is metabolised by glyoxalase 1 (Glo-1) and aldoketo reductase (AKR) isozymes, which thereby constitute an enzymatic defence against MG glycation. From studies of the level of expression of Glo-1 and AKRs [27, 28], it can be inferred that Glo-1 activity in situ exceeds that of AKR activity for MG metabolism by >30- fold in all human tissues except the renal medulla where the expression of AKR is extraordinarily high. Indeed, Glo-1 is a highly efficient and high abundance enzyme; typically 0.02 % of total protein [27] and is in the top 13 % of proteins by Glo-1 expression is also increased by copy number varia- tion (CNV) of the GLO-1 gene in human and mouse genomes. Human GLO-1 is located in chromosome 6 at locus 6p21.2 [38] and mouse Glo-1 in chromosome 17 at locus 17 a3.3 [39]. Gene cloning and bioinformatics analysis showed that human GLO-1 coding regions consists of 12 kb with introns Fig. 2 Formation of methylglyoxal, metabolism and glycation of protein and DNA in vivo. Tissue levels of MG and MG-H1 adduct residues in tissues are given for mice. PBMC DNA AGEs are for human subjects and flux of MG metabolised by the glyoxalase system and urinary excretion of MG-H1 free adduct is for healthy adduct humans. Data from [2, 63, 89, 122, 137] and Masania, J, Shafie, A, Rabbani, N and Thornalley, P.J., Unpublished observations Glycoconj J (2016) 33:513–525 516 separating six exons [40, 41]. CNV of human GLO-1 was detected by Redon et al. with a prevalence of ca. 2 %. [42]. Murine Glo-1 CNV was found in inbred strains of mice which included complete copies of Glo-1 and complete and partial copies of other genes [43, 44], giving rise to a 2–4-fold in- crease in Glo-1 expression. GLO-1 duplication appeared linked to anxiety-like behaviour in mice but may rather be due to a proximate genetic locus co-duplicated with Glo-1 [45]. physiological systems – Fig. 1b. Dicarbonyl metabolism by the glyoxalase system With lower, physiological extent of modification by MG, however, the chaperone function of αA-crystallin was not enhanced further [57]. The likely evolutionary pressure for development of Glo-1 was to evolve an enzyme that accepts the major solution species of MG, the MG-GSH hemithioacetal, which is also highly ef- ficient with kcat/KM at the diffusion limit. Glo-1 is thereby ex- quisitely suited to its function. High reactivity of arginine and cysteine residues in Glo-1 protein could be a problem for Glo-1 stability which would occur with relatively low microscopic pKa. Our computations of microscopic pKa values (as previ- ously described [48]) indicates functionally important R37, R122 and C139 of human Glo-1 have high microscopic pKa values (>12), which confers low reactivity towards MG. Also in cells with 1–4 μM MG, there is a pool of ca. 20 mM cysteinyl thiol groups and ca. 80 mM arginine residues to which MG may bind and only 2 μM and 4 μM of these, respectively, are functionally important residues of Glo-1 [27]. Therefore, Glo-1 is very resistant to inactivation in situ by MG. Early studies of specific proteins modified by MG used antibodies to the trace MG-derived AGE, argpyrimidine. In endothelial cells the heat shock protein-27 (HSP27) was de- tected as a major MG modified protein [58]. This could not be verified by ultrahigh resolution Orbitrap mass spectrometry with direct examination for MG-modification in tryptic pep- tides – even when modification of proteins was increased 10- fold in cell lysates [59], suggesting earlier studies suffered interference. Subsequent studies showed recombinant HSP27 was modified by 500 μM – 5 mM MG at multiple sites and MG-modified HSP27 was more protective than un- modified protein against apoptotic cell death [60]. The phys- iological significance is unclear, however, as recent studies were unable to find evidence of MG modification of HSP27 [59] and increased MG and Glo-1 inhibitors tend to promote rather than suppress apoptosis [61, 62]. Diabetes and diabetic vascular complications Hypotheses for the involvement of dicarbonyl stress in disease are most advanced and critically evaluated for involvement in the vascular complications of diabetes. Glo-1 activity is de- creased and MG-H1 residue content of proteins is increased in the kidney, retina and nerve of pre-clinical models of micro- vascular complications of diabetes (nephropathy, retinopathy and neuropathy) [79–83]. Functional genomics studies with Glo-1 deficient mice and Glo-1 overexpressing transgenic mice and preventive intervention such as high dose thiamine and Benfotiamine support increased MG as a risk factor linked to the development of diabetic microvascular complications [79, 84–87]. Dicarbonyl stress is also linked to diabetic car- diovascular disease – see below. Formation of MG is in- creased in cells with GLUT1 glucose transport incubated in high glucose concentration [11, 88]. Decreased Glo-1 activity synergises with increased MG formation to increase cellular Biochemical consequences of dicarbonyl stress Dicarbonyl stress produces increased in situ rates of glycation by dicarbonyls of proteins, DNA and basic phospholipids. Reaction with proteins is directed to arginine residues forming dihydroxyimidazolidine and hydroimidazolone adducts. The hydroimidazolone derived from MG, MG-H1, is one of the most quantitatively and functionally important AGEs in Glyoxal and MG are important precursors of DNA adducts in physiological systems: major adducts are imidazopurinones GdG and MGdG – nucleotide AGEs. MGdG was the major nucleotide AGE found physiologically – Fig. 1c. DNA con- tent of MGdG exceeded those of the major DNA oxidative damage adduct, 8-hydroxydeoxyguanosine. Increased 517 Glycoconj J (2016) 33:513–525 nucleotide AGEs was associated with DNA strand breaks and mutagenesis [63]. nucleotide AGEs was associated with DNA strand breaks and mutagenesis [63]. lens with age and this was linked to cataract formation [5, 68] and also in skin but to markedly lower extent [68]. Decreased Glo-1 activity was associated with age-linked impairment of wound healing [69] and increased dicarbonyl stress is associ- ated with several ageing-linked diseases – see below. Glo-1 activity declines with age so there is also increased stress on cell proteolysis and compensatory gene expression to keep AGE-modified proteins to a low tolerable level and provide replacement unmodified proteins. Dicarbonyl stress is likely a feature of proliferative senescence of fibroblasts in culture where Glo-1 expression is decreased and glycolytic flux is increased [70, 71]. It is also likely involved in senescence of plants: dicarbonyl content of broccoli increased with age [2] and MG-H1 was a major AGE in Arabidopsis leaves [72]. Obesity For many years a genetic linkage of Glo-1 to body weight in mice [73] and of GLO-1 to upper-arm circumference and supra-iliac skinfold thickness in human subjects [74] sug- gested a role for Glo-1 in obesity. In the mouse overeating model of obesity, leptin mutant (ob/ob) mice, Glo-1 protein was decreased 80 % in the liver [75]. Recent conference re- ports described increased weight gain on high fat diet (HFD)- fed mouse with through-life expression of GLO-1 siRNA and Glo-1 deficiency, compared to wild-type controls [76], and decreased weight gain in Glo-1 overexpressing transgenic mice [77], suggesting a functional role of Glo-1 and dicarbonyl stress in obesity. HFD-fed wild-type mice had in- creased MG-H1 content of heart and liver, as judged by im- munoassay [78]. Dicarbonyl stress may be a mediator of obe- sity and insulin resistance and thereby a risk factor for devel- opment of type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). Physiological consequences of dicarbonyl stress Where dicarbonyl stress occurs there is potential for increased cell dysfunction, detachment from the extracellular matrix and anoikis, and apoptosis. Cell dysfunction is driven by the ef- fects of protein glycation: loss of substrate protein activity – through inactivation and/or increased rate of proteolysis and decreased half-life (unless compensatory increased expression is activated), or by gaining a new and damaging function – for example, low density lipoproteins become small, dense and atherogenic by MG modification [64]. This affects multiple proteins – the dicarbonyl proteome - in different cell and tissue compartments [50]. The effects span multiple compartments by diffusion of increased MG or other dicarbonyl in dicarbonyl stress. MG permeates cell plasma membranes by passive diffusion of the unhydrated form. This is rate limited by MG dehydration, giving a half-life of ~4 min [2]. The half- life of MG for metabolism by the glyoxalase system to D- lactate from in situ rates of D-lactate formation in cells is ca. 10 min with free MG mostly (>95 %) reversibly bound to protein. The rate of irreversible binding to protein in plasma was ca. 3.6 h [65]. This implies that part of the MG formed inside cells leaks out from the site of formation and may dif- fuse through interstitial fluid into plasma and thereafter per- meate back into interstitial fluid and cells of other tissues. Also, MG formed from the degradation of glycated proteins in the extracellular compartment may enter cells for metabo- lism by Glo-1 and AKRs. The locus of dicarbonyl stress and related pathogenesis linked to MG accumulation is therefore likely particularly sensitive to local decrease of Glo-1 expres- sion and activity. Kinetic considerations of the rate of glycation of protein, similar to those for ROS [66], indicates a diffusion distance of MG of ca. 2–3 cm before irreversible reaction, suggesting that MG has relatively long range and half-life to locate and modify sensitive sites of proteins, often leading to protein inactivation and dysfunction. Cardiovascular disease and extracellular MG concentration. MG content of blood samples was increased by up to 5–6 fold in patients with diabetes [6]. Increased MG has been found in diabetic kidney in vivo [89] and vascular endothelial cells in high glucose concentration cultures in vitro [55] – including in mitochon- dria [90]. In locations where it is difficult to excise tissues without leaking of MG from cells and disrupting extracellular fluid – such as retina and cellular and extracellular compart- ment of peripheral nerve, evidence of dicarbonyl stress is in- creased levels of dicarbonyl–derived AGEs [83, 91]. Increased MG concentration and protein content of MG-H1 was not found in liver, skeletal muscle and brain in experi- mental models of type 1 diabetes [25, 83, 92]. MG-derived AGEs were increased in plasma protein and skin collagen of diabetic patients and were linked to risk of microvascular and macrovascular complications [26, 93–95]. A recent pre-clinical and clinical integrative genomics study revealed Glo-1 deficiency as a driver of CVD [107]. Chemical inhibition of Glo-1 induced atherosclerosis in apoE deficient mice [108]. MG-derived AGEs in plasma protein have been found to be linked to risk of CVD in diabetes [94, 109]. High levels of MG-H1 and CML were associated with rupture- prone plaques in human carotid endarterectomy, accumulating in macrophages surrounding the necrotic core. The expression of Glo-1 was decreased in ruptured compared with stable plaque segments [110]. However, overexpression of Glo-1 in mice did not affect atherosclerotic lesion size and severity in ApoE−/−mice with or without diabetes [111]. Unexpectedly plasma MG and glyoxal concentration were not decreased in Glo-1 transgenic mice. The reason for this is not clear but dicarbonyls are predominately sourced from metabolism and there was limited increase in Glo-1 activity of the liver in the Glo-1 transgenic mice [112]. Dicarbonyl stress in plasma like- ly contributes to CVD risk through induction of dyslipidaemia and vascular cell dysfunction. MG modification of LDL in- duced atherogenic transformation to small, dense LDL with increased affinity for arterial walls through binding to heparan sulfate proteoglycans [64]. MG modification of HDL induced re-structuring of the HDL particles, increasing density, de- creasing stability and plasma half-life in vivo [113]. Dicarbonyl stress also induces vascular cell dysfunction: si- lencing of Glo-1 in human aortal endothelial cells changed expression of >400 genes – including increased expression of RAGE and associated ligands [53, 107]. Chronic renal disease Experimental models of renal failure, bilateral nephrectomy and bilateral ureteral ligation - models of acute total loss and partial loss of renal function, respectively, were associated with profound dicarbonyl stress. Plasma glyoxal and MG in- creased 5 and 15 fold within 72 h [99]. Patients with end stage renal disease (ESRD) on hemodialysis and peritoneal dialysis had increased plasma MG and flux of formation of dicarbonyl-derived AGEs [4, 100]. The cause of dicarbonyl stress is unlikely due to decreased dicarbonyl excretion as there is little in normal health [23], although it is linked to renal function [99]. Decreased Glo-1 expression by hypoxia and inflammation in ESRD [101, 102], hypoxia-induced in- creased anaerobic glycolysis [103] and decreased disposal of triosephosphates by the reductive pentosephosphate pathway (enzymes of which are inhibited by uremic toxins [104]) lead- ing to increased formation of MG may produce dicarbonyl stress in ESRD. Decreased Glo-1 activity in rare GLO-1 frameshift mutation heterozygote human subjects was associ- ated with decreased glomerular filtration rate [67]. A patient with ESRD and low Glo-1 activity had a high frequency of recurrent cardiovascular disease (CVD) events [105]. Further studies showed a high mortality rate in patients with homozy- gous GLO-1 419CC mutation - reviewed in [106]. This sug- gests a link of dicarbonyl stress to development of renal failure and CVD complications of ESRD. Ageing The link of dicarbonyl stress to ageing was unequivocally established in a functional genomics study of Glo-1 in the nematode C. elegans [52]. Ageing-related decline in renal function was prevented in transgenic rats overexpressing Glo-1 [67]. Intuitively we expect this is due to AGE accumu- lation in proteins of tissues and body fluids with related pro- tein dysfunction. MG-derived AGEs were increased in human 518 Glycoconj J (2016) 33:513–525 Cardiovascular disease Dicarbonyl stress may also play a role in development of T2DM through promotion of insulin resistance [77, 96] and development of type 1 diabetes through mediation of beta-cell toxicity [97]. Recent studies have found increased levels of MG, glyoxal and 3-DG of subjects with impaired glucose tol- erance and patients with T2DM in the fasting state and during an oral glucose tolerance test challenge [98]; plasma and glyoxal levels were ca. 3-fold higher for MG and glyoxal than obtained using the reference assay protocol which control [2]. Other diseases Following discovery of Glo-1 gene duplication in some strains of mice with an anxiety phenotype [43], a link of Glo-1 to pathologic anxiety was proposed – although the anxiety phe- notype was linked to both increased and decreased Glo-1 ex- pression [119, 120]. In attempts to link Glo-1 metabolically to dysfunctional brain metabolism, MG was found to agonise the GABAA receptor in primary cerebellar granule neurons with a median effective concentration EC50 of 10.5 μM and this was proposed as mediator of sedation to explain increased GLO-1 CNV with an anxiety phenotype [121]. MG concentration in mouse brain tissue is ca. 7-fold lower than this [122] and only approached the EC50 value with dosing of 300 mg/kg MG [121] – similar to doses producing acute toxicity [123]. Transgenic mice with 2-fold, 4-fold and 5-fold increased Glo-1 expression had an anxiety phenotype with 4- and 5- fold increased expression but not with 2-fold increased ex- pression [121]. Some inconsistences remain, therefore, and further investigation is required. A better strategy is development of Glo-1 inducers through activation and binding of Nrf2 to the GLO-1 functional ARE [32]. This offers an alternative that is likely safe and effective. It also addresses a key cause of dicarbonyl stress in disease – disease-associated, tissue-specific deficiency of Glo-1. Detection of Nrf2 activators for ARE-linked induction of Glo-1 expression requires a specific screen with the GLO-1- ARE motif or similar as not all Nrf2 activators induce expres- sion of Glo-1. Nrf2 activators typically change expression of a subset of ARE-linked genes. The basis of this subset selection is unknown but it is likely determined by the level of functionally-active Nrf2 achieved in the cell nucleus in re- sponse to the Nrf2 activator, recruitment of relevant accessory proteins – such as small maf proteins [134, 135], and absence of activation of counter-signalling effects required to induce the ARE-linked gene of interest. Nrf2 regulates inducible ex- pression of ca. 890 genes [136]. The advantage of Nrf2 regu- lated, ARE-regulated genes is that they are a battery of pro- tective genes and so where co-regulated along with Glo-1, they tend to add to the health beneficial response. In this re- gard, concurrent induction of GSH synthesis for increased cellular GSH concentration to support increased in situ activ- ity of Glo-1 is particularly beneficial [137]. formation [63]. MDR tumours are susceptible to cell perme- able Glo-1 inhibitors [116, 118]. formation [63]. MDR tumours are susceptible to cell perme- able Glo-1 inhibitors [116, 118]. pyridoxamine are not efficient scavengers of MG and where associated with alleviation of dicarbonyl stress likely function by other mechanisms [128–131]. High dose thiamine supple- ments for prevention of T2DM and vascular complications of diabetes may work partly by preventing the formation of MG through increased disposal of triosephosphates in the reductive pentosephosphate pathway [132, 133]. Other diseases Dicarbonyl stress has also been linked to severe schizo- phrenia through a rare frameshift mutation of GLO-1 [124], synucleinopathies such as Parkinson’s disease in experimental pre-clinical models [122] and Alzheimer’s disease through clinical biomarker studies [125]. Carcinogenesis, tumour growth and cancer chemotherapy Dicarbonyl stress has a duality of function in cancer develop- ment and treatment. Glo-1 is a tumour suppressor protein and a mediator of multidrug resistance (MDR) in cancer chemo- therapy. The tumour suppressor function of Glo-1 was re- vealed in a genome-wide study in p53 knockout, Ras overex- pression model of liver carcinogenesis [114]. This is consis- tent with mutation arising from of dicarbonyl glycation of DNA sometimes leading to carcinogenesis. There were 14 tumour suppressor genes found and currently Glo-1 is the only one for which a readily available strategy for increased cancer prevention exists – dietary Glo-1 inducers in functional foods. The role of Glo-1 in MDR was revealed in a transcriptome- wide subtraction technique of drug-sensitive and drug- resistant tumour cell lines [115]. Increased Glo-1 expression in tumours may be mediated through GLO-1 amplification [116], and also by mutation and increased transcriptional ac- tivity of Nrf2 through ARE-linked up-regulation of Glo-1 transcription [117]. High Glo-1 activity may be permissive for growth with high glycolytic activity and flux of MG 519 Glycoconj J (2016) 33:513–525 formation [63]. MDR tumours are susceptible to cell perme- able Glo-1 inhibitors [116, 118]. Dicarbonyl stress-based therapeutics A Glo-1 inducer formulation has been optimised and eval- uated in Phase 1 clinical trial (Clinicaltrials.org; NCT02095873) in overweight and obese subjects for safety and target pharmacology at a working dose. We also did functional assessments – Phase 2 A trials for health improvement in obesity. The Glo-1 inducer is a binary com- bination of trans-resveratrol and hesperetin (tRES-HESP) and was evaluated in a randomised, placebo-controlled crossover clinical trial in 29 overweight and obese subjects. In highly overweight subjects (BMI >27.5 kg/m2), tRES-HESP co-for- mulation increased expression and activity of Glo-1, de- creased plasma methylglyoxal and total body methylglyoxal- protein glycation. It decreased fasting and postprandial plasma References glucose, increased oral-glucose-insulin-sensitivity (OGIS) in- dex – an assessment of insulin sensitivity, and improved arte- rial dilatation. In all subjects, it decreased vascular inflamma- tion marker sICAM-1. In previous clinical evaluations, tRES and HESP individually were ineffective. tRES-HESP co-for- mulation could be a suitable treatment for improved metabolic and vascular health in overweight and obese populations. It now available for evaluation in Phase 2 clinical trial against disease targets. 1. Rabbani N., Thornalley P.J.: Glyoxalase centennial conference: introduction, history of research on the glyoxalase system and future prospects. Biochem. Soc. Trans. 42(2), 413–418 (2014) 2. Rabbani N., Thornalley P.J.: Measurement of methylglyoxal by stable isotopic dilution analysis LC-MS/MS with corroborative prediction in physiological samples. Nat. Protoc. 9(8), 1969– 1979 (2014) 3. Thornalley P.J., Rabbani N.: Assay of methylglyoxal and glyoxal and control of peroxidase interference Biochem. Soc. Transit. 42(2), 504–510 (2014) g This first-in-class Glo-1 inducer trial establishes Glo-1 tar- get pharmacology for tRES-HESP. Whilst increased Glo-1 expression likely contributes to the observed health beneficial effects, changes in other gene expression occurred and their interplay may also mediate the overall health benefit achieved. Nevertheless it was pursuit and optimisation of induction of Glo-1 expression that arrived at this synergistic combination of bioactive compounds and achieved improved metabolic and vascular health in overweight and obese subjects that is unmatched by other therapy. The marked health improve- ments were achieved with expression of many antioxidant- linked genes unchanged – at least in the peripheral blood mononuclear cells analysed. 4. Rabbani, N., Thornalley, P.J.: Dicarbonyls (Glyoxal, Methylglyoxal, and 3-Deoxyglucosone). In: Uremic Toxins. pp. pp. 177–192. John Wiley & Sons, Inc., (2012) 5. Ahmed N., Thornalley P.J., Dawczynski J., Franke S., Strobel J., Stein G., Haik Jr. G.M.: Methylglyoxal-derived hydroimidazolone advanced glycation endproducts of human lens proteins. Investig. Ophthalmol. Vis. Sci. 44(12), 5287–5292 (2003) 6. McLellan A.C., Thornalley P.J., Benn J., Sonksen P.H.: The glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clin. Sci. 87(1), 21–29 (1994) 7. Matafome P., Sena C., Seiça R.: Methylglyoxal, obesity, and dia- betes. Endocr. 43(3), 472–484 (2013). doi:10.1007/s12020-012- 9795-8 8. Maessen D.E.M., Stehouwer C.D.A., Schalkwijk C.G.: The role of methylglyoxal and the glyoxalase system in diabetes and other age-related diseases. Clin. Sci. 128(12), 839–861 (2015) Cell permeable Glo-1 inhibitors which increase dicarbonyl stress may find use as anti-tumour and anti-microbial agents for treatment of Glo-1-linked MDR tumours and microbial infections. References 95(1), 50–57 (2004) Acknowledgments With thank research colleagues and collaborators who have contributed to studies covered in this review. Alleviation of dicarbonyl stress by glyoxalase 1 inducers Alleviation of dicarbonyl stress by glyoxalase 1 inducers Initial attempts to alleviate dicarbonyl stress were made through dicarbonyl scavengers and claimed for dicarbonyl scavenging properties of existing drugs in treatments for vas- cular complications of diabetes. A challenge in the design of dicarbonyl scavengers is to achieve sufficient reactivity for the low concentration of drug achieved clinically with the 1000– 10,000 fold higher concentration of arginine residues in tissues and body fluids. Aminoguanidine and phenacylthiazolium bro- mide showed some promise but their toxicity and instability prohibited further development [4, 126, 127]. Metformin and Table 1 Therapeutic agents in development targeting the glyoxalase system Therapeutic class Mechanism of action Primary target application (secondary) Stage of development Reference Glyoxalase 1 inducer Small molecule Nrf2 activator targeting GLO-1-ARE transcriptional activity Diabetic nephropathy (other microvascular complications of diabetes; obesity – NAFLD; cardiovascular disease) Clinical trial Phase 2 ready (Phase 1 complete with safety, dose and pharmacology established). [137] Glyoxalase 1 inhibitor Substrate analogue inhibitor diester (prodrug) Cancer (GLO-1 overexpressing, MDR tumours) Pre-clinical in vivo models [62, 118, 140] Glycoconj J (2016) 33:513–525 520 References Acknowledgments With thank research colleagues and collaborators who have contributed to studies covered in this review. References S-p-Bromobenzylglutathione diethyl ester was the first cell permeable Glo-1 inhibitor developed and had median growth inhibitory concentration GC50 values in the range 7– 20 μM for a range of cancer cell lines. Subsequent develop- ment of the cyclopentyl ester derivative increased potency and had anti-tumour activity in tumour-bearing mice [62, 138, 139]. A difficulty in clinical translation is identifying tumours that are sensitive to Glo-1 inhibitors. These are likely tumours with a relatively high flux of MG formation and high activity of Glo-1 such that when a Glo-1 inhibitor is delivered into the tumour, MG accumulates rapidly to toxic levels. GLO-1 am- plification in tumours is not a reliable marker of this as it is not always functional and does not report on flux of MG. 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Patient characteristics associated with being offered take home naloxone in a busy, urban emergency department: a retrospective chart review
BMC health services research
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O’Brien et al. BMC Health Services Research (2019) 19:632 https://doi.org/10.1186/s12913-019-4469-3 O’Brien et al. BMC Health Services Research (2019) 19:632 https://doi.org/10.1186/s12913-019-4469-3 (2019) 19:632 O’Brien et al. BMC Health Services Research https://doi.org/10.1186/s12913-019-4469-3 Patient characteristics associated with being offered take home naloxone in a busy, urban emergency department: a retrospective chart review Patient characteristics associated with being offered take home naloxone in a busy, urban emergency department: a retrospective chart review Daniel C. O’Brien1, Daniel Dabbs2, Kathryn Dong3, Paul J. Veugelers4 and Elaine Hyshka1* © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Overdose deaths can be prevented by distributing take home naloxone (THN) kits. The emergency department (ED) is an opportune setting for overdose prevention, as people who use opioids frequently present for emergency care, and those who have overdosed are at high risk for future overdose death. We evaluated the implementation of an ED-based THN program by measuring the extent to which THN was offered to patients presenting with opioid overdose. We analyzed whether some patients were less likely to be offered THN than others, to identify areas for program improvement. Methods: We retrospectively reviewed medical records from all ED visits between April 2016 and May 2017 with a primary diagnosis of opioid overdose at a large, urban tertiary hospital located in Alberta, Canada. A wide array of patient data was collected, including demographics, opioid intoxicants, prescription history, overdose severity, and whether a naloxone kit was offered and accepted. Multivariable analyses were used to identify patient characteristics and situational variables associated with being offered THN. Results: Among the 342 ED visits for opioid overdose, THN was offered in 49% (n = 168) of cases. Patients were more likely to be offered THN if they had been found unconscious (Adjusted Odds Ratio 3.70; 95% Confidence Interval [1.63, 8.37]), or if they had smoked or injected an illegal opioid (AOR 6.05 [2.15,17.0] and AOR 3.78 [1.32,10.9], respectively). In contrast, patients were less likely to be offered THN if they had a current prescription for opioids (AOR 0.41 [0.19, 0.88]), if they were admitted to the hospital (AOR 0.46 [0.22,0.97], or if they unexpectedly left the ED without treatment or before completing treatment (AOR 0.16 [0.22, 0.97). Conclusions: In this real-world evaluation of an ED-based THN program, we observed that only half of patients with opioid overdose were offered THN. ED staff readily identify patients who use illegal opioids or experience a severe overdose as potentially benefitting from THN, but may miss others at high risk for future overdose. We recommend that hospital EDs provide additional guidance to staff to ensure that all eligible patients at risk of overdose have access to THN. Keywords: Take home naloxone, Emergency department, Opioids, Overdose * Correspondence: ehyshka@ualberta.ca 1University of Alberta, School of Public Health, 3-300 Edmonton Clinic Health Academy, 11405 – 87 Ave, Edmonton, Alberta T6G 1C9, Canada Full list of author information is available at the end of the article Aims of the study Research on THN programs has shown that THN kits are frequently used by people who use drugs to respond to overdoses [7, 8]. Specifically, approxi- mately 25% of people who use drugs who are trained and supplied with naloxone will use it to reverse an overdose within 1 year [7]. At the population-level, reductions in overdose mortality have been observed following the implementation of THN programs, and higher rates of kit distribution lead to greater reduc- tions in mortality [9, 10]. THN distribution can be particularly effective at reducing overdose mortality when targeted at high risk populations, such as re- cently released prisoners who have lost their opioid tolerance [11]. The present study evaluates a recently-implemented THN program in a busy urban ED to determine the extent to which THN was offered to patients at high- est risk of fatal overdose: those who present with a nonfatal opioid overdose. Our specific aims are to measure the proportion of ED visits for opioid over- dose in which THN was offered, and identify patient characteristics and other situational variables associ- ated with being offered a THN kit in the ED. Ideally, 100% of individuals who present to the ED with opi- oid overdose should have an opportunity to leave with a THN kit. However, we predicted that even among this high-risk population, patients with certain demographic or clinical characteristics would have a higher likelihood of being offered THN. Additionally, we anticipated that certain situational variables, such as the time of day, length of stay in the ED, or subse- quent hospital admittance, might impact whether cli- nicians offer THN to patients. By identifying patients that may have been systematically missed, we aimed to develop new insights and recommendations for optimizing the implementation of ED-based THN dis- tribution. Finally, while the focus of our study is whether THN was offered to patients, we also de- scribe the proportion of patients who accepted THN, and any reasons that had been charted for why pa- tients declined THN. In recent years, THN programs have been increas- ingly incorporated into hospital Emergency depart- ments (EDs) in an effort to reach high risk patients [10, 12]. Hospital EDs provide a critical opportunity to reach people at risk for overdose, because people who use opioids frequently present for emergency care, and those who have overdosed are at high risk of future overdose death [13–15]. Aims of the study Indeed, ED visits may be one of the few occasions that an individual comes in contact with the health system before ex- periencing a fatal overdose. Previous investigations have demonstrated that ED-based THN distribution is feasible [16, 17], and that the majority of clini- cians are willing to provide THN in the ED. [18, 19] Further, approximately 70% of at-risk ED patients who are offered THN accept it [20]. Page 2 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 Background all ED patients at risk of overdose are offered nalox- one may be challenging [22, 23]. For instance, one evaluation found that THN was offered to only 8% of ED patients with International Classification of Disease codes for opioid overdose, misuse, or de- pendence [23]. In previous qualitative studies, ED providers who were asked to identify barriers to providing THN reported lacking clarity about which patients should be offered naloxone [23, 24]. This is problematic, because correctly identifying at-risk pa- tients to offer THN is a crucial step in providing THN to those who need it. Despite the importance of this process, the extent to which different patients are offered naloxone has not yet been studied in an ED setting. g Opioid overdose is a leading, yet preventable cause of death in North America and around the world [1, 2]. Naloxone, an opioid antagonist, is commonly used in clinical settings to reverse the potentially lethal respira- tory depression that occurs during opioid overdose [3]. “Take Home Naloxone” (THN) programs aim to prevent deaths by distributing naloxone to people likely to wit- ness an opioid overdose, such as people who use drugs and their family and friends [4–6]. Typically, THN pro- grams train people to recognize the signs of an opioid overdose and respond appropriately by providing basic life support and administering naloxone via either intra- nasal spray or intramuscular injection [2, 5, 6]. The World Health Organization has identified THN distribu- tion as a key health intervention to prevent opioid over- dose deaths [2]. Study design and setting We conducted a retrospective chart review of all ED visits for which the primary diagnosis was opioid overdose between May 1st, 2016 and April 31st 2017 at the Royal Alexandra Hospital, which is located in Edmonton, Alberta, Canada. This large, urban, ter- tiary hospital received 73,163 ED visits in 2016–2017 [25]. Additionally, the Royal Alexandra Hospital sees For ED-based THN programs to have the greatest impact, THN should be offered to all patients who are at risk of overdose, including those who report using illegal opioids, those taking high doses of pre- scribed opioids, and those using opioids who present with complications other than opioid overdose (e.g. abscesses, trauma, etc.) [20–22]. However, ensuring O’Brien et al. BMC Health Services Research (2019) 19:632 Page 3 of 12 Page 3 of 12 O’Brien et al. BMC Health Services Research the highest number of ED visits related to opioids and other substances of misuse of any hospital in the province of Alberta, with almost 4000 substance use-related ED visits occurring between 2014 and 2017 [26]. The hospital ED began offering and dis- pensing THN kits in February 2016. During the study period, patients who were identified by either a physician or a nurse as being at risk for opioid overdose would be offered a THN kit just before they were discharged from the ED. Whether a THN kit was offered was left to the discretion of ED clini- cians, as a standardized protocol of which patients to target had yet to be developed at the time of this study. policy researcher). To establish inter-rater reliability of the abstraction protocol, a second registered nurse inde- pendently reviewed a random subset of 70 (20%) medical records. Percent agreement and kappa statistics were calculated for variables collected. For all variables, kappa was above or approaching 0.8, the commonly accepted standard for excellent inter-rater agreement [28]. In addition to reviewing patient charts, each ED visit was linked to data from the provincial Pharmaceutical Information Network (PIN), which tracks prescription medication dispensations. The PIN data were obtained for all opioids dispensed to patients in the 180 days prior to their ED visit for opioid overdose, and included the date each opioid was dispensed, the type of opioid dis- pensed, and the period it was prescribed for. Our re- search protocol received ethics approval from the University of Alberta’s Health Research Ethics Board. Variables of interest Our primary outcome of interest was whether THN was offered during their hospital ED visit, or subsequent in- patient hospital stay if admitted. We also determined the number of patients that accepted a THN kit, and re- ported any reasons that were given by the patient for de- clining a THN kit. Evidence that THN was offered and accepted was identified in ED physician and nursing charts, as well as inpatient hospital records. Additionally, medication dispensation data were reviewed to confirm cases in which THN was provided. Patient variables believed to be potentially associated with being offered THN included demographics such as age (per year older) and sex (male vs. female). Addition- ally, we included several patient characteristics that indi- cate an increased risk of overdose, such as being a resident of Edmonton’s inner city area (Edmonton East- wood, yes vs. no), which is the local geographic area with the highest rate of opioid overdose in the city [26]. Other variables linked to overdose risk included having “no fixed address”, which was charted for patients with- out a permanent address and indicates unstable housing or homelessness [29], (yes vs. no), mental health disorder [13] (defined as bipolar disorder, major depressive dis- order, psychosis, personality disorder, or schizophrenia, yes vs. no), and public overdose location [30] (not in a private residence, hotel, healthcare facility or prison, yes vs. no). Study design and setting y If the patient accepted the THN kit, a registered nurse would dispense the kit and provide overdose response training, with friends and family included if possible. Registered nurses working in the ED were required to take a training module prior to distributing THN. The kits distributed are publicly funded as part of a province wide THN program, and are provided directly to non- medical persons at no cost and without a prescription. They contain 3 vials of 0.4 mg/ml naloxone, safety-engi- neered intramuscular syringes, gloves, a CPR face shield, alcohol swabs, and an instructional pamphlet. Kits can be dispensed 24 h per day, 7 days per week. Because THN kits are dispensed directly to the patient from the ED, it is not necessary for patients to fill a naloxone pre- scription after they leave. Other services for hospital pa- tients who use drugs are available through the Addiction Recovery and Community Health (ARCH) team, a spe- cialty consult service consisting of a multidisciplinary group of physicians with expertise in addiction medicine, nurse practitioners, social workers, addictions coun- selors, and peer support workers [27]. The ARCH team is available by referral to all ED and hospital inpatients, and offers a combination of harm reduction, in-hospital addiction treatment, health promotion activities, and links to appropriate community health and social sup- ports [27]. Case identification and data collection Cases were identified from the patient hospital adminis- trative system according to ICD-10 codes, as any of the following: T40.0-T40.4, and T40.6. We excluded cases if the hospital chart could not be retrieved, or if the patient died in hospital. All medical documents related to pa- tient care were subject to review and data abstraction, including ED physician charts, ED nursing charts, EMS charts, inpatient hospital charts, laboratory reports, and the medication dispensation tracking system. The data abstraction protocol was developed by DD who is an ex- perienced ED nurse, in consultation with KD (emer- gency medicine specialist) and EH (health services and Additionally, we examined whether the patient was currently prescribed opioids (yes vs. no), and the type of opioid they had overdosed with, as these characteristics may have impacted clinician perceptions of overdose risk. We identified patients who had a current prescrip- tion for any opioid medication at the time of their ED visit by using PIN data to determine whether the pre- scription period for any opioid dispensed to the patient O’Brien et al. BMC Health Services Research (2019) 19:632 Page 4 of 12 Page 4 of 12 in the 180 days prior to their ED visit overlapped with their ED visit date. was offered and not offered. To determine whether each variable was associated with offering THN, we conducted a series of analyses using Generalized Estimating Equations (GEE) regressions for binary outcomes with logit link. Initially, we fitted separate bivariate regression models for each independent variable, with offering THN kits as the outcome. Variables that appeared statistically significant in the initial bivariate analyses at the 0.1-level were in- cluded in the final multivariable analysis. The patient’s primary opioid intoxicant and route of consumption that caused their opioid overdose was abstracted in the chart review, and was coded into six categories: i) pharmaceutical opioid- oral, ii) pharmaceutical opioid- smoked, iii) pharmaceutical opioid- injected, iv) illegal opioid- oral v) illegal opioid- smoked or vi) illegal opioid- injected. The patient’s primary opioid intoxicant was confirmed by patient self-report, or else suspected by EMS or ED staff, and classified as either illegal or pharmaceut- ical. Illegal opioids included heroin, carfentanil, and illegally manufactured fentanyl. Fentanyl was as- sumed to be illegally manufactured if the patient did not have a current prescription for fentanyl at the time of their ED visit, which was determined using PIN data. Results From May 1st 2016 to April 30th 2017, there were 347 visits to the ED in which the patient received a primary diagnosis of opioid overdose. Among these visits, only 344 patient charts were reviewed, as patient records could not be retrieved for 3 ED visits. Additionally, 2 visits were excluded because the patient died while hos- pitalized. The remaining 342 ED visits were made by 297 unique patients, of whom 67.3% were males and the mean age was 38.4 (standard deviation: 14.0). Repeat ED visits for opioid overdose were made by 35 patients dur- ing the study period, with a range of 1 to 4 ED visits per patient. p Several situational variables were examined that could possibly impact whether THN was offered, including ad- mission to hospital (yes vs. no), time of day at presentation and discharge (0:01–8:00, 8:01–16:00, 16:01–24:00), length of ED stay (hours), and whether the visit occurred during a weekend (Saturday or Sunday, yes vs. no). We examined patient disposition, including whether the patient was dis- charged against medical advice (AMA, yes vs. no), or left ED without treatment or before treatment completion (yes vs. no). A patient was considered to have left the ED AMA if they disclosed to the providers that they intended to leave and signed an AMA form. In contrast, patients who left without treatment or before treatment comple- tion would have registered with triage but then left either from the waiting room or their patient care space, typic- ally without disclosing their intent to leave. Overall, THN was offered to the patient in 168 (49.1%) visits, and was accepted by patients in 128 (76.2%) visits (Fig. 1). Of the ED visits in which the patient accepted THN, a friend or family member was included during the THN training in 31 (24.2%) cases. Among the 40 visits in which the patient de- clined THN, the patients already possessed a kit in 11 (27.5%) cases, and left the ED prior to receiving naloxone in 4 (10.0%) cases (Fig. 1). Additionally, in Case identification and data collection Pharmaceutical opioids included prescribed fentanyl, and all other pharmaceutical opioids (ie. oxycodone, hydromorphone, morphine) regardless of whether they were prescribed to the patient at the time of their ED visit. We used GEE with logit link for all regression analyses because a significant portion of patients had multiple ED visits for opioid overdose within our study period, and the data collected for these patients were potentially cor- related. With GEE, standard errors are calculated that adjust for multiple observations per patient, in this case using an exchangeable correlation structure [32]. A significant portion of values were missing for pre-hospital GCS (14.9%) and primary opioid intoxi- cant (23.1%). Missing data for these and other vari- ables were imputed using chained equations with augmented regression and 30 imputations [33]. All variables were included in the imputation regres- sions, and the outcome was not imputed. We assessed the performance of the multiple imputations by conducting the same multivariate analysis without imputation (Table 3 in Appendix). In this analysis, variables with a high proportion of missingness (GCS, primary opioid intoxicant) contained an additional category for cases with missing values. Analyses were performed using STATA 14.0 IC. We anticipated that an ED provider’s decision to offer THN may be impacted by the patient’s over- dose intention (self-reported by patient, intentional vs. unintentional), as well as the severity of the pa- tient’s overdose. Overdose severity was measured using the patient’s pre-hospital Glasgow Coma Scale (GCS) [3–15], which is a standard test that measures a patient’s eye, motor, and visual responsiveness, with higher scores signifying higher consciousness [31]. GCS was measured by EMS on arrival at the overdose scene, and was therefore only available for patients who arrived at the ED via ambulance. Analysis We calculated the number and percentage of each independent variable among ED visits in which THN O’Brien et al. BMC Health Services Research (2019) 19:632 Page 5 of 12 Fig. 1 Study flow diagram showing identification of ED visits for inclusion, and the frequencies and percentages of ED visits in which take home naloxone was offered and accepted Fig. 1 Study flow diagram showing identification of ED visits for inclusion, and the frequencies and percentages of ED visits in which take home naloxone was offered and accepted (OR = 0.30 [0.13,0.65]), left the ED without treatment or before treatment completion (OR = 0.19 [0.07,0.53]), or were admitted to hospital (OR = 0.35 [0.19,0.65]), (Table 1). Additionally, older patients were less likely to be offered THN in bivariate analysis (OR = 0.97 [0.96, 0.99], per year older), (Table 1). In contrast, patients were more likely to be offered THN if they had a severe GCS score upon EMS arrival (OR = 5.60 [2.76,11.3]) for GCS 3–8 vs. 14–15), had smoked an illegal opioid (OR = 9.97 [4.66,21.3] for illegal opioid-smoked vs. pharmaceutical opioid- oral), or had injected an illegal opioid (OR = 15.1 [6.61,34.3] for illegal opioid- injected vs. pharmaceutical opioid- oral), (Table 1). two ED visits the patient declined a THN kit be- cause “it won’t happen again,” or “there won’t be a next time.” In one case they stated “I can quit,” and in one instance the patient declined because “if I hit the dirt, I can’t use it” (Fig. 1). Table 1 shows the breakdown of patient characteris- tics by ED visit. In 73 (21.4%) ED visits, the patient held a current prescription for an opioid medication at the time of their ED visit. The most common primary opioid intoxicants were smoked illegal opioids (n = 66 (25.1%)), injected illegal opioids (n = 85 (32.2%)), and pharmaceutical opioids taken orally (n = 77 (29.3%)) (Table 1). The patients’ primary opioid intoxicant was confirmed by self-report in 241 (91.6%) visits, sus- pected by EMS in 8 (3.0%) visits, suspected by ED staff in 12 (4.6%) visits, and confirmed by toxicology screening in 2 (0.8%) visits. Among ED visits in which the patient arrived by ambulance, the patient’s pre- hospital GCS was most frequently in the severe cat- egory (GCS 3–8; n = 210, 72.2%). Table 2 shows the results of the multivariable ana- lysis. Analysis Variables that were independently and posi- tively associated with being offered THN included having a severe overdose as measured by GCS (AOR = 3.70 [1.63,8.37] for GCS 3–8 vs. GCS 14– 15), and smoking or injecting an illegal opioid (AOR = 3.78 [1.32,10.9] and AOR = 6.05 [2.15,17.0] respectively for illegal opioid- smoked vs. pharma- ceutical opioid- oral and illegal opioid-injected vs. pharmaceutical opioid- oral), (Table 2). In contrast, patients were less likely to receive a THN kit if they Table 1 shows the results of the initial unadjusted bi- variate analyses. We found that patients were less likely to be offered THN if they overdosed in a public loca- tion (OR = 0.62 [0.39,0.99], intentionally overdosed O’Brien et al. *** N = 342 for all independent variables unless stated otherwise Analysis The results of the multivariate a out multiple imputation are sim with imputations (Table 3 in Appe same variables were statistically sig with the one exception being that be significantly associated with be non-imputed multivariate model (T ** Odds ratio of being offered take home nalo * Statistically significant at the level of 0.1 and a- Overall P-value < 0.001 b- Overall P-value =0.77 c- Overall P-value = 0.26 † Postal code overlaps Local Geographical Area †† Postal code was charted as “no fixed addre ¥ Anxiety disorder, Bipolar disorder, Major Dep § Methadone (liquid form, once daily ingestion Does not include prescriptions for opioid agon ¶ Oral includes oral (97%), rectal (1%), transder ‡ disclosed to the providers that they intended ‡‡ registered with triage and was assessed, bu Table 2 Multivariable associations of p overdose, with multiple imputation Visit Characteristic (n = 342) Male sex Age Mental Health disorder Opioid agonist therapy Any current opioid prescription at time of Primary opioid intoxicant illegal† Pharmaceutical opioid- oral Pharmaceutical opioid- smoked Pharmaceutical opioid- injected Illegal opioid- oral Illegal opioid- smoked Illegal opioid- injected Overdosed in public Overdosed intentionally Pre-hospital GCS†† Severe (3–8) Moderate (9–13) Mild (14, 15) Left without treatment/ before treatment c Admitted to hospital †Overall p-value = 0.009 ††Overall p value 0 008 ** Odds ratio of being offered take home naloxone for each independent variable ** Odds ratio of being offered take home naloxone for each independent variable ** Odds ratio of being offered take home naloxone for each independent variable * Statistically significant at the level of 0.1 and thus eligible for inclusion in multivariable analysis a- Overall P-value < 0.001 b- Overall P-value =0.77 c- Overall P-value = 0.26 † Postal code overlaps Local Geographical Area of Edmonton Eastwood, includes T5B, T5G, T5H, T5J, T5K, T6W †† Postal code was charted as “no fixed address,” indicating that the patient is likely unstably or homeless ¥ Anxiety disorder, Bipolar disorder, Major Depressive Disorder, Psychosis, Personality Disorder (Axis II), or Schizophrenia) § Methadone (liquid form, once daily ingestion, max period of 7 days), or Suboxone (once daily dosing, max period of 7 days) Does not include prescriptions for opioid agonist therapy ¶ Oral includes oral (97%), rectal (1%), transdermal (1%), and Percutaneous endoscopic gastrostomy (1%) ‡ disclosed to the providers that they intended to leave and signed an AMA form ‡‡ registered with triage and was assessed, but then left without warning or disclosing their intent to leave * Statistically significant at the level of 0.1 and thus eligible for inclusion in multivariable analysis O ll P l 0 001 †† Postal code was charted as “no fixed address,” indicating that the patient is likely unstably or homeless ¥ Anxiety disorder, Bipolar disorder, Major Depressive Disorder, Psychosis, Personality Disorder (Axis II), or Schizophrenia) § Methadone (liquid form, once daily ingestion, max period of 7 days), or Suboxone (once daily dosing, max period of 7 days) Does not include prescriptions for opioid agonist therapy ¶ Oral includes oral (97%), rectal (1%), transdermal (1%), and Percutaneous endoscopic gastrostomy (1%) ‡ disclosed to the providers that they intended to leave and signed an AMA form ‡‡ registered with triage and was assessed, but then left without warning or disclosing their intent to leave Analysis BMC Health Services Research (2019) 19:632 Page 6 of 12 Table 1 Characteristics of ED visits for opioid overdose, and associations with offering of take home naloxone kits Visit Characteristic (n = 342)*** Number of ED visits (%) Unadjusted OR [95% CI]** P-value Total (n = 342) Offered THN (n = 168) Not offered THN (n = 174) Patient characteristics Male sex 234 (68.4) 129 (76.8) 105 (60.3) 2.06 [1.27,3.34] 0.004* Age (n = 341) (Mean, SD) 38.2 (14.0) 35.3 (11.1) 40.9 (15.8) 0.97 [0.96,0.99] < 0.001* Resident of inner city† (n = 339) 102 (30.1) 50 (30.1) 52 (30.1) 1.04 [0.66,1.65] 0.86 No fixed address (n = 339)†† 23 (6.8) 12 (7.2) 11 (6.4) 1.03 [0.39,2.67] 0.96 Mental health disorder¥ 97 (28.4) 37 (22.0) 60 (34.5) 0.62 [0.38,1.01] 0.054* Current prescription medications Opioid agonist therapy§ 9 (2.6) 1 (0.6) 8 (4.6) 0.15 [0.02,0.91] 0.04* Any current opioid prescription 73 (21.4) 21 (12.5) 52 (29.9) 0.37 [0.22,0.65] < 0.001* Overdose details Primary opioid intoxicant (n = 263)a Pharmaceutical opioid- oral¶ 77 (29.3) 14 (10.5) 63 (48.8) ref Pharmaceutical opioid- smoked 7 (2.7) 3 (2.3) 4 (3.1) 3.60 [0.72,18.1] 0.12 Pharmaceutical opioid- injected 16 (6.1) 5 (3.8) 11 (8.5) 2.11 [0.62,7.14] 0.23 Illegal opioid- oral 12 (4.6) 5 (3.8) 7 (5.4) 3.39 [0.91,12.7] 0.070 Illegal opioid- smoked 66 (25.1) 44 (33.1) 22 (16.9) 9.97 [4.66,21.3] < 0.001* Illegal opioid- injected 85 (32.3) 62 (46.6) 23 (17.7) 15.1 [6.61,34.3] < 0.001* Overdosed in public (n = 337) 102 (30.3) 40 (24.1) 62 (36.3) 0.62 [0.39,0.99] 0.043* Overdose intentional (n = 340) 28 (8.2) 6 (3.6) 22 (12.8) 0.30 [0.13,0.65] 0.002* Glasgow Coma Scale Score (n = 291)a Severe (3–8) 210 (72.2) 127 (88.2) 83 (56.5) 5.60 [2.76,11.3] < 0.001* Moderate (9–13) 27 (9.3) 6 (4.2) 21 (14.3) 1.11 [0.37,3.30] 0.85 Mild (14-15) 54 (18.6) 11 (7.6) 43 (29.3) ref EMS & ED care Admitted to hospital 56 (16.4) 15 (8.9) 41 (23.6) 0.35 [0.19,0.65] 0.001* Left against medical advice‡ 34 (9.9) 14 (8.3) 20 (11.5) 0.72 [0.33,1.57] 0.40 Left without treatment/before treatment complete‡‡ 29 (8.5) 6 (3.6) 23 (13.2) 0.19 [0.07,0.53] 0.002* Time day at ED presentationb 0:01–8:00 94 (27.5) 47 (28.0) 47 (27.0) ref 8:01–16:00 108 (31.6) 49 (29.2) 59 (33.9) 0.92 [0.54,1.59] 0.77 16:01–24:00 140 (40.9) 72 (42.9) 68 (39.1) 1.10 [0.65,1.84] 0.73 Time of day at dischargec 0:01–8:00 121 (35.4) 58 (34.5) 63 (36.2) ref 8:01–16:00 94 (27.5) 53 (31.5) 41 (23.6) 1.43 [0.82,2.50] 0.21 16:01–24:00 127 (37.1) 57 (33.9) 70 (40.2) 0.94 [0.55,1.59] 0.82 ED visit during weekend 102 (29.8) 49 (29.2) 53 (30.5) 0.99 [0.61,1.59] 0.96 Length of ED stay (median, IQR) 5.5 (3.3,9.2) 4.52 (2.7,7.6) 6.45 (3.9,11.0) 0.98 [0.95,1.01] 0.20 *** N = 342 for all independent variables unless stated otherwise Table 1 Characteristics of ED visits for opioid overdose, and associations with offering of take home naloxone kits Visit Characteristic (n = 342)*** Number of ED visits (%) Unadjusted OR [95% CI]** P-value Total (n = 342) Offered THN (n = 168) Not offered THN (n = 174) Page 7 of 12 O’Brien et al. O’Brien et al. BMC Health Services Research (2019) 19:632 Analysis BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 left the ED without treatment or before treatme completion (AOR = 0.16 [0.05,0.48]), if they were a mitted to hospital (AOR = 0.46 [0.22,0.97]) or if th had an opioid prescription at the time of their E visit (AOR = 0.41 [0.19,0.88]). The results of the multivariate analysis conducted wit out multiple imputation are similar to those generat with imputations (Table 3 in Appendix ). In particular, t same variables were statistically significant in both mode with the one exception being that male sex was found be significantly associated with being offered THN in t non-imputed multivariate model (Table 3 in Appendix). ** Odds ratio of being offered take home naloxone for each independent v * Statistically significant at the level of 0.1 and thus eligible for inclusion in a- Overall P-value < 0.001 b- Overall P-value =0.77 c- Overall P-value = 0.26 † Postal code overlaps Local Geographical Area of Edmonton Eastwood, inc †† Postal code was charted as “no fixed address,” indicating that the patien ¥ Anxiety disorder, Bipolar disorder, Major Depressive Disorder, Psychosis, P § Methadone (liquid form, once daily ingestion, max period of 7 days), or Su Does not include prescriptions for opioid agonist therapy ¶ Oral includes oral (97%), rectal (1%), transdermal (1%), and Percutaneous ‡ disclosed to the providers that they intended to leave and signed an AMA ‡‡ registered with triage and was assessed, but then left without warning o Table 2 Multivariable associations of patient characteristics w overdose, with multiple imputation Visit Characteristic (n = 342) Male sex Age Mental Health disorder Opioid agonist therapy Any current opioid prescription at time of ED visit Primary opioid intoxicant illegal† Pharmaceutical opioid- oral Pharmaceutical opioid- smoked Pharmaceutical opioid- injected Illegal opioid- oral Illegal opioid- smoked Illegal opioid- injected Overdosed in public Overdosed intentionally Pre-hospital GCS†† Severe (3–8) Moderate (9–13) Mild (14, 15) Left without treatment/ before treatment completion Admitted to hospital †Overall p-value = 0.009 left the ED without treatment completion (AOR = 0.16 [0.05,0 mitted to hospital (AOR = 0.46 had an opioid prescription at visit (AOR = 0.41 [0.19,0.88]). Discussion left the ED without treatment or before treatment completion (AOR = 0.16 [0.05,0.48]), if they were ad- mitted to hospital (AOR = 0.46 [0.22,0.97]) or if they had an opioid prescription at the time of their ED visit (AOR = 0.41 [0.19,0.88]). In this evaluation of a recently implemented ED-based THN program, THN was offered to patients in ap- proximately half of ED visits for opioid overdose. We sought to determine whether a recently implemented ED-based THN program was missing certain patients by identifying patient characteristics and other situ- ational variables associated with being offered THN. We found that patients were more likely to be offered THN if they experienced a severe overdose (GCS of 3–8), or had consumed an illegal opioid. In contrast, patients who had an active opioid prescription at the The results of the multivariate analysis conducted with- out multiple imputation are similar to those generated with imputations (Table 3 in Appendix ). In particular, the same variables were statistically significant in both models, with the one exception being that male sex was found to be significantly associated with being offered THN in the non-imputed multivariate model (Table 3 in Appendix). Table 2 Multivariable associations of patient characteristics with offering of take home naloxone during ED visits for opioid overdose, with multiple imputation Visit Characteristic (n = 342) Adjusted OR [95% CI]** P-value Male sex 1.75 [0.97,3.16] 0.064 Age 1.00 [0.98,1.02] 0.92 Mental Health disorder 1.12 [0.59,2.12] 0.74 Opioid agonist therapy 0.37 [0.04,3.35] 0.37 Any current opioid prescription at time of ED visit 0.41 [0.19,0.88] 0.021* Primary opioid intoxicant illegal† Pharmaceutical opioid- oral ref Pharmaceutical opioid- smoked 1.32 [0.24,7.27] 0.75 Pharmaceutical opioid- injected 1.40 [0.36,5.51] 0.63 Illegal opioid- oral 1.39 [0.33,5.88] 0.66 Illegal opioid- smoked 3.78 [1.32,10.9] 0.014* Illegal opioid- injected 6.05 [2.15,17.0] 0.001* Overdosed in public 0.61 [0.33,1.11] 0.11 Overdosed intentionally 0.59 [0.21,1.62] 0.30 Pre-hospital GCS†† Severe (3–8) 3.70 [1.63,8.37] 0.002* Moderate (9–13) 2.09 [0.61,7.21] 0.24 Mild (14, 15) ref Left without treatment/ before treatment completion 0.16 [0.05,0.48] 0.001* Admitted to hospital 0.46 [0.22,0.97] 0.040* †Overall p-value = 0.009 ††Overall p-value = 0.008 *Statistically significant at the level of 0.05 **Odds ratio of being offered take home naloxone for each independent variable, adjusted for all other variables in the model ns of patient characteristics with offering of take home naloxone during ED visits for opioid Admitted to hospital Similarly, only about 13% of patients with chronic non-cancer pain report that they would be offended if offered THN [41]. Our results showed that even among patients who have recently overdosed, certain patients were more likely to be offered than others. It is likely that in the context of a busy ED, a clinician’s decision to offer THN may be driven by their perceptions of which pa- tients are most at risk for overdose death. For instance, ED staff may more readily offer THN to patients who have experienced a severe overdose because they are easily recognized as being at risk for future overdose death. Similarly, clinicians may more readily recognize people who smoke or inject illegal drugs as being more vulnerable for future overdose. This tendency is likely shaped by both clinical experience and media reports, as it is true that in the Western Canadian provinces of Alberta and British Columbia, over 80% of accidental opioid overdose deaths involved illegally manufactured fentanyl in 2017 [26, 35]. Additionally, clinicians may believe that THN is predominately meant for people who use illegal drugs, given that THN was originally developed to serve people who inject heroin [36]. Indeed, the initial rationale for THN programs was in- part to empower people who use heroin who were reluctant to call an ambulance in cases of overdose for fear of criminal prosecution [37]. Other primary care providers have expressed reluc- tance to co-prescribe THN with opioids because they felt that THN should not be necessary if opioids are prescribed properly [38]. However, offering THN to patients prescribed opioids can facilitate important conversations about overdose risk, and may even change behavior in ways that reduce the risk of fu- ture overdose and ED visits [40, 42]. For instance, at least some patients who received THN and overdose education through primary care report safer dosing, safer timing, and increased knowledge of opioids and overdose [40]. Additionally, a non-randomized trial of THN co-prescriptions in primary care found that patients who received THN had 63% less ED-related visits at 12-months compared to those that did not receive THN [42]. It was not surprising that patients who unexpectedly left the ED without treatment or before treatment completion were less likely to be offered THN. At the time of this study, ED staff typically waited until dis- charge to offer THN to patients. Admitted to hospital Statistically significant at the level of 0.05 **Odds ratio of being offered take home naloxone for each independent variable, adjusted for all other variables in the model Page 8 of 12 Page 8 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 time of their ED visit, who left the ED without treat- ment of before treatment completion, or who were admitted to the hospital were less likely to be offered THN. found that almost all ED staff who were interviewed agreed certain patients should receive THN—such as those who are have overdosed in the past, are opioid dependent, or who inject opioids [23, 24]. However, other staff disagreed on whether it was appropriate, necessary, or realistic to offer THN to all patients prescribed opioids [23, 24]. In some cases, this ap- peared be due to their perceived lower risk of over- dose [23, 24]. The proportion of patients offered THN (49%) is significantly higher than in previous studies which have evaluated the implementation of ED-based THN programs [23, 34]. This higher rate is likely attributable to our decision to only include individ- uals who were discharged with a diagnosis of opioid overdose. This decision was made based on our de- sire to examine whether the THN program was reaching the highest risk patients. These patients are likely to be readily identifiable by ED providers, and thus more likely to be offered THN compared to other patient groups at risk of overdose. In previous qualitative studies about ED-based THN programs, ED providers frequently acknowledged that it is im- portant not to miss patients who have visited the ED for an opioid overdose [23, 24]. Specific reasons as to why clinicians may be less likely to provide THN to patients taking prescription opioids have been explored in studies of primary care patients with chronic pain [38, 39]. For instance, in one qualitative study, primary care providers believed that co-prescribing THN may offend patients due to the stigma associated with substance use disorders and THN [38]. Previous research from primary care has generally refuted this perception. Indeed, the majority of patients on long-term opioid prescriptions give ei- ther positive or neutral reactions to being offered a naloxone prescription, report wanting naloxone pre- scriptions in the future, and agree that THN should be available to patients prescribed opioids for pain [40]. Admitted to hospital Therefore, patients who left the ED without disclosing their intent to leave may have been missed. Patients who have been treated for opioid overdose may be experiencing symptoms of withdrawal and be eager to leave the ED to address these symptoms. Consequently, they may be less will- ing to complete discharge paperwork or THN training [23]. These patients may be especially vulnerable to a Our finding that patients who were taking pre- scription opioids were less likely to be offered THN is consistent with previous reports. In a survey of Canadian ED physicians, it was found that while the large majority of participants (> 90%) agreed or strongly agreed that patients with a history of emer- gency care for opioid overdose would benefit from THN programs, fewer physicians (69%) agreed that patients prescribed high doses of opioids would benefit [19]. Similarly, previous qualitative studies Page 9 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 We attempted to account for the uncertainty created by missing data with multiple imputation. We ob- tained similar results from the multivariate analysis with and without the use of multiple imputation (Table 3 in Appendix). subsequent overdose immediately following their ED visit, given the relatively short half-life of naloxone [43], and the additional risk posed by consuming fur- ther doses of opioids after leaving the ED. Equipping these patients with THN (and offering opioid agonist treatment and other supports) is especially critical. Finally, our findings showed that ED patients who were admitted to the hospital were more likely to be missed. Further efforts are needed to expand THN distribution to at-risk hospital inpatients in this setting. subsequent overdose immediately following their ED visit, given the relatively short half-life of naloxone [43], and the additional risk posed by consuming fur- ther doses of opioids after leaving the ED. Equipping these patients with THN (and offering opioid agonist treatment and other supports) is especially critical. Finally, our findings showed that ED patients who were admitted to the hospital were more likely to be missed. Further efforts are needed to expand THN distribution to at-risk hospital inpatients in this setting. The data may not always be accurate for variables that were partially or fully based on patient self-re- port or clinician suspicion, such as the patient’s pri- mary intoxicant. Admitted to hospital Patients may have been reticent to disclose the complete details related to their sub- stance use, or they may have consumed a different substance from what they believed. However, given that the aim of the study was to examine clinician behavior, measuring the clinicians’ perception of the patients’ opioid intoxicant is likely more useful than the actual intoxicant. The majority (82%) of patients in our study who were offered a THN kit either accepted it or already possessed one, which confirms previous reports show- ing that ED-based naloxone distribution is acceptable to patients. This THN acceptance rate was slightly higher than the 68% reported in a previous study by Kestler et al., in which THN was offered to patients who had reported illegal drug use, were prescribed a high dose of prescription opioids, were receiving opi- oid agonist therapy, or had any clinical presentation suggestive of opioid use [20]. The higher acceptance rate among our population may suggest that people who have recently experienced an opioid overdose are more accepting of THN than other THN-eligible pa- tients. In a minority of cases, the patient’s reason for declining a THN kit had been recorded. The reasons included feeling that they were no longer at risk, intending to stop using drugs, and believing that THN is not useful if opioids are used alone. Several of these reasons are consistent with a previous evaluation of ED patient acceptance of THN, which found that the patient beliefs of being “not at risk” and “done with drugs” were the two most common rationales given for declining THN [44]. Other variables that were not collected may have also impacted a clinician’s decision to offer THN. For instance, we were unable to obtain reliable data on whether patients had been prescribed or had con- sumed any benzodiazepines. It is possible that ED providers were more likely to offer THN to patients who used opioids in combination with benzodiaze- pines, given that this combination is a strong risk factor for overdose among people receiving opioid analgesics [45, 46]. The data abstracters were not blinded to the pur- pose of the study. However, it is unlikely this affected the results significantly because there were no specific a priori hypotheses regarding which variables would be associated with THN being offered. Admitted to hospital Additionally, because our sample was limited to a single hospital site, it is possible that our results may not generalize to other geographic locations. While we have reported on reasons that patients gave for declining THN, these reasons were only recorded for a minority of patients. Consequently, it was not possible to draw conclusions based on this data. Finally, this study only captured a small subpopulation of individuals visiting the ED who are potentially at risk for overdose. Future research is needed to evaluate whether ED pro- viders are able to reach other patient groups at risk for overdose. Limitations There are several limitations to this study that are inherent to retrospective chart reviews. Because medical records are created for clinical purposes, some information may not be charted consistently. In particular, we were unable to ensure that every instance of THN being offered was documented. We attempted to ameliorate this issue by reviewing medication dispensation data to confirm cases in which THN was both offered and accepted by the patient. However, for some cases in which THN was offered but declined by the patient, ED providers may not have charted that they offered THN. There- fore, the proportion of ED visits in which THN was offered may have been underestimated. Appendix to identify high risk patients, ED providers may con- sciously or unconsciously rely on their own percep- tions as to who is at risk and overlook certain patients, such as those who use prescription opioids. To ensure optimal implementation of ED-based THN programs, ED staff should be provided with information on the importance of offering THN widely, and clear guide- lines regarding which patients should receive THN. Further, administrators or clinicians implementing ED-based THN programs should establish a reliable inpatient pathway to provide THN for eligible patients prior to discharge. As we have shown, patients admit- ted to the hospital after receiving acute care for an opioid overdose can be missed by ED-based THN programs. Another way for ED-based THN programs to im- prove their coverage to at-risk patients is to avoid waiting until discharge to offer THN. As discussed, our evaluation of an ED-based THN program showed that patients who left the ED unexpectedly were more likely to be missed by providers. Where possible, THN should be offered earlier in the patient’s ED visit, to ensure they are not missed should they leave the ED unexpectedly. p y At minimum, all patients presenting to an ED with an opioid overdose should be offered THN. However, several other groups would likely also benefit from THN distribution and training in an ED setting. In particular, the Center for Disease Control and Prevention recommends that THN be offered to patients taking higher doses of prescription opioids (≥50 OME/day), patients with concurrent opioid and benzodiazepine use, and patients with a history of a substance use disorder [47]. We suggest that in addition to targeting people who use opioids, ED providers should consider offering THN to people who use any illegal substance, including stimulants. People who use non-opioid illegal substances may be exposed to opioids through contamination [48], or may witness an opioid overdose among their peers. Future evaluations are needed to examine the extent to which these different patients can be targeted, and to develop systematic protocols to identify vari- ous individuals at risk of overdose. One such inter- vention that holds promise is electronic health record prompts, which can be triggered by terms en- tered into the patient’s initial assessment [49, 50]. Such interventions have been previously demon- strated to increase the distribution of THN to ED patients discharged after opioid overdoses [49, 50]. Authors’ contributions EH and KD conceived and designed the study, and obtained research funding. DD designed the data abstraction protocol in consultation with EH and KD. DD abstracted the data. DO planned and conducted the data analysis in consultation with PV, KD and EH. DO drafted the manuscript, and all authors contributed substantially to its revision. EH takes responsibility for the paper as a whole. All authors read and approved the final manuscript. Acknowledgements Not applicable. Appendix As evidenced by our study, without such systematic approaches, attaining complete coverage of even the highest risk patients is difficult when implementing ED-based THN programs. Abbreviations Abbreviations AMA: Against Medical Advice; AOR: Adjusted Odds Ratio; ARCH: Addiction Recovery and Community Health; ED: Emergency Department; EMS: Emergency Medical Service; GCS: Glasgow Coma Scale; GEE: Generalized Estimating Equation; OR: Odds Ratio; PIN: Pharmaceutical Information Network; THN: Take Home Naloxone Conclusions This study is the first to identify patient characteristics associated with being offered THN among a high risk population of ED patients. Our findings add to the current literature by demonstrating that implementa- tion of an ED-based THN program and accompanying staff training does not necessarily guarantee that even patients presenting with opioid overdose will be of- fered THN. In the absence of a standardized protocol Other variables were missing values for a signifi- cant percentage of ED visits, including pre-hospital GCS (14.9%), and primary opioid intoxicant (23.1%). Page 10 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 Appendix Table 3 Multivariable associations of patient characteristics with offering of take home naloxone during ED visits for opioid overdose, without multiple imputation Visit Characteristic (n = 334) Adjusted OR [95% CI]** P-value Male sex 1.94 [1.10,3.41] 0.022* Age (n = 341) 1.00 [0.98,1.02] 0.93 Mental Health disorder 1.15 [0.61,2.15] 0.67 Opioid agonist therapy 0.30 [0.04,2.44] 0.26 Any current opioid prescription at time of ED visit 0.44 [0.21,0.92] 0.029* Primary opioid intoxicant (n = 263)† Pharmaceutical opioid- oral ref Pharmaceutical opioid- smoked 1.14 [0.25,5.26] 0.87 Pharmaceutical opioid-injected 1.43 [0.34,6.01] 0.63 Illegal opioid- oral 1.26 [0.33,4.83] 0.74 Illegal opioid- smoked 3.65 [1.35,9.88] 0.011* Illegal opioid- injected 5.47 [2.07,14.5] 0.001* Missing 1.61 [0.63,4.08] 0.32 Overdosed in public (n = 337) 0.64 [0.35,1.15] 0.13 Overdosed intentionally (n = 340) 0.47 [0.17,1.31] 0.15 Pre-hospital GCS (n = 291)†† Severe (3–8) 3.57 [1.62,7.83] 0.002* Moderate (9–13) 1.39 [0.39,4.98] 0.62 Mild (14, 15) Ref Missing 3.64 [1.40,9.42] 0.008* Left without treatment/ before treatment completion 0.18 [0.06,0.57] 0.003* Admitted to hospital 0.40 [0.19,0.83] 0.014* †Overall P-value 0.003 ††Overall p-value 0.006 *Statistically significant at the level of 0.05 **Odds ratio of being offered take home naloxone for each independent variable, adjusted for all other variables in the model References 1. Dowell D, Arias E, Kochanek K, Anderson R, Guy GP Jr, Losby JL, et al. Contribution of opioid-involved poisoning to the change in life expectancy in the United States, 2000-2015. JAMA. 2017;318(11):1065–7. 1. Dowell D, Arias E, Kochanek K, Anderson R, Guy GP Jr, Losby JL, et al. Contribution of opioid-involved poisoning to the change in life expectancy in the United States, 2000-2015. JAMA. 2017;318(11):1065–7. 24. Holland TJ, Penm J, Dinh M, Aran S, Chaar B. Emergency department physicians’ and pharmacists’ perspectives on take-home naloxone. Drug Alcohol Rev. 2019;38(2):169–76. 24. Holland TJ, Penm J, Dinh M, Aran S, Chaar B. Emergency department physicians’ and pharmacists’ perspectives on take-home naloxone. Drug Alcohol Rev. 2019;38(2):169–76. 2. World Health Organization. Community management of opioid overdose. 2014. [cited 2019 May 7]. Available from: https://www.who.int/substance_ abuse/publications/management _opioid_overdose/en/ 2. World Health Organization. Community management of opioid overdose. 2014. [cited 2019 May 7]. Available from: https://www.who.int/substance_ abuse/publications/management _opioid_overdose/en/ 25. Royal Alexandra Hospital Foundation. By the heart, by the numbers- the 2016–2017 annual report. 2018 [cited 2019 May 8]. Available from: https:// www.royalalex.org/our-stories/heart-numbers-2016-17-annual-report/ 25. Royal Alexandra Hospital Foundation. By the heart, by the numbers- the 2016–2017 annual report. 2018 [cited 2019 May 8]. Available from: https:// www.royalalex.org/our-stories/heart-numbers-2016-17-annual-report/ 3. Edward W. Boyer. Management of opioid analgesic overdose. N Engl J Med. 2012;367(2):146–55. 3. Edward W. Boyer. Management of opioid analgesic overdose. N Engl J Med. 2012;367(2):146–55. 4. Minozzi S, Amato L, Davoli M. Preventing fatal overdoses: A systematic review of the effectiveness of take-home naloxone. Eur Monit Centre Drugs Drug Addict. 2015; [cited 2019 May 7]. Available from: http://www.emcdda. europa.eu/publications/emcdda-papers/naloxone-effectiveness_en. 4. Minozzi S, Amato L, Davoli M. Preventing fatal overdoses: A systematic review of the effectiveness of take-home naloxone. Eur Monit Centre Drugs Drug Addict. 2015; [cited 2019 May 7]. Available from: http://www.emcdda. europa.eu/publications/emcdda-papers/naloxone-effectiveness_en. 26. Alberta Health. Opioids and substances of misuse, Alberta report 2019 Q1: Government of Alberta; 2019. [cited 2019 July 24]. Available from: https:// open.alberta.ca/dataset/1cfed7da-2690-42e7-97e9-da175d36f3d5/resource/ dcb5da36-7511-4cb9-ba11-1a0f065b4d8c/download/opioids-substances- misuse-report-2018-q1.pdf 5. Clark A, Wilder C, Winstanley E. A systematic review of community opioid overdose prevention and naloxone distribution programs. J Addict Med. 2014;8(3):153–63. 5. Clark A, Wilder C, Winstanley E. A systematic review of community opioid overdose prevention and naloxone distribution programs. J Addict Med. 2014;8(3):153–63. 27. Salvalaggio G, Dong K, Hyshka E, Nixone L, Lavergne K, Nichols J, et al. Enhanced multidisciplinary care for inner city patients with high acute care use: study protocol. Can J Addict. Funding G & Grants & studentship funding: Alberta Innovates: Health Solutions (AIHS) Grants & studentship funding: Alberta Innovates: Health Solutions (AIHS) Page 11 of 12 Page 11 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 The Alberta Health Services Emergency Strategic Clinical Network (ESCN) The Edmonton Emergency Physicians Association (EEPA) and nasal naloxone distribution in Massachusetts: interrupted time series analysis. BMJ. 2013;346:f174. and nasal naloxone distribution in Massachusetts: interrupted time series analysis. BMJ. 2013;346:f174. and nasal naloxone distribution in Massachusetts: interrupted time series analysis. BMJ. 2013;346:f174. The Alberta Health Services Emergency Strategic Clinical Network (ESCN) The Edmonton Emergency Physicians Association (EEPA) The Edmonton Emergency Physicians Association (EEPA) Canadian Research Initiative on Substance Misuse – Prairie Node KD receives a stipend from Alberta Health Services as director of the Inner City Health and Wellness Program (ICHWP). EH receives salary support from the Royal Alexandra Hospital Foundation and Policywise for Children and Families. Canadian Research Initiative on Substance Misuse – Prairie Node Canadian Research Initiative on Substance Misuse – Prairie Node 10. Houry DE, Haegerich TM, Vivolo-Kantor A. Opportunities for prevention and intervention of opioid overdose in the emergency department. Ann Emerg Med. 2018;71(6):688–90. City Health and Wellness Program (ICHWP). EH receives salary support from the Royal Alexandra Hospital Foundation and Policywise for Children and Families. City Health and Wellness Program (ICHWP). EH receives salary support from the Royal Alexandra Hospital Foundation and Policywise for Children and Families. 11. Bird SM, McAuley A, Perry S, Hunter C. Effectiveness of Scotland’s national naloxone programme for reducing opioid-related deaths: a before (2006-10) versus after (2011-13) comparison. Addiction. 2016;111(5):883–91. The above funding bodies played no role in any of the following: The above funding bodies played no role in any of the following: The design of the study The design of the study 12. Verdier M, Routsolias JC, Aks SE. Naloxone prescriptions from the emergency department: an initiative in evolution. Am J Emerg Med. 2019;37(1):164–5. The collection, analysis, or interpretation of data The writing the manuscript. The writing the manuscript. 13. Brady JE, DiMaggio CJ, Keyes KM, Doyle JJ, Richardson LD, Li G. Emergency department utilization and subsequent prescription drug overdose death. Ann Epidemiol. 2015;25(8):619.e2. Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request. 14. Caudarella A, Dong H, Milloy MJ, Kerr T, Wood E, Hayashi K. Non-fatal overdose as a risk factor for subsequent fatal overdose among people who inject drugs. Drug Alcohol Depend. 2016;162:51–5. 14. Caudarella A, Dong H, Milloy MJ, Kerr T, Wood E, Hayashi K. Non-fatal overdose as a risk factor for subsequent fatal overdose among people who inject drugs. Drug Alcohol Depend. 2016;162:51–5. Received: 11 February 2019 Accepted: 27 August 2019 22. Pedigo JR, Seifert CF. Rate of patients at elevated risk of opioid overdose visiting the emergency department. Am J Emerg Med. 2018;36(12):2161–5. 23. Drainoni M, Koppelman EA, Feldman JA, Walley AY, Mitchell PM, Ellison J, et al. Why is it so hard to implement change? A qualitative examination of barriers and facilitators to distribution of naloxone for overdose prevention in a safety net environment. BMC Research Notes. 2016;9:465. Consent for publication Not applicable. 17. Samuels E. Emergency department naloxone distribution: a Rhode Island department of health, recovery community, and emergency department partnership to reduce opioid overdose deaths. R I Med J. 2014;97(10):38. Competing interests 18. Samuels EA, Dwyer K, Mello MJ, Baird J, Kellogg AR, Bernstein E, et al. Emergency department-based opioid harm reduction: moving physicians from willing to doing. Acad Emerg Med. 2016;23(4):455–65. 18. Samuels EA, Dwyer K, Mello MJ, Baird J, Kellogg AR, Bernstein E, et al. Emergency department-based opioid harm reduction: moving physicians from willing to doing. Acad Emerg Med. 2016;23(4):455–65. The authors declare that they have no competing interests. Author details 1 19. Lacroix L, Thurgur L, Orkin AM, Perry JJ, Stiell IG. Emergency physicians’ attitudes and perceived barriers to the implementation of take-home naloxone programs in Canadian emergency departments. CJEM. 2018;20(1):46–52. 1University of Alberta, School of Public Health, 3-300 Edmonton Clinic Health Academy, 11405 – 87 Ave, Edmonton, Alberta T6G 1C9, Canada. 2University of Alberta, Faculty of Medicine and Dentistry, 2J2.00 WC Mackenzie Health Sciences Centre, 8440 112 St. NW, Edmonton, Alberta T6G 2R7, Canada. 3University of Alberta, Faculty of Medicine and Dentistry, 790 University Terrace Building, 8303 112 St. NW, Edmonton, Alberta T6G 2T4, Canada. of Alberta, Faculty of Medicine and Dentistry, 2J2.00 WC Mackenzie Health Sciences Centre, 8440 112 St. NW, Edmonton, Alberta T6G 2R7, Canada. 3University of Alberta, Faculty of Medicine and Dentistry, 790 University Terrace Building, 8303 112 St. NW, Edmonton, Alberta T6G 2T4, Canada. 20. Kestler A, Buxton J, Meckling G, Giesler A, Lee M, Fuller K, et al. Factors associated with participation in an emergency department–based take-home naloxone program for at-risk opioid users. Ann Emerg Med. 2017;69(3):340. 21. Ellison J, Walley AY, Feldman JA, Bernstein E, Mitchell PM, Koppelman EA, et al. Identifying patients for overdose prevention with ICD-9 classification in the emergency department, Massachusetts, 2013-2014. Public Health Rep. 2016;131(5):671–5. 4University of Alberta, School of Public Health, 33-50 University Terrace, 8303 - 112 Ave, Edmonton, Alberta T6G 2T4, Canada. 4University of Alberta, School of Public Health, 33-50 University Terrace, 8303 - 112 Ave, Edmonton, Alberta T6G 2T4, Canada. Received: 11 February 2019 Accepted: 27 August 2019 Ethics approval and consent to participate 15. Wagner KD, Liu L, Davidson PJ, Cuevas-Mota J, Armenta RF, Garfein RS. Association between non-fatal opioid overdose and encounters with healthcare and criminal justice systems: identifying opportunities for intervention. Drug Alcohol Depend. 2015;153:215–20. Our research protocol received ethics approval from the University of Alberta’s Health Research Ethics Board. The Research Ethics Board agreed that it was not necessary or feasible to obtain individual consent because our study used secondary use of extracted and anonymized administrative health data. Therefore, there was no contact with human subjects. 16. Dwyer K, Walley AY, Langlois BK, Mitchell PM, Nelson KP, Cromwell J, et al. Opioid education and nasal naloxone rescue kits in the emergency department. West J Emerg Med. 2015;16(3):381–4. References 2016;6(3):34–41. 6. Mueller SR, Walley AY, Calcaterra SL, Glanz JM, Binswanger IA. A review of opioid overdose prevention and naloxone prescribing: implications for translating community programming into clinical practice. Subst Abus. 2015;36(2):240–53. 6. Mueller SR, Walley AY, Calcaterra SL, Glanz JM, Binswanger IA. A review of opioid overdose prevention and naloxone prescribing: implications for translating community programming into clinical practice. Subst Abus. 2015;36(2):240–53. 28. McHugh ML. Interrater reliability: the kappa statistic. Biochemia medica. 2012;22(3):276–82. 29. Briggs D, Rhodes T, Marks D, Kimber J, Holloway G, Jones S. Injecting drug use and unstable housing: scope for structural interventions in harm reduction. Drugs: Education, Prevention and Policy. 2009;16(5):436–50. 7. Siegler A, Huxley-Reicher Z, Maldjian L, Jordan R, Oliver C, Jakubowski A, et al. Naloxone use among overdose prevention trainees in new York City: a longitudinal cohort study. Drug Alcohol Depend. 2017;179:124–30. 7. Siegler A, Huxley-Reicher Z, Maldjian L, Jordan R, Oliver C, Jakubowski A, et al. Naloxone use among overdose prevention trainees in new York City: a longitudinal cohort study. Drug Alcohol Depend. 2017;179:124–30. 30. Small W, Rhodes T, Wood E, Kerr T. Public injection settings in Vancouver: physical environment, social context and risk. Int J Drug Policy. 2007;18(1):27–36. 8. McAuley A, Aucott L, Matheson C. Exploring the life-saving potential of naloxone: a systematic review and descriptive meta-analysis of take home naloxone (THN) programmes for opioid users. Int J Drug Policy. 2015;26(12):1183–8. 8. McAuley A, Aucott L, Matheson C. Exploring the life-saving potential of naloxone: a systematic review and descriptive meta-analysis of take home naloxone (THN) programmes for opioid users. Int J Drug Policy. 2015;26(12):1183–8. 31. Teasdale G, Jennett B. Assessment of coma and impaired consciousness. A practical scale. Lancet. 1974;2(7872):81–4. 32. Hosmer DW, Lemeshow S, Sturdivant RX. Applied logistic regression. Third ed. Hoboken: Wiley; 2013. 9. Walley AY, Xuan Z, Hackman HH, Quinn E, Doe-Simkins M, Sorensen-Alawad A, et al. Opioid overdose rates and implementation of overdose education 32. Hosmer DW, Lemeshow S, Sturdivant RX. Applied logistic regression. Third ed. Hoboken: Wiley; 2013. Page 12 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 33. StataCorp. STATA Multiple-Imputation Reference Manual, Release 13. 2013; Available from: https://www.stata.com/manuals13/mi.pdf. 34. Barbour K, McQuade M, Somasundaram S, Chakravarthy B. Emergency physician resistance to a take-home naloxone program led by community harm reductionists. Am J Emerg Med. 2018;36(11):2110–2. 35. British Columbia Coroners Service. References CDC guideline for prescribing opioids for chronic pain - United States, 2016. MMWR Recomm Rep. 2016;65(1):1–49. 48. Tupper KW, McCrae K, Garber I, Lysyshyn M, Wood E. Initial results of a drug checking pilot program to detect fentanyl adulteration in a Canadian setting. Drug Alcohol Depend. 2018;190:242–5. 48. Tupper KW, McCrae K, Garber I, Lysyshyn M, Wood E. Initial results of a drug checking pilot program to detect fentanyl adulteration in a Canadian setting. Drug Alcohol Depend. 2018;190:242–5. 49. Devries J, Rafie S, Ajayi TA, Kreshak A, Edmonds KP. Results of a naloxone screening quality-improvement project in an academic emergency department. J Emerg Med. 2019;56(4):378–85. 49. Devries J, Rafie S, Ajayi TA, Kreshak A, Edmonds KP. Results of a naloxone screening quality-improvement project in an academic emergency department. J Emerg Med. 2019;56(4):378–85. 50. Marino R, Landau A, Lynch M, Callaway C, Suffoletto B. Do electronic health record prompts increase take-home naloxone administration for emergency department patients after an opioid overdose? Addiction. 2019;114(9):1575–81. References Fentanyl-detected illicit drug overdose deaths, January 1, 2009 to January 31, 2019. Government of British Columbia Ministry of Public Safety and Solicitor General; 2019 [cited 2019 July 24]. Available from: https://www2.gov.bc.ca/assets/gov/birth-adoption- death-marriage-and-divorce/deaths/coroners-service/statistical/illicit-drug.pdf 36. McDonald R, Campbell ND, Strang J. Twenty years of take-home naloxone for the prevention of overdose deaths from heroin and other opioids- conception and maturation. Drug Alcohol Depend. 2017;178:176–87. 37. Strang J, Darke S, Hall W, Farrell M, Ali R. Hero home naloxone. BMJ. 1996;312(7044):1435–6. 37. Strang J, Darke S, Hall W, Farrell M, Ali R. Heroin overdose: the case for take- home naloxone. BMJ. 1996;312(7044):1435–6. 38. Binswanger IA, Koester S, Mueller SR, Gardner EM, Goddard K, Glanz JM. Overdose education and naloxone for patients prescribed opioids in primary care: a qualitative study of primary care staff. J Gen Intern Med. 2015;30(12):1837–44. 39. Behar E, Bagnulo R, Coffin PO. Acceptability and feasibility of naloxone prescribing in primary care settings: a systematic review. Prev Med. 2018;114:79–87. 40. Behar E, Rowe C, Santos GM, Murphy S, Coffin PO. Primary care patient experience with naloxone prescription. Ann Fam Med. 2016;14(5):431–6. 41. Nielsen S, Peacock A, Lintzeris N, Bruno R, Larance B, Degenhardt L. Knowledge of opioid overdose and attitudes to supply of take-home naloxone among people with chronic noncancer pain prescribed opioids. Pain Med. 2018;19(3):533–40. 42. Coffin PO, Behar E, Rowe C, Santos G, Coffa D, Bald M, et al. Nonrandomized intervention study of naloxone coprescription for primary care patients receiving long-term opioid therapy for pain. Ann Intern Med. 2016;165(4):245. 43. Rzasa Lynn R, Galinkin JL. Naloxone dosage for opioid reversal: current evidence and clinical implications. Ther Adv Drug Saf. 2018;9(1):63–88. 44. Kestler A, Giesler A, Buxton J, Meckling G, Lee M, Hunte G, et al. Yes, not now, or never: an analysis of reasons for refusing or accepting emergency department-based take-home naloxone. CJEM. 2019;21(2):226–34. 45. Park TW, Saitz R, Ganoczy D, Ilgen MA, Bohnert AS. Benzodiazepine prescribing patterns and deaths from drug overdose among US veterans receiving opioid analgesics: case-cohort study. BMJ. 2015;350:h2698. 46. Sun EC, Dixit A, Humphreys K, Darnall BD, Baker LC, Mackey S. Association between concurrent use of prescription opioids and benzodiazepines and overdose: retrospective analysis. BMJ. 2017;356:j760. 47. Dowell D, Haegerich TM, Chou R. CDC guideline for prescribing opioids for chronic pain - United States, 2016. MMWR Recomm Rep. 2016;65(1):1–49. 47. Dowell D, Haegerich TM, Chou R. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Didaktisches zur Aberration
Astronomische Nachrichten
1,920
public-domain
3,023
Didaktisches zur Aberration. Von F. Hayn. I I1 111 x’/u = c cos a. cos a0 + 2 or4 7 sin Q - or3 2 cosy sin e y‘/v = csinorocosdO -20.47 COSQCOSE +0.3zcos~cost1 z’/v = c sin So -20.47 cosOsins. I I1 111 x’/u = c cos a. cos a0 + 2 or4 7 sin Q - or3 2 cosy sin e y‘/v = csinorocosdO -20.47 COSQCOSE +0.3zcos~cost1 z’/v = c sin So -20.47 cosOsins. Das Folgende mag vielleicht vielen als langst bekanntes oder selbstverstandliches erscheinen; dafi ich es trotzdem dep Lesern der A. N. unterbreite, dam haben mich Mifiverstand- nisse veranlaflt, die ihren Grund in der Behandlungsweise zu haben scheinen, die das Problem der Aberration in ver- schiedenen Lehrbuchern der Astronomie und Physik und auch anderswo erfahren hat und noch erfahrt. Dabei Ieitete mich die Ansicht, dafl vieles dem Manne der Praxis als selbst- verstandlich erscheint, dem Lernenden jedoch nicht, und dafl dieser beim Studium der Aberration in den verschiedenen Bucliern und Schriften zu der Anslcht kommen kann, als bote das Problem der Aberration gewisse Schwierigkeiten und enthielte Widerspruche, die noch zu losen waren. Den absoluten Betrag des von I abhangigen Teils der Aberration werden wir nie bestimmen konnen, da sich kein‘ im Raume festes System definieren lant. Man pflegt die von I, 11, I11 abhangigen Teile der Aberration der Reihe nach rnit sakularer, jahrlicher, taglicher Aberration zu bezeichnen. In jedem Zeitpunkte ist demnach gleich die sakulare Aberration fur alle Bewohner des Sonnensystems, n jahrliche )) >) )) )) der Erde, )) tagliche )) )> )> )) eines gewissen Be- zirkes auf der Erde. Ich mufl zur Vervollstandigung des Bildes meinen Be- trachtungen in aller Kurze die Berechnungsweise der Aber- ration vorausschicken. Die Aberration wird bedingt durch die relative Be- wegung gegen den Ort, von dem das Licht ausging, aber nicht durch die relative Bewegung Gestirn -Erde. Deshalb mussen auch alle Sterne dieselbe Aberrationskonstante er- geben. Wir werden aus dem Folgenden sehen, dafl wir nirgends irgend eine Lichttheorie vorauszusetzen brauchen ; das ganze Problem la& sich rein geometrisch behandeln. Zwei Erfahrungstatsachen miissen vorausgesetzt werden : I . Das Licht pflanzt sich im leeren Raume geradlinig rnit gleichformiger Geschwindigkeit (n) fort. 2. Die Fortpflanzung ist unabhangig von der Ge- schwindigkeit der Lichtquelle, wenigstens wenn diese klein ist gegenuber der Lichtgeschwindigkeit. Wir betrachten der Einfachheit halber Be- wegungsvorgange in einer Ebene. ASTRONOMISCHE NACHRICHTEN. Band 211. Nr. 5042. Band 211. Didaktisches zur Aberration. Von F. Hayn. Gegeben in einein ruhenden System 0 zwei Punkte A und B, deren Verbindungslinie rnit der Nullrichtung den Winkel 0 y’ bildet. Geht vom Punkte B Licht aus, so ~ trifft es in A ein nach BA/v Sek. Die schein- A A‘ bare Richtung von AB ist gleich der wahren y‘. Wird jetzt das System in gleichformige Bewegung in der Richtung der Nullinie rnit der Geschwindigkeit (I ver- setzt, dann trifft ein Lichtblitz voni Punkte B den Punkt A zwar im Orte A’, aber der Strahl ist gewandert auf der sich gleichzeitig parallel verschiebenden Geraden AB. In eineni engen Rohre, das diese Gerade einhullt, wiirde man also das Licht B erblicken, ganz gleich, ob das ganze System ruht oder sich bewegt; d. h. die scheinbare Richtung im ruhenden und bewegten System ist dieselbe. AbsoIut im Raume hat das Licht, das in das Auge des Beobachters A gelangt, den Weg BA’ zuruckgelegt, relativ aber, d. h. fur den Beobachter, der an der Bewegung des Systems teilnimmt, den Weg BA. 0 A A‘ 0 A A‘ Angenommen werde ein rechtwinkliges, im Raume festes Koordinatensystem, das nach dem Aquator orientiert ist. Die Koordinaten des Punktes, von dem das Licht aus- ging, seien X, 1; 2, die des Beobachters x, y, 3. Sind nun die Koordinaten der Sonne xo, yo, zo, die der Erde gegen die Sonne xl, yl, zL und die des Beobachters gegen den Erdmittelpunkt xz, y2, z2, dann ist x = xo+x,+x* y =yo+y1+yz z = zo+z,+z, und die 3 Geschwindigkeitskomponenten desBeobachtungsortes x’ = x’o +x’, +x’, y’ = y’o +y’, +y’2 2’ = z’o +z’, +z’* . x = xo+x,+x* y =yo+y1+yz z = zo+z,+z, x = xo+x,+x* y =yo+y1+yz z = zo+z,+z, und die 3 Geschwindigkeitskomponenten desBeobachtungsortes und die 3 Geschwindigkeitskomponenten desBeobachtungsortes x’ = x’o +x’, +x’, y’ = y’o +y’, +y’2 2’ = z’o +z’, +z’* . Der Rurze halber werde die Fortpflanzungsrichtung des Lichtes mit ))Strahl(( bezeichnet. Der vom Punkte (X’YZ) ausgehende Strahl trifft den Beobachter im Punkte (xyz) ; die 3 Richtungskosinus dieses absoluten Strahles (wahre Richtung) seien cos S cos a cos 6 sin a sin 6 die Richtungskosinus des relativen Strahles (scheinbare Rich- tung) cos d’ cos a’ cos 6’ sin a’ sin d’ . die Richtungskosinus des relativen Strahles (scheinbare Rich- tung) cos d’ cos a’ cos 6’ sin a’ sin d’ . Didaktisches zur Aberration. Von F. Hayn. 2 . Beziehen sie sich auf den Erdmittelpunkt, dann ist die Richtung frei von sakularer und jahrlicher Aberration. 3. Alle auf der Erdoberflache beobachteten Richtungen sind frei von jeder Aberration. Diese letzte, bekannte und naturgeniatl als selbstver- standlich angesehene Tatsache sagt . aber nichts anderes aus als der bekannte Interferenzversuch von Michelson, daD nam- lich, wie oben ausgesprochen, die Lichtvorgange zwischen z Punkten auf der Erde fur den mitbewegten Beobachter sich genau so abspielen, als waren die Punkte in Ruhe. Aus den Uberlegungen, die Michelson. zu seinem beruhmten Ver- such veranlanten, mufite man ganz ebenso folgern, daO irdische Azimute alle Arten von Aberration zeigen mufiten. Die obigen Betrachtungen aber beweisen, daf3 das ein arger Fehl- schluD ware; damit sind auch die Jfichelsanschen Voraus- setzungen wideriegt. Man sieht aus diesem Beispiele, daO Selbstverstandlichkeit ein sehr relativer Begriff ist. Von zwei Folgerungen, die auf genau denselben Pramissen aufgebaut sind, wird die eine ohne weiteres als unrichtig 'nicht in den Kreis der Erwagung gezogen, wahrend die andere groOes Aufsehen erregt hat und AnlaD zu allerlei Hypothesen ge- wesen ist. Diese Umrechnung der Zeit oder der Richtung ist gleichbedeutend mit einer 'Aberration, hervorgerufen durch die relative Bewegung der Erde gegen ein System, das sich auf den lichtaussendenden Ort bezieht. Daher stamint wohl auch der Name Planetenaberration. Wenn geozentrische Koordinaten fur die Zeit t2 gegeben sind, wie es bei Sonne und Mond iiblich ist, dann ist nur die tagliche Aberration zu berucksichtigen. Der Fall der Mondephemeride wird ubrigens in keinem Buche erwahnt. Mein Bestreben ging dahin, in dieser kurzen Darlegung zu zeigen, rnit wie einfachen Uberlegungen man auf alle Fragen des Aberrationsproblems bestimmte Antworten geben kann, ohne 211 irgend welchen Theorien und ohne zum schwersten Rustzeug moderner Wissenschaft greifen zu mussen. Ich habe in der Einleitung dieses Aufsatzes von MiDver- standnissen gesprochen, die durch die Behandlungsweise des vorliegenden Problems herbeigefuhrt werden konnten. Zur Rechtfertigung meiner Ansicht sei noch folgendes angefuhrt. Aus dem obigen Beispiele sehen wir: Licht, das im bewegten System vom Punkte R ausgeht, verhalt sich geradeso, als kame es von einem Fixsterne, dessen wahre Richtung y gefunden wird aus y ' - y = -(.I.) s h y ; d. h. es besteht kein Unterschied zwischen diesen beiden relativen Strahlen. Didaktisches zur Aberration. Von F. Hayn. Dann ist nach bekannten Formeln, wenn man sich auf die Hauptglieder beschrankt, Dann ist nach bekannten Formeln, wenn man sich auf die Hauptglieder beschrankt, a’-a = (~/n)[y’cosa-x’sina] secS 8-8 = -(~/v)[.r’cosasinS+y’sinas a’-a = (~/n)[y’cosa-x’sina] secS 8 8 a’-a = (~/n)[y’cosa-x’sina] secS 8-8 = -(~/v)[.r’cosasinS+y’sinasin~-z’cosd]. 8-8 = -(~/v)[.r’cosasinS+y’sinasin~-z’cosd]. Wir musen vorlaufig annehmen,, die Sonne bewege sich geradlinig und gleichformig nach eineni Punkte (ao do); wenn wir uns auf die Hauptglieder beschranken, ist g Man kann also den Satz aussprechen: D i e L i c h t - v o r g a n g e z w i s c h e n 2 P u n k t e n e i n e s b e w e g t e n 2 5042 I 9 2 0 S y s t e m s spielen sich fur d e n m'itbewegten Beob- a c h t e r genau so ab, als ware das System in Ruhe. Dieser Satz gilt auch fur den Fall, wenn das System sich in gleichformiger Rotation befindet, vorausgesetzt, daO die Bewegung der Punkte A und.R innerhalb der Zeit 2x1. als geradlinig betrachtet werden darf. S y s t e m s spielen sich fur d e n m'itbewegten Beob- a c h t e r genau so ab, als ware das System in Ruhe. Dieser Satz gilt auch fur den Fall, wenn das System sich in gleichformiger Rotation befindet, vorausgesetzt, daO die Bewegung der Punkte A und.R innerhalb der Zeit 2x1. als geradlinig betrachtet werden darf. Zu den obigen Behauptungen I . und 2. ist noch einiges hinzuzufugen. Das Licht geht aus vom (XI'Z) zur Zeit t, und trifft zur Zeit tz in ( x y z ) . ein. Die Richtung dieses Strahls war gegeben durch a und 6, die beobachtete schein- bare Rlchtung durch a' und 8'. Bell den groOen Entfernungen der Fixsterne interessiert die Zeit tl nicht; wir rechnen bei Fixsternen nicht rnit Raumkoordinaten, sondern nur mit Rich- tungen, die fur die Zeit t2 gelten. Didaktisches zur Aberration. Von F. Hayn. g g Den gefundenen Satz konnen wir auch in der Form aussprechen: Richtungen in einem b e w e g t e n System s i n d f r e i v o n d e r A b e r r a t i o n , d i e d u r c h d i e B e - we g u n g e r ze u g t wird. 1. Im Falle heliozentrischer Koordinaten von Licht- quelle und Beobachtungsort unterscheiden sich die beiden Richtungen um den Betrag : jahrl. ttagl. Aberration. Alle Erscheinungen in diesem Bezugssystem sind von diesen beiden Aberrationen beeinfluat. g g g Hieraus ergeben sich weiter folgende Tatsachen : I . Beziehen sich die Koordinaten von Lichtquelle und Beobachter auf die Sonne, so ist die Richtung des Strahles frei von der sakularen Aberration. I . Beziehen sich die Koordinaten von Lichtquelle und Beobachter auf die Sonne, so ist die Richtung des Strahles frei von der sakularen Aberration. 2 . Geozentrische Koordinaten .sind gegeben. Die Ephe- meride gibt die wahre Richtung von der r u h e n d e n Erde fur die Zeit tl. Sol1 die Reobachtung zur Zeit f2 mit der Ephemeride verglichen werden, so ist t2 rnit der Lichtzeit und derjenigen Entfernung, die fur die Zeit 1/z(tl+t2) gilt, auf t l zu reduzieren. Oder man kann auch rnit den aus der Ephemeride folgenden Geschwindigkeiten der geozentrischen Koordinaten und der Zeit tz -tl den Ephemeridenort auf die Zeit tz bringen. Beides kommt auf dasselbe hinaus. Bei Sonne und Mond ist i2 - tl nahezu konstant, dem entspricht eine konstante Richtungsanderung, die in der Ephemeride schon enthalten ist ; die Reduktion unterbleibt daher. 2 . Geozentrische Koordinaten .sind gegeben. Die Ephe- meride gibt die wahre Richtung von der r u h e n d e n Erde fur die Zeit tl. Sol1 die Reobachtung zur Zeit f2 mit der Ephemeride verglichen werden, so ist t2 rnit der Lichtzeit und derjenigen Entfernung, die fur die Zeit 1/z(tl+t2) gilt, auf t l zu reduzieren. Oder man kann auch rnit den aus der Ephemeride folgenden Geschwindigkeiten der geozentrischen Koordinaten und der Zeit tz -tl den Ephemeridenort auf die Zeit tz bringen. Beides kommt auf dasselbe hinaus. Bei Sonne und Mond ist i2 - tl nahezu konstant, dem entspricht eine konstante Richtungsanderung, die in der Ephemeride schon enthalten ist ; die Reduktion unterbleibt daher. Ioo(p-pFLO)/,uO = -0!~04sina-0~14 cosa Ioo(p-pFLO)/,uO = -0!~04sina-0~14 cosa Ioo(p-pFLO)/,uO = -0!~04sina-0~14 cosa wo mit a die vom Jahresanfang in BogenmaD gerechnete Zeit, niit po der mittlere und mit p der momentane Wert der Refraktionskonstante bezeichnet wird. wo mit a die vom Jahresanfang in BogenmaD gerechnete Zeit, niit po der mittlere und mit p der momentane Wert der Refraktionskonstante bezeichnet wird. Zwischen den beobachteten Zenitdistanzen und den zu bestimmenden GroDen besteht. folgende Beziehung : Es schien wunschenswert eine ahnliche Untersuchung fur Pulkovo auszufuhren, um festzustellen, ob wir es hier mit einer lokalen Anomalie oder mit einer allgemeingultigen Refraktionserscheinung zu tun haben. Zu diesem Zwecke wurde eine Bearbeitung folgender groi3en Beobachtungsreihen mit dem Pulkovoer Vertikalkreise vorgenommen : Die Beob- achtungen der absoluten DekIinationen fur die Epoche 1845 von GyZa'Ln, die fur die Epochen 1865, 1885 und 1900 von Nyrkn, sowie die fur 1905 von Kma'vawtzef. Alle Beob- achtungen mit Z> 30' wurden mitaufgenommen. Von je z Be- obachtungen eines Sternes wurde eine Gleichung zur gleich- zeitigen Ableitung des Ausdehnungskoeffizienten der Luft, sowie der als Sinussoide angenomnienen Jahresperiode der Re- fraktion gebildet. Ich erhielt folgende Refraktionsperiode (siehe Publ. d. 1'Obs. Centr. Nic. XXVII): g g AZ = ~ / l o o ~ z ~ ( A ~ K + A ( ~ , - ti).i+dz .p +Asinor. a+LIcosa.b) wo k = I O O ( ~ - ~ , ) / ( I + Y ' . ~ ~ Z ~ ) i = ioo.m-s und to = ti+s(t,-ti) p = roox/8B wenn Bo = B-'/sxrr ~ o o ( p - p ~ ) / p ~ = -asina-bcosa. k = I O O ( ~ - ~ , ) / ( I + Y ' . ~ ~ Z wo p = roox/8B wenn Bo = B-'/sxrr ( p - p ~ ) / p ~ = -asina-bcosa. Mit a, z, q, Y,, p und m werden bezeichnet: die in Winkelma8 angegebene Zeit der Beobachtung, die Zenit- distanz, die absolute Feuchtigkeit der Luft, die Refraktion in Zenitdistanz z, die Refraktionskonstante und der Aus- dehnungskoeffizient der Luft. Alle Sterne rnit 2130' wurden in die Untersuchung mithineingezogen. ' Beobachtungen mit noch kleineren Zenit- distanzen wurden nicht beriicksichtigt, weil bei diesen die kleinen Refraktionsglieder von unbedeutenden systematischen Fehlern anderen Ursprungs leicht iibertroffen werden und dadurch eine Verzerru,ng der Resultate zu befiirchten ist. Didaktisches zur Aberration. Von F. Hayn. Wir wissen, daD ein Strahl von B aus seine Richtung bei- behalt, gleichviel ob man ein )) wassergefulltes Fernrohra in den Strahlengang AB einschaltet oder nicht; demnach gilt dasselbe auch fur den relativen Strahl vom Fixsterne. Beide Strahlen folgen in gleicher Weise allen optischeh Gesetzen uber Rrechung, Reflexion usw., die ja aus den auf der Erde allein moglicheh Beobachtungen von relativen Strahlen ab- geleitet worden sind. Nunmehr la& sich ohne weiteres an- geben, bis zu welchem Betrage die beobachtete Aberration von der Atmosphare der Erde abhangig sein kann. Man braucht nur den Punkt B an die Grenze der Atmosphare zu verlegen und fur diesen Punkt die Aberration zu berechnen. So interessant Beweise, die auf dem Umwege uber irgend eine Lichttheorie dartun, daO die Aberration vom bewegten Medium unabhangig ist, sein mogen, notwendig sind sie nicht. Alle unsere optischen Gesetze beziehen sich auf bewegte Medien, an deren Bewegung der Beobachter teilninimt. Die Darstellungsweise, die zu der SchluDfolgerung ver- leitete, die Aberration sei vom Fernrohrinnern abhangig, ist noch nicht verschwunden. 1st doch in einer mir vorliegen- den Vorlesung die Frage offen gelassen, warum die Aber- ration vom Medium am Ende des Lichtwegs unabhangig sei. Es ist qn mehr als einer Stelle zu lesen, unsere Vorsteliungen reichten noch nicht aus, um die Aberration vollig zu erklaren. Es ist verstandlich, wenn in speziellen Lehrbuchern nur so- vie1 von der Aberration gegeben wird, wie zur regelrechten Reduktion der in Frage komnienden Beobachtungen not- wendig ist. Aber in Lehrbuchern der allgemeinen und spha- rischen Astronoinie und in denen der Physik sucht man doch eine erschop€ende Erklarung der Erscheinungen. So- lange einzelne Punkte noch nicht vollig geklart sind oder von anderer Seite unrichtig dargestellt werden, erscheint es 2 2 5042 5042 2 1 voraussetzte und deren Ausbleiben nicht erklart werden konnte. mir didaktisch nicht richtig, stillschweigend solche Punkte zu ubergehen ; eine Widerlegung ist dann notwendig. Uber das Fehlen jeder Aberration bei terrestrischen Richtungen habe ich nirgends eine Bemerkung gefunden, trotzdem es in vollem Widerspruch steht zu Erscheinungen, die man Die Vorschriften zur Berucksichtigung der Aberration werden durch meine Darlegungen naturgemat3 nicht beruhrt. .E Nayn. Leipzig, 1920 Jan. zy. Leipzig, 1920 Jan. zy. Die jahrlichen Refraktionsanderungen in Odessa und Pulkovo. 2. Beobachtungen mit dem Ertelschen Kreise im Me- ridiansaale, 2. Beobachtungen mit dem Ertelschen Kreise im Me- ridiansaale, Die in Odessa 1908-1910 mit dem Repsoldschen Vertikalkreise ausgefuhrten Deklinationsbeobachtungen fuhren zu einer ausgesprochenen Jahresperiode der Refraktionskon- stante. Die empirisch ohne jegliche Voraussetzungen uber den Ausdehnungskoeffizienten der Luft oder uber den ana- lytischen Ausdruck der Refraktionsanderungen erhaltene Jahreskurve (siehe Publ. d. 1'Obs. Centr. Nic. XXIV) kann durch folgende Sinussoide gut dargestellt werden : 3. Beobachtungen mit dem Ertelschen Kreise in der Siidsternwarte. 3. Beobachtungen mit dem Ertelschen Kreise in der Siidsternwarte. Aus den Beobachtuegen sollten gleichzeitig folgende Grohen abgeleitet werden : die Korrektion des angewandten Ausdehnungskoeffizienten der Luft (m,= 0.003689), die fur die Refraktion mafigebende Lufttemperatur (to) als Funktion der Temperatur ram Instrument (ti) und in freier T,uft (ta), der Einflui3 der Feuchtigkeit auf die Refraktion, die Ko- effizienten der als Sinussoide angenomrnenen Jahresperiode der Refraktion. Ioo(p-pFLO)/,uO = -0!~04sina-0~14 cosa ~ o o ( p - p ~ ) / p ~ = -or01 sinac-oYIocosa f o . 0 2 -+0.*03 Bei der Bearbeitung der Deklinationsbeobachtungen in Pulkovo 19 I 1-16 habe ich die Untersuchung der jahrlichen Refraktionsaidderungen wieder aufgenommen. Diese uber I 3 ooo Beobachtungen von zum Teil tief kulminierenden Sternen enthaltende Reihe ist zu derartigen Untersuchungen besonders geeignet, weil mit zwei Instrumenten (dem Repsold- schen und dem Ertelschen Vertikalkreis) und in zwei sehr verschiedenen Beobachtungsraumen (Pulkovoer grofier Meri- diansaal und der Westturm der Sudsternwarte) beobachtet worden ist. I In jeder der 3 obengenannten Gruppen wurde aus den Beobachtungen je eines Katalogsternes eine Gleichung ge- bildet. Auf diese Weise. wurden 6 Beobachtungen von jedem Sterne verwertet. In solchen Fallen, wo mehrere Beobach- tungen von dem Sterne vorhanden waren, wurden die beiden gewahlt, die in nioglichst verschiedenen Temperaturen beob- achtet worden waren Von den tief kulminierenden Sternen, die speziell fur Refraktionsuntersuchung beobachtet worden sind, wurden alle Beobachtungen berucksichtigt. Die Gewichtseinheit entspricht dem mittleren Fehler =t oY42 eines Unterschieds Az. Wir teilen die Untersuchung in drei Gruppen: I . Beobachtungen mit dem Repsoldschen Kreise im Meridiansaale, Die Koeffizienten der Normalgleicbungen fur die Be- stimmung voni R, i, p, a und b sind folgende:
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Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitor Dapagliflozinstabilizes Diabetes-Induced Atherosclerotic Plaque Instability
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Methodology Sodium glucose co-transporter 2 (SGLT2) inhibitor dapagliflozin stabilizes diabetes-induced atherosclerotic plaque instability Yung Chih Chen1,2,3, Karin Jandeleit-Dahm3,4,5*, Karlheinz Peter1,2,3,6* 1Baker Heart & Diabetes Institute, Melbourne, Australia 2Central Clinical School, Monash University, Melbourne, Australia 3Department of Cardiomentabolic Health, University of Melbourne, Melbourne, Australia 4Department of Diabetes, Monash University, Melbourne, Australia 5German Diabetes Centre, Leibniz Centre for Diabetes Research at Heinrich Heine University, Duesseldorf, Germany 6Department of Cardiology, The Alfre Hospital, Melbourne, Australia *equally contributing senior authors Corresponding authors: Karlheinz Peter, MD PhD Atherothrombosis & Vascular Biology, Baker Heart and Diabetes Institute PO Box 6492, St Kilda Road Central Melbourne, VIC 8008, Australia Phone: +613 8532 1490; Fax: +613 8532 1100 Email: karlheinz.peter@baker.edu.au Karin Jandeleit-Dahm, MD, PhD Department of Diabetes Results: Diabetic mice showed an increase in the size of unstable atherosclerotic plaques in the TS model. Plaque instability markers such as MCP-1, CD68, and necrotic core (NC) size were significantly increased. Mice treated with the sodium glucose co-transporter 2i (SGLT2i) dapagliflozin demonstrated attenuated glucose levels. Importantly, these mice demonstrated plaque stabilization with enhanced collagen accumulation, increased fibrosis, increased cap- to-lesion ratios, and significant upregulation of plaque NADPH oxidase 4 (NOX4) expression. Aims/hypothesis: Diabetes is known to accelerate the progression of atherosclerosis and increase plaque instability. However, there has been a lack of suitable animal models to study the effect of diabetes on plaque instability. We hypothesized that the tandem stenosis (TS) mouse model, which reflects plaque instability and rupture as seen in patients, can be applied to study the effects of diabetes and its respective therapeutic approaches on plaque instability/rupture. Methods: ApoE-/- mice at 7 weeks of age were injected with streptozotocin (STZ) for 5 consecutive days. 5 weeks after STZ injection, mice were surgically subjected to TS in the right carotid artery and fed with a high-fat diet for an additional 7 weeks. To validate this newly developed animal model, administration of the interventional drug dapagliflozin was provided via drinking water (25 mg/kg) 3 days after TS surgery. Conclusions/interpretation: The TS mouse model in combination with the application of STZ represents a highly suitable and unique mouse model for studying plaque destabilization under diabetic conditions. Furthermore, for the first time, we provide evidence of plaque-stabilizing effects of SGLT2i. Our data also suggest that this newly developed mouse model is an attractive preclinical tool for testing anti-diabetic drugs for their highly sought-after potential to stabilize atherosclerotic plaques. Keyword: Animal model, Streptozotocin; Sodium glucose co-transporter 2 inhibitor; Plaque instability. Background Myocardial infarction (MI) is a major cause of mortality/morbidity in patients with diabetes mellitus and typically atherosclerosis is the underlying pathology [1]. Diabetes has been postulated to accelerate the development of atherosclerosis and increase plaque instability/vulnerability, ultimately causing plaque rupture and MI. Therapeutic approaches to stabilizing vulnerable plaques in diabetic patients are thus highly sought-after. Until recently there was no conclusive proof that any of the antidiabetic drugs provided cardiovascular protection. However, the advent of new antidiabetic agents, such as the sodium glucose co- transporter 2 inhibitor (SGLT2i) and GLP-1 receptor agonists, has substantially changed this scenario, adding certain antidiabetic drugs to the standard repertoire of cardiologists. This paradigm shift is based on several randomized, controlled clinical trials demonstrating that SGLT-2 inhibitors confer beneficial effects on cardiovascular outcomes in patients with type 2 diabetes mellitus [2]. There is clinical evidence that SGLT2i protects against heart failure; however, although postulated as a mechanism, there is no direct clinical proof of anti- atherosclerotic and/or plaque-stabilizing effects of SGLT2i. We have previously validated a unique mouse model of plaque instability/rupture by introducing a tandem stenosis (TS) to the carotid artery [3]. This TS model closely reflects plaque instability/rupture as seen in patients and has recently been successfully used to define the plaque-stabilizing effects of a myeloperoxidase inhibitor [4] and anti-CD47 antibodies [5]. We now systematically use this model to investigate whether diabetes promotes plaque instability and, most importantly, whether SGLT2i exhibits plaque-stabilizing effects in this newly developed animal model. Animal studies ApoE-/- mice of C57/Bl6 background (Australia Resource Centre, Western Australia) at 7 weeks of age were injected with streptozotocin (STZ) at a dose of 55 mg/kg for 5 consecutive days. At 12 weeks of age, 5 weeks after STZ, mice were anesthetized intraperitoneally by a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg). An incision was made in the neck and the right common carotid artery was dissected from circumferential connective tissues. TS with 150 µm outer diameters was introduced, with the distal point 1 mm from the carotid artery bifurcation and the proximal point 3 mm from the distal stenosis. The stenosis diameter was obtained by placing a 6-0 blue braided polyester fiber suture around the carotid artery together with a 150 µm needle that was tied to it and later removed as described previously [3, 4]. Animals were sacrificed at 7 weeks after surgery. Histology and immunohistochemistry Histology and immunohistochemistry The left and right common carotid arteries and brachiocephalic trunk were sectioned as described previously [3]. Serial cryosections were obtained at 100 µm intervals at 1,500– 2,000 µm proximal to the proximal suture in the right common carotid artery and from the brachiocephalic trunk and stored at −80 °C until staining was performed. Picrosirius red staining was performed to highlight collagen within the atherosclerotic plaque. Hematoxylin and eosin (H&E) staining was used for necrotic core determination and atherosclerotic plaque burden. Oil red o staining was used for lipid content quantification in the plaque. Images were obtained using a light microscope (Olympus BX50). Sections were incubated with rat anti- mouse TER-119 biotin-labeled antibody (eBioscience 13-5921) 1:400; rat anti-mouse CD68 (AbD Serotech MCA1957GA) 1:100 or MOMA-2 (AbD Serotech MCA519G); rabbit polyclonal anti-NOX4 (Sigma-Aldrich ABC459) 1:100; and anti-monocyte chemoattractant protein-1 antibody (Abcam ab7202) 1:200 at 4 °C overnight. After washing steps and incubation in ABC reagent, DAB reagent was applied to each section. Conversion of the DAB substrate into a colored product was monitored and slides were immersed in dH2O to stop the reaction. Sections were dehydrated and covered with coverslips using DPX mounting media. Quantification of images was performed on Fiji Image J[6]. polyclonal anti-NOX4 (Sigma-Aldrich ABC459) 1:100; and anti-monocyte chemoattractant protein-1 antibody (Abcam ab7202) 1:200 at 4 °C overnight. After washing steps and incubation in ABC reagent, DAB reagent was applied to each section. Conversion of the DAB substrate into a colored product was monitored and slides were immersed in dH2O to stop the reaction. Sections were dehydrated and covered with coverslips using DPX mounting media. Quantification of images was performed on Fiji Image J[6]. Statistical analyses Quantitative results are expressed as mean±SEM. Comparisons of parameters among two groups were made by the unpaired Student’s t-test or Mann–Whitney test. Comparisons of table statistics were made by Fisher's exact test. Comparisons of parameters among three groups were made by one-way ANOVA followed by post hoc analysis using the Newman– Keuls test. A value of P<0.05 was considered to be statistically significant. Results We induced insulin-deficient diabetes using the beta-cell toxin STZ in ApoE-/- mice on a high- fat diet who underwent TS surgery after confirming diabetes. The diabetic mice showed significantly elevated glucose and glycated hemoglobin levels (Figure 1A, 1B). Under diabetic conditions, the locally defined unstable atherosclerotic plaque area in the TS model showed a significant increase (Figure 1C). Importantly, several typical markers of plaque instability, such as the monocyte chemoattractant protein-1 (MCP-1; Figure 1D), macrophages (CD68; Figure 1E), and necrotic core size (Figure 1F), were significantly increased as quantified as a percentage of plaque area. As a direct proof of plaque instability, intraplaque hemorrhage (IH) increased from 52% of untreated mice to 81% in STZ-treated mice with diabetes (Figure 1G) and plaque rupture increased from 32% to 56%, respectively (Figure 1H) as measured in a total of 226 mice, whereby 116 were untreated mice with TS and 100 were STZ-treated mice with TS. These data confirm that diabetic conditions cause plaque instability and demonstrate the suitability of the TS mouse model for detecting diabetes-associated changes in plaque instability. Next, we examined the potential of the SGLT2 inhibitor dapagliflozin to stabilize plaques. Repeated STZ protocol as previously described, dapagliflozin was given to ApoE-/- mice via drinking water 3 days after TS surgery. 2 weeks after the start of dapagliflozin treatment, STZ- induced hyperglycemia was modestly attenuated (Figure 2). The effect on lowering glucose was sustained for another 6 weeks; however, normoglycemia was never achieved. We focused on investigating markers of plaque instability. Interestingly, we did not see changes in inflammatory markers such as the macrophage markers (CD68 and MOMA-2) or MCP-1 levels (data not shown). Although the strongest markers of plaque instability such as plaque rupture and IH (data not shown) were unchanged, early signs of plaque stabilization such as an overall reduction in lipid content (Figure 3A) and increases in collagen content and the cap-to-core ratio were significant in the dapagliflozin-treated group (Figure 3B, 3C). These data are of substantial translational relevance as the cap-to-core ratio is a particularly important clinical marker that reflects plaque stability and is closely linked to improved clinical outcomes in patients. Increased formation of reactive oxygen species (ROS) is associated with plaque instability in diabetes. Discussion SGLT2i has emerged as a new therapeutic target for lowering glucose. Several clinical trials have demonstrated the efficacy of the glucose lowering of SGLT-2 inhibitors (EMPA-REG OUTCOME, CANVAS, DECLARE-TIMI) in association with improved cardiovascular outcomes [8-10]. The EMPA-REG OUTCOME trial of empagliflozin demonstrated a reduction in cardiovascular outcomes and death from other causes [10]. Nevertheless, direct evidence that SGLT2i stabilizes atherosclerotic plaques is still lacking, partly due to the fact that animal models of atherosclerosis typically do not exhibit plaque instability and rupture [11]. Currently, only a few experimental studies have investigated the effect of SGLT2i in classical mouse models of atherosclerosis. Al-Sharea et al. treated LDLR-/- mice on a high-fat diet and after STZ application with dapagliflozin and reported a reduction in peripheral monocyte count and a lower degree of atherosclerosis, postulating a change in plasma lipoproteins as the major cause [12]. In ApoE-/- mice on a high-fat diet, empagliflozin reduced blood glucose, aortic plaque area, insulin resistance, and inflammation[13]. These studies provide the first experimental indication that SGLT2i exhibits anti-atherosclerotic effects. Our studies go beyond and demonstrate the plaque-stabilizing capability of SGLT2i and suggest that changes in the pro-oxidant enzyme NOX4 are mechanistically involved in plaque stabilization. Results NADPH oxidases, particularly increased expression of NADPH oxidase1 (NOX1) and reduced expression of the vasculoprotective NOX4 isoform in advanced plaques, have been shown to contribute to plaque development and remodeling in diabetes [7]. Interestingly, while expression of pro-atherosclerotic NOX-1 (data not shown) was not upregulated, the expression of anti-atherosclerotic NOX-4 (Figure 3D) was upregulated in the dapagliflozin group. Therefore, the regulation of certain NOX isoforms by SGLT2i therapy represents a potential mechanistic explanation for the beneficial effect seen in patients treated with SGLT2i. Consent for publication Not applicable Authors' contributions YCC collected and analysed and interpreted the data. YCC performed the histological examination of the atherosclerotic plaques. YCC, KP, KJD co-designed the animal experiment and discussed the data, and jointly wrote the manuscript. All authors have read and have approved the final manuscript. Funding Yung-Chih Chen is supported by the Heart Foundation Future leader fellowship (No:102068). Karlheinz Peter is supported by an NHMRC L3 investigator fellowship. The work was partially supported by an NHMRC project grant (APP1163233, K Jandeleit-Dahm). Ethics approval and consent to participate All experimental protocols and animal experiments have been approved by the Animal Ethics Committees of the Alfred Research Alliance approved number (E/1838/2018/B). Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding authors on reasonable request. Conclusion In conclusion, the tandem stenosis mouse model in conjunction with STZ-induced diabetes reflects plaque destabilization as seen in patients with diabetes and importantly provides initial proof of the plaque-stabilizing effects of SGLT2i. Indeed, these effects on plaque stability could explain at least in part the reduction of cardiovascular events seen in diabetic patients treated with SGLT2i and as such bears direct translational relevance. Our data also establish the TS mouse model in combination with STZ-treatment as a preclinical tool for testing of anti- diabetic drugs for highly sought-after plaque-stabilizing effects. proof of the plaque-stabilizing effects of SGLT2i. Indeed, these effects on plaque stability could explain at least in part the reduction of cardiovascular events seen in diabetic patients treated with SGLT2i and as such bears direct translational relevance. Our data also establish the TS mouse model in combination with STZ-treatment as a preclinical tool for testing of anti- diabetic drugs for highly sought-after plaque-stabilizing effects. Competing interests All authors declare no competing interests. Acknowledgments Not applicable Not applicable Figures Figures Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of the TS model. Quantification was performed on H&E stained sections, where the plaque was measured from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per mouse at 200 µm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2 sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quantified as described in D. (F) Unstable carotid plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major characteristic of plaque vulnerability in Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels c Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of the TS model. Quantification was performed on H&E stained sections, where the plaque was measured from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per mouse at 200 µm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2 sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quantified as described in D. (F) Unstable carotid plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Not applicable Quantification was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic mice. IH was defined by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Plaque rupture (PR) was increased in diabetic mice. PR was defined by visible defects of the fibrous cap with or without luminal thrombi. PR in the TS and TS+STZ groups were 25 out of 96 and 48 out of 87, respectively. Non-PR in the TS and TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact test. All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t-test. Figure 2: The SGLT2 inhibitor dapagliflozin reduces hyperglycemia in TS mice treated with STZ. Dapagliflozin was given via drinking water at 25 mg/kg after TS. 2 weeks after the initiation of daily dapagliflozin, glucose levels were significantly reduced to 24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice 28.61±1.13 mmol/L and remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t-test. ure 2: The SGLT2 inhibitor dapagliflozin reduces hyperglycemia in TS mice treate with STZ. Dapagliflozin was given via drinking water at 25 mg/kg after TS. 2 weeks after the initiation of daily dapagliflozin, glucose levels were significantly reduced to 24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice 28.61±1.13 mmol/L and remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t-test. Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the collagen in the unstable atherosclerotic plaque area in the TS model. Oil Red O staining and Picrosirius red staining were used to quantify the total lipid and collagen content, respectively. (C) Diabetes significantly reduces the cap-to-core ratio, whereas dapagliflozin restores it, indicating a more stable plaque phenotype. The cap-to-core ratio was determined by dividing the total fibrous cap area by the maximal lesion height in every cross-section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple comparisons test. (D) Dapagliflozin increases NOX4 expression in unstable plaques of the TS model. The NOX4 expression in plaques was investigated by immunohistochemistry and each data point represents the mean of 2 sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test. Not applicable Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases th Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the collagen in the unstable atherosclerotic plaque area in the TS model. Oil Red O staining and Picrosirius red staining were used to quantify the total lipid and collagen content, respectively. (C) Diabetes significantly reduces the cap-to-core ratio, whereas dapagliflozin restores it, indicating a more stable plaque phenotype. The cap-to-core ratio was determined by dividing the total fibrous cap area by the maximal lesion height in every cross-section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple comparisons test. (D) Dapagliflozin increases NOX4 expression in unstable plaques of the TS d l Th NOX4 i i l i i d b i hi h i d Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the TS model. The NOX4 expression in plaques was investigated by immunohistochemistry and each data point represents the mean of 2 sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test. References [1] Cavallari I, Bhatt DL, Steg PG, et al. (2021) Causes and Risk Factors for Death in Diabetes: A Competing-Risk Analysis From the SAVOR-TIMI 53 Trial. J Am Coll Cardiol 77(14): 1837-1840. 10.1016/j.jacc.2021.02.030 [1] Cavallari I, Bhatt DL, Steg PG, et al. (2021) Causes and Risk Factors for Death in Diabetes: A Competing-Risk Analysis From the SAVOR-TIMI 53 Trial. J Am Coll Cardiol 77(14): 1837-1840. 10.1016/j.jacc.2021.02.030 ( ) j j [2] Furtado RHM, Bonaca MP, Raz I, et al. (2019) Dapagliflozin and Cardiovascular Outcomes in Patients With Type 2 Diabetes Mellitus and Previous Myocardial Infarction. Circulation 139(22): 2516-2527. 10.1161/CIRCULATIONAHA.119.039996 [3] Chen YC, Bui AV, Diesch J, et al. (2013) A Novel Mouse Model of Atherosclerotic Plaque Instability for Drug Testing and Mechanistic/Therapeutic Discoveries Using Gene and MicroRNA Expression Profiling. Circ Res 113(3): 252-265. [2] Furtado RHM, Bonaca MP, Raz I, et al. (2019) Dapagliflozin and Cardiovascular Outcomes in Patients With Type 2 Diabetes Mellitus and Previous Myocardial Infarction. Circulation 139(22): 2516-2527. 10.1161/CIRCULATIONAHA.119.039996 [3] Chen YC, Bui AV, Diesch J, et al. (2013) A Novel Mouse Model of Atherosclerotic Plaque Instability for Drug Testing and Mechanistic/Therapeutic Discoveries Using Gene and MicroRNA Expression Profiling. Circ Res 113(3): 252-265. Circulation 139(22): 2516-2527. 10.1161/CIRCULATIONAHA.119.039996 [3] Chen YC, Bui AV, Diesch J, et al. (2013) A Novel Mouse Model of Atherosclerotic Plaque Instability for Drug Testing and Mechanistic/Therapeutic Discoveries Using Gene and MicroRNA Expression Profiling. Circ Res 113(3): 252-265. [4] Rashid I, Maghzal GJ, Chen YC, et al. (2018) Myeloperoxidase is a potential molecular imaging and therapeutic target for the identification and stabilization of high-risk atherosclerotic plaque. Eur Heart J 39(35): 3301-3310. 10.1093/eurheartj/ehy419 molecular imaging and therapeutic target for the identification and stabilization of high-risk atherosclerotic plaque. Eur Heart J 39(35): 3301-3310. 10.1093/eurheartj/ehy419 [5] Kojima Y, Volkmer JP, McKenna K, et al. (2016) CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature 536(7614): 86-90. 10.1038/nature18935 [6] Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675. 10.1038/nmeth.2089 atherosclerotic plaque. Eur Heart J 39(35): 3301 3310. 10.1093/eurheartj/ehy419 [5] Kojima Y, Volkmer JP, McKenna K, et al. (2016) CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature 536(7614): 86-90. References 10.1038/nature18935 [6] Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of i l i N t M th d 9(7) 671 675 10 1038/ th 2089 p q j y [5] Kojima Y, Volkmer JP, McKenna K, et al. (2016) CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature 536(7614): 86-90. 10.1038/nature18935 [6] Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675. 10.1038/nmeth.2089 [7] Gray SP, Di Marco E, Kennedy K, et al. (2016) Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling. Arterioscler Thromb Vasc Biol 36(2): 295-307. [8] Wiviott SD, Raz I, Bonaca MP, et al. (2019) Dapagliflozin and Cardiovascular Outcomes in Type 2 Diabetes. N Engl J Med 380(4): 347-357. 10.1056/NEJMoa1812389 [9] Neal B, Perkovic V, Mahaffey KW, et al. (2017) Canagliflozin and Cardiovascular [8] Wiviott SD, Raz I, Bonaca MP, et al. (2019) Dapagliflozin and Cardiovascular Outcomes in Type 2 Diabetes. N Engl J Med 380(4): 347-357. 10.1056/NEJMoa1812389 [9] Neal B, Perkovic V, Mahaffey KW, et al. (2017) Canagliflozin and Cardiovascular and Renal Events in Type 2 Diabetes. N Engl J Med 377(7): 644-657. 10.1056/NEJMoa1611925 [10] Zinman B, Wanner C, Lachin JM, et al. (2015) Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N Engl J Med 373(22): 2117-2128. 10.1056/NEJMoa1504720 [11] Getz GS, Reardon CA (2012) Animal models of atherosclerosis. Arterioscler Thromb Vasc Biol 32(5): 1104-1115. 10.1161/ATVBAHA.111.237693 [12] Al-Sharea A, Murphy AJ, Huggins LA, Hu Y, Goldberg IJ, Nagareddy PR (2018) SGLT2 inhibition reduces atherosclerosis by enhancing lipoprotein clearance in Ldlr(-/-) type 1 diabetic mice. Atherosclerosis 271: 166-176. 10.1016/j.atherosclerosis.2018.02.028 [13] Han JH, Oh TJ, Lee G, et al. (2017) The beneficial effects of empagliflozin, an SGLT2 inhibitor, on atherosclerosis in ApoE (-/-) mice fed a western diet. Diabetologia 60(2) 364 376 10 1007/ 00125 016 4158 2 [11] Getz GS, Reardon CA (2012) Animal models of atherosclerosis. Arterioscler Thromb Vasc Biol 32(5): 1104-1115. 10.1161/ATVBAHA.111.237693 [12] Al-Sharea A, Murphy AJ, Huggins LA, Hu Y, Goldberg IJ, Nagareddy PR (2018) SGLT2 inhibition reduces atherosclerosis by enhancing lipoprotein clearance in Ldlr(-/-) type 1 diabetic mice. Atherosclerosis 271: 166-176. 10.1016/j.atherosclerosis.2018.02.028 [13] Han JH, Oh TJ, Lee G, et al. (2017) The beneficial effects of empagliflozin, an SGLT2 inhibitor, on atherosclerosis in ApoE (-/-) mice fed a western diet. Diabetologia 60(2): 364-376. References 10.1007/s00125-016-4158-2 Figures Figures Figure 1 (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS m (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid arter the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measu from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections pe mouse at 200 μm intervals (D) MCP-1 is upregulated in diabetic mice with TS The percentage of p Figure 1 (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measured from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per mouse at 200 μm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2 sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quanti¦ed as described in D. (F) Unstable carotid plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Figure 1 (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measured from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per mouse at 200 μm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2 sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quanti¦ed as described in D. (F) Unstable carotid plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measured from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per mouse at 200 μm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2 sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quanti¦ed as described in D. Figure 1 All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t- test. Plaque rupture (PR) was increased in diabetic mice. PR was de¦ned by visible defects of the ¦brous cap with or without luminal thrombi. PR in the TS and TS+STZ groups were 25 out of 96 and 48 out of 87, respectively. Non-PR in the TS and TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact test. All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t- test. Figure 2 The SGLT2 inhibitor dapagli§ozin reduces hyperglycemia in TS mice treated with STZ. Dapagli§ozin was given via drinking water at 25 mg/kg after TS. 2 weeks after the initiation of daily dapagli§ozin, glucose levels were signi¦cantly reduced to 24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice 28.61±1.13 mmol/L and remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t- test. Figure 1 (F) Unstable carotid plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Plaque rupture (PR) was increased in diabetic mice. PR was de¦ned by visible defects of the ¦brous cap with or without luminal thrombi. PR in the TS and TS+STZ groups were 25 out of 96 and 48 out of 87, respectively. Non-PR in the TS and TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact test. All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t- test. Plaque rupture (PR) was increased in diabetic mice. PR was de¦ned by visible defects of the ¦brous cap with or without luminal thrombi. PR in the TS and TS+STZ groups were 25 out of 96 and 48 out of 87, respectively. Non-PR in the TS and TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact test. Figure 2 The SGLT2 inhibitor dapagli§ozin reduces hyperglycemia in TS mice treated with STZ. Dapagli§ozin was given via drinking water at 25 mg/kg after TS. 2 weeks after the initiation of daily dapagli§ozin, glucose levels were signi¦cantly reduced to 24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice 28.61±1.13 mmol/L and remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t- test. Figure 3 Figure 3 (A) Dapagli§ozin signi¦cantly reduces the lipid content and (B) increases the collagen in the unstable atherosclerotic plaque area in the TS model. Oil Red O staining and Picrosirius red staining were used to quantify the total lipid and collagen content, respectively. (C) Diabetes signi¦cantly reduces the cap-to- core ratio, whereas dapagli§ozin restores it, indicating a more stable plaque phenotype. The cap-to-core ratio was determined by dividing the total ¦brous cap area by the maximal lesion height in every cross- section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple comparisons test. (D) Dapagli§ozin increases NOX4 expression in unstable plaques of the TS model. The NOX4 expression in plaques was investigated by immunohistochemistry and each data point represents the mean of 2 sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test. (A) Dapagli§ozin signi¦cantly reduces the lipid content and (B) increases the collagen in the unstable atherosclerotic plaque area in the TS model. Oil Red O staining and Picrosirius red staining were used to quantify the total lipid and collagen content, respectively. (C) Diabetes signi¦cantly reduces the cap-to- core ratio, whereas dapagli§ozin restores it, indicating a more stable plaque phenotype. The cap-to-core ratio was determined by dividing the total ¦brous cap area by the maximal lesion height in every cross- section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple comparisons test. (D) Dapagli§ozin increases NOX4 expression in unstable plaques of the TS model. The NOX4 expression in plaques was investigated by immunohistochemistry and each data point represents the mean of 2 sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test. Dapagli§ozin increases NOX4 expression in unstable plaques of the TS model. The NOX4 expression in plaques was investigated by immunohistochemistry and each data point represents the mean of 2 sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test.
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Employing plant functional groups to advance seed dispersal ecology and conservation
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Employing plant functional groups to advance seed dispersal ecology and conservation Lowell Street, Tucson, AZ 85721, USA 5Dynamic Macroecology, Landscape Dynamics, Swiss Federal Research Institute WSL, Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland 6Faculty of Biology and Pre-Clinical Medicine, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany 7Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA 8Department of Fish, Wildlife and Conservation Biology, Colorado State University, 1474 Campus Delivery, Fort Collins, CO 80523, USA 9Biology Department Woods Hole Oceanographic Institution 266 Woods Hole Road Woods Hole MA 02543 USA Faculty of Biology and Pre Clinical Medicine, University of Regensburg, Universitätsstraße 31, D 93053 Regensburg, Germany 7Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA gy p , g p , , , , 10Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA y p g p las School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA gy p g p 10Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA 11Department of Biology, University of Washington, Seattle, WA 98195, USA epartment of Biology, University of Washington, Seatt 12Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 318 W 12th Avenue, Columbus, OH 43210, USA 12Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 318 W 12th Aven USA rtment of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA C i i C l b Ni i 13Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA 14CRS F C i i C l b Ni i 13Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA 14CRS Forestry Commission, Calabar, Nigeria Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA 14CRS Forestry Commission, Calabar, Nigeria 4CRS Forestry Commission, Calabar, Nigeria y g 15National Socio-Environmental Synthesis Center, 1 Park Place, Annapolis, MD 21401, USA y p andschaftsökologie, Albert-Ludwigs-Universität Freiburg, Tennenbacher Str. 4, D-79106 Freiburg, Germany 16Naturschutz & Landschaftsökologie, Albert-Ludwigs-Universität Freiburg, Tennenbacher Str. 4, D-79106 Freiburg, Germany 17Department of Geography, Texas A&M University, College Station, TX 77843, USA 18Centre for Ecology & Hydrology, Wallingford, UK 19Wildlife Biology Program, University of Montana, 32 Campus Drive, Missoula, MT 59812, USA 20Department of Mathematics, University of Miami, 1365 Memorial Drive, Coral Gables, FL 33146, USA 21University of California-Davis, Davis, CA 95616, USA 22Department of Mathematics, Howard University, Washington, DC 20059, USA 23Physics Department, California Polytechnic State University, 1 Grand Avenue, San Luis Obispo, CA 93407, USA 24Department of Biology, College of Charleston, Charleston, SC 29424, USA 25Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269, USA 26Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA 27Department of Mathematics, University of Tennessee, Knoxville, TN 37996, USA 28Department of Mathematics, Lafayette College, 730 High Street, Easton, PA 18042, USA Received: 15 August 2018  Editorial decision: 24 January 2019  Accepted: 5 February 2019  Published: 7 February 2019 Associate Editor: Kim McConkey Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Point of View SPECIAL ISSUE: The Role of Seed Dispersal in Plant Populations: Perspectives and Advances in a Changing World Employing plant functional groups to advance seed dispersal ecology and conservation Clare Aslan1*, Noelle G. Beckman2, Haldre S. Rogers3, Judie Bronstein4, Damaris Zurell5, Florian Hartig6, Katriona Shea7, Liba Pejchar8, Mike Neubert9, John Poulsen10, Janneke HilleRisLambers11, Maria Miriti12, Bette Loiselle13, Edu Effiom14, Jenny Zambrano15, Geno Schupp2, Gesine Pufal16, Jeremy Johnson17, James M. Bullock18, Jedediah Brodie19, Emilio Bruna13, Robert Stephen Cantrell20, Robin Decker21, Evan Fricke3, Katie Gurski22, Alan Hastings21, Oleg Kogan23, Onja Razafindratsima24, Manette Sandor25, Sebastian Schreiber21, Rebecca Snell26, Christopher Strickland27 and Ying Zhou28 1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA 2Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA 3Department of Ecology, Evolution, and Organismal Biology, Iowa State University, 251 Bessey Hall, Ames, IA 50011, USA 4Department of Ecology and Evolutionary Biology, University of Arizona, 1041 E. Lowell Street, Tucson, AZ 85721, USA 5Dynamic Macroecology, Landscape Dynamics, Swiss Federal Research Institute WSL, Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland 6Faculty of Biology and Pre-Clinical Medicine, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany 7Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA 8Department of Fish, Wildlife and Conservation Biology, Colorado State University, 1474 Campus Delivery, Fort Collins, CO 80523, USA 9Biology Department, Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, MA 02543, USA 10Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA 11Department of Biology, University of Washington, Seattle, WA 98195, USA 12Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 318 W 12th Avenue, Columbus, OH 43210, USA 13Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA 14CRS Forestry Commission, Calabar, Nigeria 15National Socio-Environmental Synthesis Center, 1 Park Place, Annapolis, MD 21401, USA 16Naturschutz & Landschaftsökologie, Albert-Ludwigs-Universität Freiburg, Tennenbacher Str. Employing plant functional groups to advance seed dispersal ecology and conservation Clare Aslan1*, Noelle G. Beckman2, Haldre S. Rogers3, Judie Bronstein4, Damaris Zurell5, Florian Hartig6, Katriona Shea7, Liba Pejchar8, Mike Neubert9, John Poulsen10, Janneke HilleRisLambers11, Maria Miriti12, Bette Loiselle13, Edu Effiom14, Jenny Zambrano15, Geno Schupp2, Gesine Pufal16, Jeremy Johnson17, James M. Bullock18, Jedediah Brodie19, Emilio Bruna13, Robert Stephen Cantrell20, Robin Decker21, Evan Fricke3, Katie Gurski22, Alan Hastings21, Oleg Kogan23, Onja Razafindratsima24, Manette Sandor25, Sebastian Schreiber21, Rebecca Snell26, Christopher Strickland27 and Ying Zhou28 1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA 1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA 2Department of Biology Utah State University 5305 Old Main Hill Logan UT 84322 USA 1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA 2Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA p , y, g , , 2Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA 3Department of Ecology, Evolution, and Organismal Biology, Iowa State University, 251 Bessey Hall, Ames, IA 50011, USA 4Department of Ecology and Evolutionary Biology, University of Arizona, 1041 E. Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Citation: Aslan C, Beckman NG, Rogers HS, Bronstein J, Zurell D, Hartig F, Shea K, Pejchar L, Neubert M, Poulsen J, HilleRisLambers J, Miriti M, Loiselle B, Effiom E, Zambrano J, Schupp G, Pufal G, Johnson J, Bullock JM, Brodie J, Bruna E, Cantrell RS, Decker R, Fricke E, Gurski K, Hastings A, Kogan O, Razafindratsima O, Sandor M, Schreiber S, Snell R, Strickland C, Zhou Y. 2019. Employing plant functional groups to advance seed dispersal ecology and conservation. AoB PLANTS 11: plz006; doi: 10.1093/aobpla/plz006 Abstract. Seed dispersal enables plants to reach hospitable germination sites and escape natural enemies. Understanding when and how much seed dispersal matters to plant fitness is critical for understanding plant population and community dynamics. At the same time, the complexity of factors that determine if a seed will be successfully dispersed and subsequently develop into a reproductive plant is daunting. Quantifying all fac- tors that may influence seed dispersal effectiveness for any potential seed-vector relationship would require an unrealistically large amount of time, materials and financial resources. On the other hand, being able to make dispersal predictions is critical for predicting whether single species and entire ecosystems will be resilient to global change. Building on current frameworks, we here posit that seed dispersal ecology should adopt plant functional groups as analytical units to reduce this complexity to manageable levels. Functional groups can be used to distinguish, for their constituent species, whether it matters (i) if seeds are dispersed, (ii) into what context they are dispersed and (iii) what vectors disperse them. To avoid overgeneralization, we propose that the utility of these functional groups may be assessed by generating predictions based on the groups and then testing those predictions against species-specific data. We suggest that data collection and analysis can then be guided by robust functional group definitions. Generalizing across similar species in this way could help us to better understand the population and community dynamics of plants and tackle the complexity of seed dis- persal as well as its disruption. Keywords:  dependency; directed dispersal; dispersal vectors; generalization; mutualism; seed dispersal effectiveness. Employing plant functional groups to advance seed dispersal ecology and conservation 4, D-7 17Department of Geography, Texas A&M University, College Station, TX 77843, USA 17Department of Geography, Texas A&M University, College Station, TX 77843, USA 7Department of Geography, Texas A&M University, Co 18Centre for Ecology & Hydrology, Wallingford, UK 19Wildlife Biology Program, University of Montana, 32 Campus Drive, Missoula, MT 59812, USA 19Wildlife Biology Program, University of Montana, 32 Campus Drive, Missoula, MT 59812, USA 19Wildlife Biology Program, University of Montana, 32 gy g y p 20Department of Mathematics, University of Miami, 1365 Memorial Drive, Coral Gables, FL 33146, USA 0Department of Mathematics, University of Miami, 13 20Department of Mathematics, University of Miami, 1365 Memorial Drive, Coral Gab 21University of California-Davis, Davis, CA 95616, USA 22Department of Mathematics, Howard University, Washington, DC 20059, USA 22Department of Mathematics, Howard University, Washington, DC 20059, USA 22Department of Mathematics, Howard University, Wa 22Department of Mathematics, Howard University, Washington, DC 20059, USA 24Department of Biology, College of Charleston, Charleston, SC 29424, USA 24Department of Biology, College of Charleston, Charleston, SC 29424, USA 25Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269, USA 25Department of Ecology and Evolutionary Biology, University of Connecticut, Stor 26Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA 26Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA gy y 27Department of Mathematics, University of Tennessee, Knoxville, TN 37996, USA 27Department of Mathematics, University of Tennessee, Knoxville, TN 37996, USA 28Department of Mathematics, Lafayette College, 730 High Street, Easton, PA 18042, USA Received: 15 August 2018  Editorial decision: 24 January 2019  Accepted: 5 February 2019  Published: 7 February 2019 Associate Editor: Kim McConkey *Corresponding author’s e-mail address: clare.aslan@nau.edu This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommo licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work i © The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. 1 © The Author(s) 2019 AoB PLANTS  https://academic.oup.com/aobpla Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Introduction: Seed Dispersal Is Fundamental to Populations and Communities, Yet Complex – Defining functional groups relevant to the importance of seed dispersal behaviours and physiology of biotic dispersers amplify the complexity of seed dispersal. Seed handling, for example, can affect the condition of the seed and change the likelihood of germination and subsequent survival and growth after seed deposition (Ladley and Kelly 1996; Traveset and Verdú 2002; Fricke et al. 2013). Some plant species exhibit extreme specialization in micro- habitats and require dispersers to move seeds to these locations (e.g. desert mistletoe requires dispersal to the branches of a very limited range of host trees; Aukema 2004). The preferences and physiology of dispersers may influence the direction and distance of seed dispersal (Beckman and Rogers 2013) (Fig. 1). Stochastic events may include rare, long-distance dispersal events, which are difficult to observe and measure but can be critical for colonization of new geographic regions and provide connectivity among habitat patches across a landscape (Muller-Landau et  al. 2003; Jordano et  al. 2007; Shea 2007; Auffret et  al. 2017). Behavioral aspects of biotic dispersers, such as local aggregation, social organiza- tion, mating system, competition and territoriality, can influence both spatial and temporal dispersal of seeds, with potential ramifications for seed aggregation and competition between seeds (reviewed in Karubian and Durães 2009). A given disperser may also disperse seeds of certain shapes or sizes, depending on disperser body or gape sizes (McConkey and Drake 2006; Muñoz et al. 2017). An extensive literature has explored the dispersal syndromes, or seed and fruit traits (e.g. size, shape, col- our, chemistry, dormancy) that appear predictive of the primary dispersers of a given plant species, with investi- gation into the roles of co-evolution, secondary disper- sal and specialization (e.g. Vander Wall and Beck 2012; much it matters by what vector they are dispersed. We list such functional groups and discuss their potential value in achieving general insights. We close by consid- ering key knowledge gaps that this proposed functional group approach may address. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Introduction: Seed Dispersal Is Fundamental to Populations and Communities, Yet Complex It is evident that a quantitative understanding of dis- persal is key for predicting how environmental changes, and consequent changes in dispersal vectors, will impact plant populations and communities. Operationalizing this goal and moving seed dispersal ecology towards a predictive science, however, requires confronting a wide array of interacting factors and stochastic elements (Robledo-Arnuncio et al. 2014). Here, we discuss how a functional group approach may help simplify the com- plexity of seed dispersal ecology and boost our predic- tive capacity. Plants rely on dispersal vectors—for example, animals, wind and water—to move across the landscape. We focus here on the dispersal of seeds, although many of the arguments we make could be generalized to other forms of dispersal. Dispersal occurs when a seed is moved from its origin and deposited elsewhere (Schupp et  al. 2010). Through dispersal, plants may experience reduced exposure to competition, predation and parasitism (Janzen 1970; Connell 1971; Howe and Miriti 2004); colonize open habitats after distur- bance (Wunderle 1997; Puerta-Piñero et al. 2013); reach potential suitable microsites in otherwise unsuitable landscapes (Wenny 2001); track cli- mate fluctuations and environmental change (Corlett and Westcott 2013); and contribute to gene flow within and between populations (Bacles et  al. 2006). As a result of these processes, seed dispersal is a fundamental driver of the diversity, structure, composition and spatial arrangement of plant communities. Seed dispersal ecology thus elucidates mechanisms of species coexistence, implications of species extinctions and impacts of global environmental change. Functional group frameworks, in which species are categorized by ecological functions and the result- ing groups treated as analytical units, have helped researchers confront complexity in other ecological sub- disciplines and have been tentatively explored in seed dispersal (e.g. Dennis and Westcott 2006; Brodie et al. 2009b; Bastazini et al. 2017). However, they have not yet been developed sufficiently to link empirical patterns of seed dispersal with theoretical predictions. In this Viewpoint, we discuss the complexity of seed dispersal and the need to reach generalities about it. We propose that to better understand the importance of seed dis- persal in plant populations and communities, it would be useful to identify functional groups that distinguish plant species based on (i) how much it matters if their seeds are dispersed at all, (ii) how much it matters into what ecological context they are dispersed and (iii) how 2 © The Author(s) 2019 AoB PLANTS  https://academic.oup.com/aobpla Aslan et al. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Where and when a seed is deposited are clearly influ- enced by many sources of variability (Robledo-Arnuncio et  al. 2014). Additionally, the spatial pattern of seed deposition on the landscape can then influence subse- quent interspecific interactions (e.g. pollination, mycor- rhizal associations, competition, predation, herbivory). Such interactions are important to the fitness of the newly established plant and determine the likelihood of survival and growth, access to limiting resources, the likelihood of mortality due to natural enemies and the probability of successful reproduction (Beckman and Rogers 2013). As a result of these interactions, the re- sulting plant community may more or less closely reflect the initial template established by seed deposition. ; ) A full utilization of the SDE framework involves quanti- fying the effectiveness of dispersal for interacting pairs of seed and disperser species, taking into account pre-, mid- and post-dispersal factors that might affect seed survival and germination and the growth and fecundity of the re- sulting plant (e.g. Fig. 1). However, parameterization of SDE requires immense investment of empirical resources and includes up to 15 different measurable quantities for a given seed-disperser pair (Schupp et al. 2010). In one study, plant species were dispersed by an average of just over seven different disperser species (Aslan et al. 2013); parameterization of SDE for such a plant would therefore require a minimum of 7 × 15 = 105 separately measured parameters—a degree of complexity that would exhaust the resources of most scientific endeavours. Nevertheless, SDE has guided impressive efforts to measure subsets of these parameters, generating important insights. For example, McConkey et al. (2014) measured disperser ef- fectiveness as a combination of the percent of monitored fruit dispersed by each disperser species combined with the distance of dispersal and survival of seedlings at each distance. Nogales et al. (2017) compared the number of seeds dispersed and effect of gut treatment by reptile vs. bird frugivores in the Galápagos. González-Castro et  al. (2015) combined the number of seeds dispersed with con- dition of seeds after dispersal and seedling emergence/ survival probabilities to compare SDE for birds and lizards. As these studies illustrate, different dispersers contribute in different ways to the template constraining an even- tual plant community. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 To understand these roles across many more sites and for many more species, we require approaches that build off the SDE framework while sim- plifying the complexity inherent in biologically diverse systems. The complexity of seed dispersal Due to their complexity, seed dispersal processes are dif- ficult to quantify empirically (Fig. 1). Since the quantifica- tion of these processes forms the basis for understanding plant population and community dynamics, methods to reduce this complexity are essential. Both biotic and abiotic dispersal vectors can influence which seeds are dispersed, the risks and costs of dispersal, the spatial direction and distance that seeds travel, the probability that seeds will encounter specific microhabitats and the probability of seed aggregation (Howe and Miriti 2004; Côrtes and Uriarte 2013; Morales et al. 2013). For seeds transported by abiotic vectors, wind and water speeds and turbulence determine the distance and direction of seed movement (Katul et al. 2005; Nathan et al. 2011): not only are these factors intrinsically variable, but that variation interacts with the physical structure of the envi- ronment and the size and shape of the seed. In biotic dispersal, the set of disperser animals interacting with a seed may dictate its survival, growth and eventual reproduction (García and Martínez 2012). Dispersal vec- tors vary in their interactions with landscape structure, implying that the mechanism of dispersal may dictate the composition and arrangement of a plant community (Metzger 2000; Albrecht et al. 2012; Effiom et al. 2013; Razafindratsima and Dunham 2016; Chen et al. 2017). We largely focus on biotic seed dispersal because the Figure 1. Seed dispersal exemplifies ecological complexity. Survival to adulthood and the fitness of individual adults are influenced by pre-, mid- and post-dispersal variables including the availability of abiotic and biotic vectors; the behaviours, preferences, morphology and physi- ology of dispersers; the spatio-temporal heterogeneity in seed deposition locations; and the probability of encountering other mutualists, facilitators, predators, pathogens and competitors following dispersal. Figure 1. Seed dispersal exemplifies ecological complexity. Survival to adulthood and the fitness of individual adults are influenced by pre-, mid- and post-dispersal variables including the availability of abiotic and biotic vectors; the behaviours, preferences, morphology and physi- ology of dispersers; the spatio-temporal heterogeneity in seed deposition locations; and the probability of encountering other mutualists, facilitators, predators, pathogens and competitors following dispersal. 3 3 © The Author(s) 2019 AoB PLANTS  https://academic.oup.com/aobpla Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Howe 2016). Below, we briefly touch on the importance of dispersal syndromes as a form of functional group- ing that categorizes dispersal adaptations. The complexity of seed dispersal However, our proposed framework focuses instead on functional group delineations that distinguish the importance of dispersal in plant populations and communities.l combined with measures of habitat quality across the landscape (Schupp et  al. 2010). The SDE framework examines the complexity of variation within and among seeds, dispersers and other interactors determining the likelihood that a seed grows into a seedling (and, ultimately, reproductive adult), given a specific vector moving that seed to a specific location (e.g. Alvarez- Buylla and Martinez-Ramos 1990; Godinez-Alvarez and Jordano 2007; Escribano-Avila 2014; Rey and Alcántara 2014; Rother et al. 2016). Seeking predictive capacity in light of global change: adapting current frameworks for functional groups As established above, the plant community in a given lo- cation is constrained by the template established by seed deposition, but the post-deposition interactions within the seedscape (i.e. the full environmental context into which the seed is dispersed) determine which subset of those seeds succeeds. Empirically quantifying all relevant pre- and post-dispersal variables is a complex task for even one plant-disperser pair, and impossible for the thousands of species pairs that participate in seed dispersal mutualisms worldwide (Howe and Smallwood 1982; Aslan et al. 2013; Beckman and Rogers 2013). Nevertheless, without an at- tempt to understand these processes, their variability and the drivers of that variability, our understanding of system dynamics is hamstrung. The Seed Dispersal Effectiveness (SDE) framework is a comprehensive framework to summarize the full suite of variables affecting the dispersal service provided to any particular plant species by any particular vector (Schupp et al. 2010). The SDE framework summarizes the contribution of each dispersal vector (whether bi- otic or abiotic) to the production of new adult plants by evaluating variables influencing the quantity of seeds dispersed and the quality of the seed dispersal event. Quantity metrics within SDE include, for example, the frequency of visits from the disperser to the plant spe- cies and the number of seeds dispersed per visit. Quality metrics include, for example, the condition of the de- posited seed (which depends, e.g., on an animal’s seed- handling behaviour) or a disperser’s movement patterns To achieve this goal, we propose using plant func- tional groups in place of individual species in the SDE framework (Table 1). Functional groups are employed in many fields of ecology and have proven to be use- ful (e.g. functional group classifications yielded insights into plant species responses to climate change in Africa, Scheiter and Higgins 2009; successional dynamics in a Costa Rican forest, Chazdon et al. 2010; and global vege- tation patterns, Sato et al. 2007). By identifying relevant 4 AoB PLANTS  https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Table 1. Functional groups relevant to the importance of seed dispersal for seed survival and thereby plant population and community dynamics. We propose that researchers and conservation planners determine whether target plant species belong to functional groups for which dispersal disruption is likely to significantly decrease fitness vs. have only minor effects on fitness. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Functional group category Characteristics of functional groups likely significantly affected by seed dispersal disruption Characteristics of functional groups likely less affected by seed dispersal disruption Sample references Groups for which plant fitness is affected by whether seeds are dispersed. High colonization ability High competitive ability Coomes and Grubb (2003) Long-distance dispersal adaptations Local dispersal adaptations Muller-Landau et al. (2003) Density-dependent survival Density-independent survival Rey and Alcántara (2014) Groups for which plant fitness is affected by where or when dispersal occurs. Thin/vulnerable seed coats Thick/hard/spiky seed coats Notman and Gorchov (2001) Shade-intolerant Shade-tolerant Alvarez-Clare and Kitajima (2007) Fire-intolerant Fire-tolerant Wenny (2001) Self-incompatible Self-compatible Bond (1994) Reproduction by seed only Reproduces asexually Bond (1994) Intolerant of low nutrients Tolerant of low nutrients Wenny (2001) Low phenotypic plasticity High phenotypic plasticity Goh et al. (2013) Metapopulation-dependent Continuous population distribution Bohrer et al. (2005) Negative distance-dependent mortality No negative distance-dependent mortality Beckman et al. (2012) Inability to seed bank Seed banking Gutterman (2000) Seasonal dispersal Low dispersal seasonality Ruggera et al. (2015) Groups for which plant fitness is affected by the vector of dispersal Seed size* Seed size* Tamme et al. (2014) Intraspecific competitor/ non-facilitator Intraspecific facilitator Martorell and Freckleton (2014) Seed coat with germination inhibitors No seed coat germination inhibitors Traveset and Verdú (2002) aded o ps //acade c oup co /aobp a/a c e abs ac / / /p 006/5308909 by U a S a e U e s y traits, functional group frameworks unite species shar- ing those traits under a common lens allowing gener- alization across diverse organisms. Because functional groups by definition describe the ecological functions present in a given site, functional group diversity has in some cases been found to predict whole-ecosystem function almost as well (or better than) species diver- sity (Dı́az and Cabido 2001). At the same time, func- tional groups are conceptual constructs and thereby subject to the perspective of ecologists identifying traits they deem important to particular questions. Beginning with SDE allows us to anchor our functional group rec- ommendations in a robust and established guiding traits, functional group frameworks unite species shar- ing those traits under a common lens allowing gener- alization across diverse organisms. Seeking predictive capacity in light of global change: adapting current frameworks for functional groups These groups are categorized based on how much it matters whether a seed is dispersed (shown in red; groups 1–3); how much it matters where or when dispersal occurs (shown in blue; groups 4–14); and how much it matters what vector disperses the seed (shown in green; groups 15–17). Applying vulnerability assessments and SDE calculations at the level of these functional groups may enable us to achieve a predictive understanding of seed dispersal ecology in the face of combined global change and complexity. *For species exhibiting a measurable fitness boost from dispersal, seed size may dictate which abiotic or biotic vectors are effective. Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Previous uses of functional groups in seed dispersal ecology have been narrow in scope (focusing on single systems or a small number of focal functions) but are indicative of the usefulness of this approach. For ex- ample, Dennis and Westcott (2006) distilled 26 detailed measurements of seed disperser traits into 10 trait di- mensions. They used these dimensions to identify 15 functional groups in a suite of 65 Australian seed dis- perser species; their mathematical approach could be more broadly applied to reduce complexity in other sys- tems (Dennis and Westcott 2006). Rodríguez-Rodríguez et al. (2017) categorized plant–animal interactions into typologies and evaluated links between these typolo- gies and plant fitness. Zamora (2000) explored how the consistency of fitness benefits offered by seed dis- persal across systems and groups of species permits generalization within functional groups. A key benefit of a functional group approach is that it could provide an understanding of the functions that may be lost when extinctions occur (Blondel 2003; Bastazini et al. 2017). This conservation-oriented conceptual application was highlighted by Schleuning et al. (2014) in their call for more work examining the linkage between trait-based approaches such as functional group delineation and structural approaches such as network analysis (Ruggera et al. 2015). Functional groups can be used to predict the role of suites of species in an ecosystem and the re- sponse of those species to drivers of global change. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Because functional groups by definition describe the ecological functions present in a given site, functional group diversity has in some cases been found to predict whole-ecosystem function almost as well (or better than) species diver- sity (Dı́az and Cabido 2001). At the same time, func- tional groups are conceptual constructs and thereby subject to the perspective of ecologists identifying traits they deem important to particular questions. Beginning with SDE allows us to anchor our functional group rec- ommendations in a robust and established guiding comprehensive framework. Thus, the three broad cat- egories of functional groups described below have been selected to distil the comprehensive SDE framework into straightforward conceptual bins. We acknowledge that other approaches to simplifying matters are possible, perhaps based on different criteria, or on different quan- tifications of the same criteria. However, as we demon- strate here, considerable insights can be obtained with our approach. It is also important to acknowledge that, compared with a species-level SDE analysis, a functional group-level SDE analysis carries a risk of overgeneraliza- tion, whereby meaningful sources of variation are dis- missed due to limited understanding. 5 © The Author(s) 2019 AoB PLANTS  https://academic.oup.com/aobpla Functional groups distinguishing how much it matters if a seed is dispersed at all Species may be categorized based on the importance of dispersal for their survival and reproduction. Well- established frameworks examining fitness benefits that may be derived from seed dispersal can guide functional group determination in this arena, since functional groups can be defined by traits linked to such fitness benefits. The escape hypothesis states that seeds will experience fitness boosts as a result of removal from the neighbourhood of natural enemies, including patho- gens, parasites, herbivores and competitors (Howe and Smallwood 1982; Howe and Miriti 2004). Escape from be- neath the canopy of a parent tree reduces the chances of pathogens and herbivores finding a seed (Janzen 1970; Connell 1971), as well as the chance that a seed will be deposited immediately adjacent to a close rela- tive and thus compete for necessary resources. A logical extension of this hypothesis suggests that species can be assembled into functional groups by traits indicating dependence upon such escape (i.e. susceptible to infec- tion or herbivory; exhibiting negative density-depend- ence in survival and growth) vs. those less dependent upon escape (i.e. exhibiting thick seed coats or other protections against infection and herbivory; exhibiting low negative density-dependence). As an example, in a study of olive (Olea europaea) regeneration in human- altered vs. unaltered landscapes in Spain, proximity to maternal trees was associated with elevated seedling mortality; O. europaea thus appears to occupy a func- tional group characterized by escape dependence and negative density-dependence (Rey and Alcántara 2014) (Table 1). Similarly, fungal pathogens led to strong den- sity-dependent mortality in Pleradenophora longicuspis in Belize, evidence that functional group categorization based on density-dependence is appropriate for this species (Bagchi et al. 2010). By contrast, species with low density-dependence, and thus likely to be classified into functional groups with reduced dispersal-dependence, include a suite of common species in a Panamanian rainforest, where density-dependence varies consider- ably among tree species (Comita et  al. 2010). Species with greater seed mass exhibited reduced negative density-dependence on Barro Colorado Island (Lebrija- Trejos et al. 2016). Previous species-specific studies have examined density dependent damage and mortality in seeds and Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Functional groups distinguishing how much it matters where or when a seed is dispersed Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 A large fraction of the ‘quality’ element of the SDE frame- work centres on where and when a seed is dispersed. Fundamentally, this will dictate which abiotic and bi- otic resources and threats are encountered by the seed and subsequent plant (Schupp et al. 2010; Beckman and Rogers 2013). Abiotic resources may include nutrients, moisture, space and light. Abiotic threats could include drought, nutrient deficiencies, frost and fire. On the bi- otic side, resources could include mutualists such as soil mycorrhizae, pollinators, seed dispersers and facilita- tors, and threats could include herbivores, competitors, predators and pathogens. Functional groups that cat- egorize species by whether deposition setting matters to a seed may include (i) groups of plants that are par- ticularly susceptible to abiotic stressors/disturbances or natural enemies (e.g. plants with low competitive ability or thin seed coats) vs. (ii) those tolerant of threats (e.g. shade-tolerant, fire-tolerant, drought-tolerant, etc.). Other relevant functional groups would include spe- cies dependent on mutualists or facilitators (Calvo and Horvitz 1990; Onguene and Kuyper 2002; Hoehn et al. 2008; Teste et al. 2009), frost-intolerant species that re- quire nurse plants, species dependent on forest gaps to escape shading and species that require a narrow range of soil nutrient content. Interspecific interactions can affect parameters of matrix population models, enabling their effect on fit- ness to be examined using elasticity and sensitivity analyses (McGraw and Caswell 1996; Horvitz et al. 1997; Benton and Grant 1999; Mills et al. 1999; Carslake et al. 2009; Jongejans et al. 2011). The effect of seed dispersal failure can be explored via elasticity analyses simulating loss of dispersers and resulting failure to escape from natural enemies or encounter recruitment sites (Howe and Miriti 2004; Brodie et  al. 2009a; Rodríguez-Pérez and Traveset 2012; Traveset et al. 2012; Caughlin et al. 2015; Pérez-Méndez et al. 2015). Applying such analyses to functional groups that enable generalization beyond a few carefully measured surrogates to other species within a group might greatly expand the predictive cap- acity of such analyses across systems.ii Plant species with plastic phenotypes may be rela- tively generalized with regard to their interspecific inter- action requirements, suggesting that functional groups defined by plasticity may be appropriate. Plasticity may influence dependence upon certain abiotic conditions or interspecific interactions. Meaningful Functional Groups in Seed Dispersal Ecology We define plant functional groups based on traits influ- encing the importance of seed dispersal for plants (Table 1). Our proposed functional groups categorize plants based on: (i) how important it is to plant recruitment if seeds are dispersed at all, (ii) how important the lo- cation and timing of seed deposition are and (iii) how much vector identity matters. These groups thus define important points in the dispersal process at which seed fate may be influenced, with a focus on the fitness bene- fits derived from dispersal events. Applying SDE to these functional groups will enable researchers to predict how populations of the species within a group will be af- fected by total or partial dispersal disruption, changes in phenology or habitat conditions, or entry of non-native species into dispersal networks. A  given plant species may display traits that make them likely vulnerable to dispersal disruption based on one functional group cat- egory and less vulnerable based on another; in such a case, these categories will help to pinpoint sources of such vulnerability. Previous species-specific studies have examined density-dependent damage and mortality in seeds and seedlings encountering abundant herbivores, patho- gens and predators in close proximity to parent trees (the Janzen–Connell effect) (e.g. Petermann et  al. 2008; Bagchi et al. 2010; Liu et al. 2012). Study results have been mixed, but largely show increased success 6 © The Author(s) 2019 AoB PLANTS  https://academic.oup.com/aobpla Aslan et al. – Defining functional groups relevant to the importance of seed dispersal at greater distances from one another (Snyder and Chesson 2003; Snyder 2011). of seeds and seedlings after removal from the parent, with various explanatory mechanisms (e.g. Thomas 1990; Blundell and Peart 1998; Packer and Clay 2000; Petermann et al. 2008; Bagchi et al. 2014). These studies suggest that assigning seeds to functional groups based on whether they exhibit negative density-dependence is important. For those groups that do exhibit such den- sity-dependence (Table 1), loss of dispersers may be expected to affect plant fitness significantly. Assigning plants to functional groups may in some cases be pos- sible through observational studies, generalizing from what we know about similar species, and in other cases may require experimental assessments—which are still far less extensive than a traditional species-specific SDE assessment (Table 1).i Functional groups distinguishing how much it matters where or when a seed is dispersed Gutterman 2000). In other cases, the timing of dispersal interacts with char- acteristics that determine habitat quality (e.g. ephem- eral environmental conditions or seasonally migratory dispersers) (Ruggera et al. 2015). Timing can matter on the plant side, too: in a study of Pistacia lentiscus dis- persal in Spain, seed viability was found to vary during the fruiting season, such that dispersers interacting with the species when viability is high were more effective than those handling fruits at other times (González-Varo et al. 2018). as seed size sets a lower limit on the type and size of dispersal vector that can lift (e.g. wind) or ingest (e.g. animals) the seed (Wheelwright 1985; Ganeshaiah and Shaanker 1991; McConkey and Drake 2002). In some cases, dispersal syndromes explain some variation in dispersal distances and can be used to predict dispersal distances (Tamme et al. 2014), but the variation within dispersal syndromes can be very high (Clark et al. 2005; Muller-Landau et  al. 2008). Dispersal syndromes tend to be broad categories (e.g. large mammal vs. small mammal vs. wind). Even within these categories, spe- cies may be dispersed by a diversity of vectors, and in some cases secondary dispersal is performed by an al- together different class of vector than primary dispersal (Böhning-Gaese et al. 1999; Vander Wall and Beck 2012). Whether the identity of the vector matters to the even- tual success of the seed is an important component of understanding the role of dispersal in eventual plant population and community dynamics. In spite of these successful attempts to achieve gen- eral insights, there are certain risks associated with generalizing across vectors (or dispersers). Identifying a dispersal syndrome may suggest that a broad cate- gory of vector is the likely disperser, but such categories could include many potential disperser species varying in effectiveness (Jordano et al. 2007; Howe 2016). Thus, dispersal syndromes are not sufficient to predict the effects of losing certain vectors. Nor do dispersal syn- dromes give us information on the likelihood of being dispersed by a ‘non-standard’ dispersal vector—that is, a vector other than the most common vector or vec- tors interacting with a particular plant—which might be more influential than ‘standard’ vectors in long-disper- sal events (Higgins et al. 2003; Jordano et al. 2007) and therefore exert larger effects on plant populations (Kot et al. 1996; Neubert and Caswell 2000). Functional groups distinguishing how much it matters where or when a seed is dispersed As an important lesson for dispersal ecology, the concept of syndromes has faced substantial criticism in pollination ecology (Ollerton et al. 2009). Careful empirical study has dem- onstrated that in most cases both plants and pollinators are much more opportunistic and interact with a much broader suite of partners than morphological pollination syndromes would suggest (Waser et  al. 1996; Fenster et al. 2004; Ollerton et al. 2009; Waser et al. 2018). If syndromes are similarly uninformative in dispersal, this carries implications for conservation and management, since incorrect generalization stemming from syn- dromes could lead to fallacious assumptions about the redundancy of dispersers within interaction networks and, consequently, about restoration and conservation needs (Howe 2016). In theory, the functional groups most dependent upon dispersal include species in patchy habitats, those with strong density-/distance-dependent mortality, those lacking the ability to maintain a seed bank and those with specific requirements for the timing and lo- cation of the dispersal event (Table 1). Functional groups distinguishing how much it matters where or when a seed is dispersed For example, mycorrhizal asso- ciations could provide critical assistance to plant individ- uals with delicate or small root systems, but individuals with plastic growth (e.g. those able to divert resources towards robust root growth as required) might be less affected by an absence of root symbionts (Valladares et al. 2007; Goh et al. 2013). Other fitness benefits of seed dispersal may arise from colonization of unpredictable and newly available germi- nation sites and directed dispersal to hospitable micro- sites located within a non-hospitable matrix (Wenny 2001; Howe and Miriti 2004). Dispersal is likely to mat- ter most to functional groups of species with specialized spatio-temporal germination and growth site require- ments or low competitive ability and thus high depend- ence on vacant establishment sites. Identification of such species may be informed by competition/coloniza- tion trade-off theory, which predicts that species exhibit a trade-off between dispersal ability and competitive ability (for example, plant species may trade off the production of a few large, well-provisioned seeds for the production of many smaller seeds) (e.g. Bolker and Pacala 1999; Dalling and Hubbell 2002; but see Coomes and Grubb 2003). Similarly, theoretical ecologists have investigated when long-distance dispersal vs. local dis- persal is evolutionarily advantageous, given the fitness advantages of colonizing new sites and the lower prob- ability of finding habitats sharing specific characteristics Dispersal also matters for plants living in habitats that are temporally or spatially variable. Important functional groups include those with specific habitat requirements that are spatially heterogeneous (e.g. species dependent on metapopulation processes for persistence; Bohrer et al. 2005) vs. general habitat re- quirements that are widespread and homogeneous. For example, in a human-disturbed, patchy landscape, affinity of dispersers for seedling habitat leads to in- creased germination of the relic Chinese yew (Taxus 7 AoB PLANTS  https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal chinensis), indicating that directed dispersal matters for this endangered plant species (Li et al. 2016) (Table 1). In another example, seeds of the shrub Daphne rod- riguezii dispersed to sites below nurse plants exhibit higher seedling survival (Rodríguez-Pérez and Traveset 2010) (Table 1). Functional groups distinguishing how much it matters where or when a seed is dispersed Lower location specificity can be found in, for example, shade-tolerant species that exhibit physical defence mechanisms and are thus able to sur- vive and reproduce in conditions of high competition; eight such species were studied in Panama and their physical traits documented (Alvarez-Clare and Kitajima 2007). This indicates that material characteristics can be identified to classify such species into functional groups with less dependence on dispersal (Table 1). Some func- tional groups may be affected by positive or negative distance- or density-dependent mortality (e.g. if seeds must be dispersed in clumps to germinate and grow; Beckman et al. 2012), or may require rare micro-condi- tions (Pufal and Garnock-Jones 2010). Temporally, some species can protect themselves against poor dispersal years by living many years as adults or remaining viable in a seed bank for a long time (e.g. Gutterman 2000). In other cases, the timing of dispersal interacts with char- acteristics that determine habitat quality (e.g. ephem- eral environmental conditions or seasonally migratory dispersers) (Ruggera et al. 2015). Timing can matter on the plant side, too: in a study of Pistacia lentiscus dis- persal in Spain, seed viability was found to vary during the fruiting season, such that dispersers interacting with the species when viability is high were more effective than those handling fruits at other times (González-Varo et al. 2018). chinensis), indicating that directed dispersal matters for this endangered plant species (Li et al. 2016) (Table 1). In another example, seeds of the shrub Daphne rod- riguezii dispersed to sites below nurse plants exhibit higher seedling survival (Rodríguez-Pérez and Traveset 2010) (Table 1). Lower location specificity can be found in, for example, shade-tolerant species that exhibit physical defence mechanisms and are thus able to sur- vive and reproduce in conditions of high competition; eight such species were studied in Panama and their physical traits documented (Alvarez-Clare and Kitajima 2007). This indicates that material characteristics can be identified to classify such species into functional groups with less dependence on dispersal (Table 1). Some func- tional groups may be affected by positive or negative distance- or density-dependent mortality (e.g. if seeds must be dispersed in clumps to germinate and grow; Beckman et al. 2012), or may require rare micro-condi- tions (Pufal and Garnock-Jones 2010). Temporally, some species can protect themselves against poor dispersal years by living many years as adults or remaining viable in a seed bank for a long time (e.g. Functional groups enabling us to distinguish how much the identity of the dispersal vector matters 2007; Bueno et al. 2013; Escribano-Avila et al. 2014; González-Varo et al. 2017). Plant functional groups of interest when deter- mining whether a specific vector is important include groups defined by seed size, seed coat thickness (e.g. groups of species with thick coats requiring substantial gut treatment for germination), presence of germin- ation inhibitors, and intraspecific facilitation or positive density-dependence. scale, straightforward functional groups founded on seed morphology and physiology (e.g. determined by seed size and shape) may dictate potential disperser suites and indicate how important different dispersal vectors may be, relative to one another, for a given plant species (Table 1). Identifying plant species at risk from dispersal disruption (e.g. McConkey et  al. 2018) may be possible when the importance of vector iden- tity is understood. Vectors may differ in the number of seeds dispersed, the condition of dispersed seeds, dis- persal distances and dispersal spatial arrangements. Wind, for example, is most likely to move seeds that are small in mass (Shea 2007; Nathan et al. 2011). Disperser animals with large gape sizes are more likely than small dispersers to disperse greater numbers of larger seeds over longer distances (Cox et al. 1991). Since large dis- persers with low reproductive rates are often most threatened by direct human exploitation coupled with low reproductive rates (Farwig and Berens 2012), the plant functional group that includes large-seeded spe- cies is of particular interest in seed dispersal conserva- tion. Losses of key large dispersers can threaten plant species and functional group diversity in seed dispersal networks (Donoso et al. 2017). Dispersers with special- ized habitat requirements may aggregate seeds by re- turning frequently to a limited number of sites (Howe 1989). Because different vectors may provide dispersal services in different ways, plant species may experience complementary dispersal services from them, with a greater diversity of vectors maximizing the success of a plant (Levin et al. 2003; Jordano et al. 2007; Bueno et al. 2013; Escribano-Avila et al. 2014; González-Varo et al. 2017). Plant functional groups of interest when deter- mining whether a specific vector is important include groups defined by seed size, seed coat thickness (e.g. groups of species with thick coats requiring substantial gut treatment for germination), presence of germin- ation inhibitors, and intraspecific facilitation or positive density-dependence. to the functional groups we propose (Table 1) (McConkey et al. 2018). Functional groups enabling us to distinguish how much the identity of the dispersal vector matters By contrast, SDE of a suite of bird species was studied for two Miconia species in Brazil (Santos et  al. 2017). Although the birds varied in the quantity of seeds they dispersed, they did not vary in quality of dispersal (Santos et al. 2017). Miconia species with their small seeds and large disperser suites (e.g. Levey and Byrne 1993) therefore appear to fall into a proposed functional group for which vector identity is less impor- tant (Table 1). Dispersal vector identity has been shown to af- fect population growth rates for some but not all of the few vertebrate-dispersed plant species that have been studied (e.g. Godinez-Alvarez and Jordano 2007; Brodie et al. 2009b; Loayza and Knight 2010). However, the importance of different vectors is unknown for most plant species, and that lack of clarity ham- pers our ability to predict the outcomes of changes in vectors. Predictions are better-developed for bal- listic- and wind-mediated dispersal than for animal- mediated dispersal (Skarpaas and Shea 2007; Nathan et al. 2011; Bullock et al. 2012), in large part because of the complexity of animal behaviour and movement and the diffuse nature of most seed dispersal systems, wherein multiple animals disperse any given plant (Shea 2007). Even when detailed information about the role of specific vectors has been obtained for a given plant species, studies are often narrow in spa- tial and temporal extent and thus context-dependent (i.e. information is specific to a particular time and place, given a particular disturbance history), and the importance of individual vectors may change under different contexts. Functional groups enabling us to distinguish how much the identity of the dispersal vector matters Dispersal syndromes are used to categorize plants by the type of vector known or assumed to best disperse their seeds. Syndromes are the most common functional group classifications used in seed dispersal ecology. Illustrating the potential value of generalization across similar dispersers, Tamme et al. (2014) successfully used plant traits to predict dispersal distances for over 500 species. Dispersal distances could then be related to dis- persal syndromes, growth form and other plant traits, such as plant height and seed size (Thomson et al. 2011; Tamme et al. 2014). Previous studies have reported an interaction between seed size and dispersal vector size, Although the use of dispersal syndromes per se thus carries a risk of drawing conclusions at too crude a 8 AoB PLANTS  https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal scale, straightforward functional groups founded on seed morphology and physiology (e.g. determined by seed size and shape) may dictate potential disperser suites and indicate how important different dispersal vectors may be, relative to one another, for a given plant species (Table 1). Identifying plant species at risk from dispersal disruption (e.g. McConkey et  al. 2018) may be possible when the importance of vector iden- tity is understood. Vectors may differ in the number of seeds dispersed, the condition of dispersed seeds, dis- persal distances and dispersal spatial arrangements. Wind, for example, is most likely to move seeds that are small in mass (Shea 2007; Nathan et al. 2011). Disperser animals with large gape sizes are more likely than small dispersers to disperse greater numbers of larger seeds over longer distances (Cox et al. 1991). Since large dis- persers with low reproductive rates are often most threatened by direct human exploitation coupled with low reproductive rates (Farwig and Berens 2012), the plant functional group that includes large-seeded spe- cies is of particular interest in seed dispersal conserva- tion. Losses of key large dispersers can threaten plant species and functional group diversity in seed dispersal networks (Donoso et al. 2017). Dispersers with special- ized habitat requirements may aggregate seeds by re- turning frequently to a limited number of sites (Howe 1989). Because different vectors may provide dispersal services in different ways, plant species may experience complementary dispersal services from them, with a greater diversity of vectors maximizing the success of a plant (Levin et al. 2003; Jordano et al. AoB PLANTS  https://academic.oup.com/aobpla Acknowledgements Our discussion emerged from a National Science Foundation-funded Seed Dispersal Workshop held at the National Socio-Environmental Synthesis Center in May 2016. All workshop participants are co-authors on this paper and have contributed to the development of these ideas. Contributions by the authors C.A. led the development of the concepts, writing, and revising of the manuscript with input from N.G.B. and H.S.R. All authors contributed to the development of concepts and are listed in order of contribution and alphabetical order within each level of contribution. Sources of funding Ideas for this manuscript initiated during the Seed Dispersal Workshop held in May 2016 at the Socio- Environmental Synthesis Center in Annapolis, MD and supported by the US National Science Foundation Grant DEB-1548194 to N.G.B. and the National Socio‐ Environmental Synthesis Center under the US National Science Foundation Grant DBI‐1052875. D.Z. received funding from the Swiss National Science Foundation (SNF, grant: PZ00P3_168136/1) and from the German Science Foundation (DFG, grant: ZU 361/1- 1). Compiling large amounts of data from multiple systems to test functional group approaches can be resource-intensive, although increasingly global databases are becoming available and serving as common data hubs (e.g. COMPADRE Plant Matrix Database and COMADRE Animal Matrix Database; Max Planck Institute for Demographic Research (Germany); available at www.compadre-db.org). One promising approach is to standardize data col- lection by many research groups working across many systems (e.g. NutNet; Borer et  al. 2014). If groups collect the same data across systems, those data can be introduced to a common modelling plat- form to explore patterns that hold across systems. By assembling data sets in this way, the costs are spread across research groups, and data collection can be useful even when sample sizes within a spe- cific system are limited. As an example, standard- ized data collection across systems has enabled researchers to identify consistent patterns of grass- land responses to land use change (Garnier et  al. 2007). We thus recommend that research teams join forces to collect standardized data exploring vary- ing effectiveness of dispersal by different agents, the role of spatio-temporal dynamics and the influ- ence of interspecific interactions pre-, mid- and post-dispersal. Once data are collected in many sys- tems, it will be necessary to bring them together to make them available for broad analysis. Depositing seed dispersal data into public-access repositories is therefore important. Useful repositories include Dryad (http://datadryad.org) and KNB (http://knb. ecoinformatics.org). AoB PLANTS  https://academic.oup.com/aobpla Using Functional Groups to Close Our Knowledge Gaps The use of functional groups defined by dispersal- related traits can reduce the amount of data needed to parameterize models (Mokany et al. 2014). The digital availability of trait data is increasing (e.g. via publicly ac- cessible databases such as TRY; Kattge et al. 2011) but continued empirical research is needed to relate those data to dispersal processes. Even so, certain functional groups can now be defined and used to distinguish spe- cies that are relatively more or less strongly dispersal- dependent (Table 1). If a particular plant species belongs to a group for which fitness is strongly linked to dispersal (Table 1, column 2), we can predict that this species is likely to be vulnerable in the face of dispersal disruption, based strictly on functional group. Exemplifying the importance of this functional group delineation, different behaviours of large mammal dis- persers resulted in differential contributions to dispersal of the large-seeded Platymitra macrocarpa in Thailand, with some species dispersing higher quantities of seeds with poor survival outcomes and others dispersing fewer seeds with greater success per seed (McConkey et  al. 2018). In this case, dispersers contributed differentially to the dispersal of the plant but overall plant regenera- tion was poor, leading researchers to speculate that there may be important dispersers that are missing or rare (McConkey et al. 2018). In that context, the large seed size of the plant suggests that the identity of the dispersal vector in this example is important according While functional groups may enable us to generalize across full plant communities, overgeneralization could 9 AoB PLANTS  https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal plant populations and communities. We invite the eco- logical community to join us in this effort. cause us to lose sight of meaningful sources of varia- tion. It is thus necessary to test the value of functional group approaches in seed dispersal ecology by generat- ing predictions based on them and then testing those predictions against species-specific data. 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The main directions in the implementation of organic agriculture
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* Corresponding author: zuev_1960_nikolai@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). The main directions in the implementation of organic agriculture N.P. Zuev*, N.N. Shvetsov, Y.P. Breslavets, V.A. Shumsky, and Y.P. Masalykina Belgorod State Agrarian University named after V. Y. Gorin, Belgorod, Russia N.P. Zuev*, N.N. Shvetsov, Y.P. Breslavets, V.A. Shumsky, and Y.P. Masalykina Belgorod State Agrarian University named after V. Y. Gorin, Belgorod, Russia Abstract. Measures for the introduction of organic agriculture, along with the use of appropriate techniques in agriculture, imply the rejection of the use of chemical-synthetic growth regulators or hormones; limited, strictly tied to the area, livestock; feeding animals, if possible, with their own feed, minimum purchase of feed and avoiding antibiotics. However, there is currently no way to permanently stop using antibiotics. Therefore, a gradual decrease in doses, frequency and timing of their application is relevant. The work is devoted to the implementation of this direction. A synergistic relationship of gentamicin and tylosin was established with an increase in the efficiency of using the created composite preparation with a decrease in the dose and the possibility of the formation of drug-resistant populations of bacteria - causative agents of animal diseases. BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 https://doi.org/10.1051/bioconf/20213905003 2 Materials and methods In relation to organic livestock are needed [6]: - description of premises in which animals are raised, areas for outdoor walking, pastures, outdoor trails, etc., as well as premises for slaughter of animals and premises for packaging and storage of animal products, incoming materials and raw materials; - description of premises in which animals are raised, areas for outdoor walking, pastures, outdoor trails, etc., as well as premises for slaughter of animals and premises for packaging and storage of animal products, incoming materials and raw materials; - a complete description of the manure warehouses or storage areas that are obtained from the breeding of animals; - a complete description of the manure warehouses or storage areas that are obtained from the breeding of animals; - contracts concluded with other producers (if necessary), when applying manure, i h b i d d i h b di f i l - contracts concluded with other producers (if necessary), when applying manure, which was obtained during the breeding of animals; - contracts concluded with other producers (if neces which was obtained during the breeding of animals; - information on breeding or origin of livestock; - information about the purchase or sale of livestock; - a veterinary care plan used to prevent and treat disease, injury and reproductive problems; - information on all medical procedures and medicines used for any purpose, including - information on all medical procedures and medicines used for any purpose, including for the purposes of ensuring the quarantine period and processing of animals; information on all medical procedures and medicines used for any purpose, inclu for the purposes of ensuring the quarantine period and processing of animals; e purposes of ensuring the quarantine period and processing of animals; - information about purchased feed and its sources, animal rations; - information on the movement of livestock within the production unit; - information on the transportation, slaughter and sale of animals outside the production unit. 1 Introduction The main idea of organic farming is to manage the farming in accordance with the laws of nature. At the same time, an agricultural enterprise is considered, first of all, as an organism with its constituent parts - man, animal, plant and earth. It is planned to increase the consumption of organic products in the Belgorod region. At the same time, the speed of this process is not sufficient and does not correspond to the needs of society. In 2020, this economic and social direction accounted for 1-3% of the total volume of livestock production of all types of property. Purpose: development and implementation of the main elements of organic agriculture in animal husbandry in the system of organic-rational relationship of its constituent parts: land, plants, animals and humans. The main directions of the implementation of organic agriculture include: rejection of plant protection products with chemical-synthetic means; the use of slow-acting natural fertilizers (encapsulated and nanostructured pesticides of various directions); refusal to use chemical-synthetic growth regulators or hormones; livestock associated with land; production of animal feed directly on the farm; development of antibiotic substitution measures and minimization of their harmful effect on the earth-plant-animal-human system (the main components of organic agriculture). https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 2 Materials and methods An important component of the relationship in the earth-plant-animal-human system is the production of micronutrient-enriched food products: the production of fortified human food products up to 25-35% (by 2030); the fortification of dairy products, flour and bread with minerals and vitamins [6] An important social moment is the possibility of consumption of food products enriched with micronutrients by adults with low and low incomes and children in social institutions, as well as their use in the prevention of pathologies associated with a lack of biologically active substances in humans (vitamins, vitamin-like substances, macro- and microelements). Organizational and economic support for the implementation of organic agriculture necessarily provides for the associated and coordinated activities in agricultural technology, fodder production, plant and animal protection, processing industry technologies at the regional, municipal level of action [6]. In the presence of livestock on the farm, the necessary need for feed (coarse, juicy, green, concentrated) is determined, and how much feed will be obtained from natural lands. The missing amount of forage must be produced on arable land [6]. Determine which crops will cover the need for hay, silage, haylage, green fodder and concentrates. 2 Materials and methods As an example, the specific implementation of one of the areas of organic farming, and organic agriculture in general, we present the technology for the development of the production and use of composite antimicrobial drugs, which determine their use in lower doses and thereby reduce their intake into the body of animals during their treatment and prevention of diseases, people with food, together with manure into the soil, reducing the possibility of the formation of drug-resistant populations of bacteria - causative agents of diseases of animals and humans and their harmful effect on the biological components of the soil. The use of such funds helps to increase the rate of elimination (excretion) of them from the body of animals. BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 The proposed invention relates to animal husbandry and can be used for therapy with Escherichiosis gastroenteritis of piglets. The proposed invention relates to animal husbandry and can be used for therapy with Escherichiosis gastroenteritis of piglets. Known drugs for therapy with gastroenteritis of pigs using various antibacterial agents: tanibel, phosphopag, combinations of tylosin, nitrofurans, and nitazole [7, 8, 9, 12]. Therapeutic efficacy in gastroenteritis of piglets has fradizin itylosin tartrate [1, 2, 3, 4]. Tylosin tartrate is one of the preparations for microbiological synthesis. The disadvantages of tylosin-containing preparations and other above-mentioned preparations, including in relation to E. coli, are: a narrow spectrum of activity in relation to microorganisms that cause gastroenteritis in pigs, the rapid emergence of pathogens of resistance to them. Based on the foregoing, the task of the invention is set: the creation of a drug for therapy for Escherichiosis gastroenteritis of pigs by increasing the prophylactic activity of tylosin for gastroenteritis of pigs by increasing its antimicrobial activity and the creation of its composite forms. p When solving the task, it was necessary: 1. Expansion of the range of antimicrobial drugs in severe infections against microbial pathogens of gastroenteritis in piglets that require immediate initiation of treatment before bacteriological diagnosis, or in mixed infection, when pathogens of microbial association have different susceptibility to antibacterial drugs. 2. To prevent the development of toxic effects by achieving a rapid and more complete effect with the simultaneous introduction of two or more drugs in smaller than usual, course doses. This is important when using antibiotics that cause nephrotoxic (aminoglycosides, polymyxin, griseofulvin), hepatotoxic (tetracycline, erythromycin, novobicin) effects, adverse effects on the gastrointestinal tract (tetracycline (chloracycline, erythioramine, erythiram). 3. Achieving a potential antibacterial effect. 4. Reduction or elimination of the possibility of the acquired drug resistance of pathogenic microorganisms. Based on the above requirements, macrolide, nitrofuran and aminoglycoside (gentamicin) drugs are promising as means for creating composite antibacterial drugs with a wide spectrum of action. Based on the above requirements, macrolide, nitrofuran and aminoglycoside (gentamicin) drugs are promising as means for creating composite antibacterial drugs with a wide spectrum of action. Tylosin-containing drugs have high antimicrobial activity against gram-positive and relatively low - gram-negative microflora. 3 Results and discussion Example No. 1 The drug for therapy with Escherichiosis gastroenteritis of piglets 2 https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 Nitrofuran compounds exhibit a broad range of antimicrobial activity Previously, we found that a synergistic combination of tylosin tartrate with furazonal in a ratio of 3: 1 is effective in treating sick animals and preventing the development of gastroenteritis in piglets. This combination is effective when administered with feed at a dose of 10 mg / kg body weight [5, 6, 7]. However, due to the limited use of nitrofuran drugs and the insufficient effectiveness of the above drugs, as well as the need to prevent the formation of bacterial resistance, the use of antibiotics from the aminoglycoside group (gentamicin) is more promising. We have carried out laboratory studies that determine the optimal ratio of components in the created composite agent while studying their antimicrobial action against bacteria that play an etiological role in the occurrence of gastroenteritis in pigs. The results of the studies are presented in table 1. 3 3 https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 Table 1. Antimicrobial activity of the composition of tylosin tartrate with gentamicin. Type of micro- organisms Minimum bacteriostatic concentration (MBK, µg/ml) Fractional inhibitory concentration (μg / ml) in a 1: 1 ratio FIC- index of the compoun d tylosin tartrate gentami cin compositions of tylosin tartrate with gentamicin tylosin tartrate Gentam icin 3:1 2:1 1:1 Escherichia coli 15±0,17 10±0,12 7,03,1±0,7 6,0±0,8 5,0±0,4 0,15 0,25 0,40 Table 1. Antimicrobial activity of the composition of tylosin tartrate with gentamicin. The information presented in Table 1 on the search for the most effective proportions of the constituent ingredients of the composite preparation indicate the potentiation of the antimicrobial activity of the combination of tylosin tartrate with gentamicin in a 1: 1 ratio. The fractional inhibitory concentration (FIC) of tylosin tartrate in relation to E. coli when interacting with gentamicin was 0.15, and that of gentamicin - 0.25, and the fractional inhibitory index of the composite compound in relation to a microorganism was respectively equal to 0.40, which predetermined the prospects for further drug development. Prevention of adaptation in causative agents of gastroenteritis to tylosin tartrate, which occurs after 30 passages, 30 was studied in a separate experiment, the results of which are reflected in table 2. Table 2. Prevention of adaptation of microorganisms to causative agents of gastroenteritis in piglets. BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 The disadvantages of tylosin-containing drugs are: a narrow spectrum of activity in relation to microorganisms that cause gastroenteritis in piglets, the rapid emergence of pathogens of resistance to them. The objective of the invention: increasing the therapeutic activity of tylosin in dysentery gastroenteritis of pigs. Based on the above requirements, the macrolide preparations tylosin and erythromycin are promising for the creation of complex antibacterial agents with a wide spectrum of action. Tylosin-containing drugs have high antimicrobial activity against gram-positive and relatively low - against gram-negative microflora. Erythromycin exhibits a wide range of antimicrobial activity [1, 2, 3, 4, 5]. A synergistic combination of tylosin tartrate with erythromycin in a 1: 1 ratio is effective in treating sick animals. This combination is effective when administered with feed at a dose of 10 mg / kg body weight. Laboratory studies were carried out to determine the optimal ratios of the components in the created composite agent while studying their antimicrobial action against bacteria that play an etiological role in the occurrence of gastroenteritis in pigs. The studies carried out to find the most effective proportions of the constituent ingredients of the composite preparation indicate the potentiation of the antimicrobial activity of the combination of tylosin tartrate with erythromycin in a 1: 1 ratio. The fractional inhibitory concentration (FIC) of tylosin tartrate in relation to Brachispira hyodysenteriae when interacting with erythromycin was 0.4, for erythromycin, respectively, 0.1 μg / ml. FIC - the index of the composite compound for the above microorganisms, respectively, equal to 0.2; 0.3; 0.3. In an experiment to study the therapeutic efficacy of a composite preparation at a dosage of 5 mg / kg of body weight (animals received the preparation with food for 10 days), its high therapeutic efficacy (90%) was established in comparison with its components (80%) and in control - 45%. The data obtained indicate that the therapeutic efficacy of the composite preparation increases due to the synergistic interaction of tylosin tartrate and erythromycin. The present invention is a composite preparation for the prevention and treatment of piglet dysentery by administering to animals a composition of tylosin tartrate and erythromycin in a 1: 1 ratio. The compositional preparation is effective for piglets from 2 weeks to 4 months of age. The drug is effective at a dose of 5 mg / kg of body weight of piglets for 10 days in the treatment of dysentery. BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 Bacteriostatic concentration of tylosin tartrate, μg / ml nutrient medium Background (MBK after the first passage) Аfter 30 passages on culture media containing gentamicin Colibacillus Colibacillus 15±0.14 15±0.14 Table 2. Prevention of adaptation of microorganisms to causative agents of gastroenteritis in piglets. From the data in Table 2, it can be seen that the antimicrobial activity of tylosin tartrate against Escherichia coli did not change even after 30 passages. In an experiment to study the therapeutic effect of a composite drug at a dosage of 10 mg / kg of body weight (animals received the drug with food for 10 days), the results of which are presented in Table 3, its high therapeutic efficacy (90%) was established, compared with its components (80%). Table 3. Comparative therapeutic efficacy of drugs for gastroenteritis of pigs. Table 3. Comparative therapeutic efficacy of drugs for gastroenteritis of pigs. Group of animals Composite preparation Positive control Control Tylosin Gentamicin The number of sick pigs at the beginning of the experiment (heads) 20 20 20 20 Recovered - piglets (head) 18 16 16 15 - therapeutic effectiveness, % 90 80 80 75 The present invention is a compositional preparation for therapy for colorectal gastroenteritis in pigs by administering to piglets a composition of tylosin tartrate and gentamicin in a 1: 1 ratio. The compositional preparation is effective for piglets from 2 4 BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 https://doi.org/10.1051/bioconf/20213905003 weeks to 4 months of age. The use of a developed and tested composite preparation with feed is the most technologically advanced and economically feasible method. Example No. 2 weeks to 4 months of age. The use of a developed and tested composite preparation with feed is the most technologically advanced and economically feasible method. Example No. 2 Composite preparation for the treatment of gastroenteritis in dysentery. The present invention relates to veterinary medicine and can be used for the treatment of piglet gastroenteritis caused by brachispira. Known drugs for the treatment of gastroenteritis of pigs using various antibacterial agents: tetracyclines, oxamycins, macrolides and fluoroquinolones, as well as immunoglobulin-C. It is known that fradizin and other tylosin preparations have therapeutic efficacy in piglet gastroenteritis [1. 2, 3, 4, 5, 10, 11]. However, they have a narrowly targeted antibacterial effect [1]. Tylosin is one of the preparations for microbiological synthesis. BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 The introduction of the composite preparation can be started when the first signs of gastroenteritis appear in piglets. The use of a developed and tested composite preparation with feed is the most technologically advanced and economically feasible method. 4 Conclusions Thus, the development of composite prophylactic and therapeutic agents realizes the possibility of reducing the doses of individual pharmacological pharmacological agents and thereby reduces the drug load on the main components of organic farming and agriculture: soil - plants - feed - animals - humans. 5 5 https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021) MTSITVW 2021 References 1. V.A. Antipov, B.C. Grishkevich, Technical preparations of antibiotics - effective therapeutic and prophylactic means for veterinary medicine and animal husbandry, Collection of scientific works Pharmacology and toxicology of new drugs and feed additives in veterinary medicine, 171-173 (L., 1990) 2. V.D. Bukhanov, Therapy and prevention of pig dysentery, 24 (Author. Dis ... Cand. vet. n. M., 1986) 3. N.P. Zuev, V.D. Bukhanov, Preparation and development of antimicrobial compositions based on tylosin-containing preparations, Materials of the first congress of veterinary pharmacologists of Russia, 311-316 (Voronezh, RAAS, VNIVIPF and T. - S., 2007) 4. N.P. Zuev, V.D. Bukhanov, Therapeutic efficacy of composite tylosin-containing drugs in acute experience, Materials of the first congress of veterinary pharmacologists of Russia, 307-311 (Voronezh, RAAS, VNIVIPF, 2007) 5. N.P. Zuev, V.A. Shumsky, A.M. Kovalenko, V.Yu. Kovaleva, E.E. Zueva, V.V. Aristov, V.V. Kontsevenko, The use of tylosin preparations in animal husbandry and veterinary medicine, (Monograph Belgorod, 2018) 6. Concept on organic agriculture, (Belgorod, 2013) 7. RU, 2 033 172, C1, A61K 35/60 (1995.01), (20.04.1995) 8. RU 2 412 702, C1, A61K31 / 351 (2006.01), A61K 31/70 (2006.01), A61 K31 / 341 (2006.01), A61 K31 / 4196 (2006.01), A61 P31 / 09 (2006.01) ( ), ( ), ( ) 9. RU 2 337 670 C2, A61 K31 / 00 (2006.01), A61 9/48 (2006.01), A61D 7/00 (2006.01) 9. RU 2 337 670 C2, A61 K31 / 00 (2006.01), A61 9/48 (2006.01), A61D 7/00 (2006.0 10. I.Ye. Mozgov, Pharmacology, 416 (M.: Agropromizdat, 1985) 11. A.M. Makukhina, A.G. Rezvykh, О.А. Gavrilova et al., Description of the invention to the copyright certificate (11), 681598 (1981) 12. Z. Sevryuk, Environmental problems of pathology, pharmacology and therapy of animals. - International coordination meeting, 346-348 (Voronezh) 12. Z. Sevryuk, Environmental problems of pathology, pharmacology and therapy of animals. - International coordination meeting, 346-348 (Voronezh) 6
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https://sui-generis.ch/article/download/sg.173/1773
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Mehrheitsfähige Klimapolitik
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sui generis Atila Stocker Mehrheitsfähige Klimapolitik Ein Kommentar zu «Whistleblowing für das Weltklima» von Marc Thommen und Jascha Mattmann, erschienen im Januar 2021 in sui generis Am 13. Januar 2020 hat das Bezirksgericht Lausanne ein aufsehenerregendes Urteil gefällt, indem es zwölf Klimaaktivisten, die in einer Credit-Suisse-Filiale Tennis gespielt hatten, in allen Anklagepunkten freigesprochen hat. Das Gericht wurde scharf dafür kritisiert, dass es einen rechtfertigenden Notstand (Art. 17 StGB) angenommen hat. Einige Autoren beliessen es jedoch nicht bei der blossen Kritik, sondern brachten gleich eine alternative Begründung vor, um von der Bestrafung der Aktivisten abzusehen. Der Autor geht hier auf den Rechtfertigungs- bzw. Schuldausschlussgrund der Wahrung berechtigter Interessen ein, welcher von Marc Thommen und Jascha Mattmann ins Feld geführt wurde. I. Zugrunde liegendes Urteil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Gegenstand des Kommentars .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Wahrung berechtigter Interessen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Berechtigtes Ziel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Eignung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Erforderlichkeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Fazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 104 104 104 105 105 106 Zitiervorschlag: Atila Stocker, Mehrheitsfähige Klimapolitik, sui generis 2021, S. 103 Atila Stocker, stud. iur., juristischer Mitarbeiter Switzerlend AG (atila.stocker@uzh.ch). URL: sui-generis.ch/173 DOI: https://doi.org/10.21257/sg.173 Dieses Werk ist lizenziert unter einer Creative Commons Namens­ nennung — Weitergabe unter gleichen Bedingungen 4.0 International Lizenz. 103 I. 1 Zugrunde liegendes Urteil Durch das Urteil des Bezirksgerichts Lausanne PE19.000742/PCL/llb vom 13. Januar 2020 wurde ein Dutzend Klimaaktivisten, die in einer Credit-Suisse-­ Filiale Tennis gespielt hatten, vom Vorwurf des Hausfriedensbruchs, der Hinderung von Amtshandlungen und der unbewilligten Demonstration freigesprochen.1 Die Aktivisten wollten mit ihrer Aktion auf die Beteiligung der Banken an der Klimaerwärmung aufmerksam machen. Der Richter nahm an, dass sämtliche Taten durch Notstand gerechtfertigt waren. Er begründete diesen Schluss damit, dass der Klimawandel eine imminente Gefahr für die Menschheit und somit auch für die Beschuldigten darstelle und der Tennis-Match das mildest mögliche geeignete Mittel war, um diese Gefahr abzuwenden. Der Entscheid wurde vielfach kritisiert.2 Das Urteil wurde durch die Folgeinstanz aufgehoben.3 II. Gegenstand des Kommentars 2 Am 20. Januar 2021 haben Marc Thommen und Jascha Mattmann eine Besprechung des Lausanner Bezirksgerichtsurteils unter dem Titel «Whistleblowing für das Weltklima» publiziert. Die Autoren gehen darin zuerst auf den Vorwurf des Ungehorsams gegenüber der Polizei ein und besprechen anschliessend die möglichen Rechtfertigungs- und Schuldausschlussgründe für die Tat. Sie stellen richtigerweise fest, dass entgegen dem Bezirksgericht nicht von einem rechtfertigenden Notstand ausgegangen werden kann, da die begangene Tat nicht geeignet war, das Leben der Aktivisten oder Dritter zu retten.4 Auch die Voraussetzungen eines übergesetzlichen Notstands sehen sie als nicht erfüllt an, weil die Aktion auch nicht geeignet war, kollektive Rechtsgüter wie das 1 2 Urteil des Bezirksgerichts Lausanne PE19.000742/PCL/llb vom 13. Januar 2020; grosses Medienecho in den darauffolgenden Tagen vgl. NZZ vom 14. Januar 2020, S. 1 (Freispruch für Klimaaktivisten) und Tages-Anzeiger vom 15. Januar 2020, S. 4 («Das Lausanner Urteil lässt aufhorchen»). Vgl. Lucien Scherrer, Richter im Aktivistenmodus, NZZ vom 15. Januar 2020, S. 11; Hansueli Schöchli, «Das würde zum Chaos führen», NZZ vom 15. Januar 2020, S. 13; Katharina Fontana, Freipass für den Rechtsbruch, Weltwoche vom 16. Januar 2020, S. 31; Markus Melzl, Rechtsprechung mit Zeitgeist verwechselt, Basler Zeitung vom 20. Januar 2020, S. 2; Grace Schild Trappe / Felix Schöbi, Not kennt kein Gebot beim Hausfriedensbruch?, Jusletter vom 11. Mai 2020. Mit abweichender Argumentation kamen einige Autoren jedoch ebenfalls zur Straflosigkeit: Andrés Payer, Klimawandel als strafrechtlicher Notstand, sui generis 2020, S. 226; Marc Thommen / Jascha Mattmann, Whistleblowing für das Weltklima, sui generis 2021, S. 13; Martino Mona, in: Watson vom 15. Januar 2020 (Weshalb sich Experten nach dem Freispruch der Klimaaktivisten an den Kopf fassen). Weltklima zu retten.5 In der Folge prüfen die Autoren die Wahrung berechtigter Interessen sowohl als Rechtfertigungs- als auch als Schuldausschlussgrund und kommen zum Schluss, dass die Aktivisten kein Schuldvorwurf trifft.6 Diese Argumentation soll im Folgenden näher beleuchtet und deren Schwachstellen aufgezeigt werden. III. Wahrung berechtigter Interessen Das Bundesgericht hat die Wahrung berechtigter Interessen als Rechtfertigungsgrund anerkannt, wenn die Tat das mildest mögliche Mittel darstellt, ein berechtigtes Ziel zu erreichen (Eignung, Erforderlichkeit) und sie offenkundig weniger schwer wiegt als die gewahrten Interessen (Verhältnismässigkeit).7 1. Berechtigtes Ziel Die Autoren führen aus, dass ein berechtigtes Interesse vorliegt, wenn das angestrebte Ziel entweder sozial erwünscht ist oder in der Ausübung verfassungsmässiger Freiheitsrechte liegt.8 Es wird argumentiert, dass sich aus der Ratifizierung des Pariser Klimaübereinkommens9 ergibt, dass es sich beim Klimaschutz um ein sozial erwünschtes Ziel handelt. Dem ist grundsätzlich nicht zu widersprechen, jedoch ist die Formulierung des Ziels derart allgemein gefasst, dass wohl kaum singuläre, und nicht unmittelbar themenbezogene Handlungen direkt darauf hinstreben können. Vielmehr stellt sich die Frage, auf welchem Weg dieses grossmehrheitlich anerkannte Ziel umgesetzt wird. Die Klimaaktivisten im vorliegenden Fall möchten jegliche Investitionen in fossile Energien durch Schweizer Banken unterbinden.10 Ein solch radikales Vorgehen wird von der Mehrheit der Bevölkerung nicht unterstützt und es ist somit fraglich, ob es noch als sozial erwünscht betrachtet werden kann.11 Sozial erwünscht bedeutet von der Gesellschaft gewollt. 5 Thommen/Mattmann (Fn. 2), N 28. 6 Thommen/Mattmann (Fn. 2), N 36 in fine. 7 BGE 134 IV 216 E. 6.1. 8 Thommen/Mattmann (Fn. 2), N 29; so auch Payer (Fn. 2), N 23. 9 Übereinkommen von Paris vom 12. Dezember 2015 (SR 0.814.012), in Kraft getreten für die Schweiz am 5. November 2017. 10 «Ensemble pour […] pousser CS à cesser ses investissements dans les énergies fossiles !» Lausanne Action Climat auf Facebook am 23. November 2018. 4 Thommen/Mattmann (Fn. 2), N 27; so auch Trappe/Schöbi (Fn. 2), N 17; Payer (Fn. 2), N 18; Urteil des Kantonsgerichts Waadt Nr. 371 PE19.000742/PCL vom 22. September 2020 E. 6.3. 11 Auf eine staatliche Regulierung des Finanzplatzes Schweiz in Bezug auf klimaschädliche Investitionen wurde im neulich verabschiedeten CO2 -Gesetz (Bundesgesetz über die Verminderung von Treibhausgasemissionen vom 25. September 2020, BBl 2020 7847) bewusst zugunsten der Selbstregulierung verzichtet (Ablehnung Antrag Levrat, AB S 2019 925 ff.). Auch der Bundesrat zeigt in seinem Bericht «Nachhaltigkeit im Finanzsektor Schweiz: Eine Auslegeordnung und Positionierung mit Fokus auf Umweltaspekte» vom 24. Juni 2020 (insb. Ziff. 4) verschiedene Massnahmen auf, die auf die Eigenverantwortung der Finanzinstitute und der Anleger setzen und verzichtet klar auf staatliche Investitionsverbote. Atila Stocker, Mehrheitsfähige Klimapolitik, sui generis 2021, S. 103 104 3 Urteil des Kantonsgerichts Waadt Nr. 371 PE19.000742/PCL vom 22. September 2020. 3 4 Der Wille einer Gesellschaft bestimmt sich aus dem Willen der Einzelnen, welcher sich in den politischen Mehrheiten widerspiegelt. 5 Den Autoren zufolge ist das Vorgehen der Aktivsten zudem grundrechtlich geschützt durch Art. 16 und 22 BV12, sowie Art. 10 und 11 EMRK13.14 Sie verkennen dabei die bundesgerichtliche Rechtsprechung, aus der klar hervorgeht, dass die Versammlungsfreiheit nicht das Recht beinhaltet, sich auf fremdem Boden zu versammeln.15 Dies ergibt sich bereits daraus, dass die Credit Suisse mangels Drittwirkung nicht an die genannten Grundrechte gebunden ist.16 Auch Art. 10 und 11 EMRK sehen ein solches Recht nicht vor.17 6 Das von den Aktivisten verfolgte Ziel scheint somit weder sozial erwünscht, noch liegt es in der Ausübung verfassungsmässiger Freiheitsrechte. 2. 7 Die Rechtfertigung der Aktivisten scheitert zusätzlich an der Eignung ihrer Handlungen, denn nur Handlungen, die geeignet sind, das berechtigte Interesse zu wahren, können gerechtfertigt sein. Es steht fest, dass der unbe­ willigte Aufenthalt in der Credit-Suisse-Filiale weder geeignet ist, die Klimaerwärmung zu stoppen, noch geeignet ist, einen Investitionsstopp für fossile Energien herbeizuführen. Die Autoren umgehen diese Frage, indem sie prüfen, ob der Tennis-Match geeignet war, die Aufmerksamkeit der Öffentlichkeit auf die Klimapolitik zu richten.18 Sie legen jedoch nicht dar, inwieweit es sich beim Aufmerksammachen der Öffentlichkeit um ein sozial erwünschtes oder grundrechtlich geschütztes Ziel handelt. Dies geht nicht aus dem Pariser Klimaabkommen hervor, das darauf abzielt, dass die Mitgliedstaaten einen direkten Beitrag zum Klimaschutz leisten und nicht bloss öffentlich darauf aufmerksam machen. 3. 8 Eignung Erforderlichkeit Bei der Frage nach der Erforderlichkeit der Handlungen ist zu prüfen, ob mildere, insbesondere legale Mittel hät12 Bundesverfassung der Schweizerischen Eidgenossenschaft vom 18. April 1999 (BV; SR 101). 13 Konvention zum Schutze der Menschenrechte und Grundfreiheiten, abgeschlossen in Rom am 4. November 1950 (EMRK; SR 0.101). 14 Thommen/Mattmann (Fn. 2), N 30. 15 «La liberté de réunion [...] ne comprend en tout cas pas le droit de s’assembler sur le fonds d’autrui.» (BGE 97 I 911 E. 3a). 16 Ulrich Häfelin / Walter Haller / Helen Keller / Daniela Thurnherr, Schweizerisches Bundesstaatsrecht, 10. Auflage, Zürich 2020, N 279. ten gewählt werden können, um das berechtigte Ziel zu erreichen.19 «In einem demokratischen Rechtsstaat sind politische und ideelle Anliegen grundsätzlich auf politischem Wege bzw. auf dem Rechtsweg zu verfolgen»20, hält das Bundesgericht zutreffend fest. Die Autoren folgen hier der Argumentation des Bezirksrichters, dass die Aktivisten bereits alle legalen Mittel, insbesondere den Weg über die Politik, ausgeschöpft hätten und diese erfolglos geblieben seien.21 Es ist jedoch fraglich, ob dies in Bezug auf einen Investitionsstopp in fossile Energien tatsächlich zutrifft. Das Bezirksgericht führt aus, dass der Bundesrat ungenügend auf entsprechende Vorstösse von Bundesparlamentariern eingegangen ist.22 Es wurden etliche Interpellationen zu diesem Thema eingereicht, welche allesamt vom Bundesrat ordnungsgemäss beantwortet wurden.23 Das einzige eingereichte Postulat wurde vom Nationalrat abgelehnt und zwei Motionen wurden abgeschrieben, weil sie nicht innert zwei Jahren behandelt wurden, und von den entsprechenden Parlamentsmitgliedern nicht wieder aufgegriffen wurden, wodurch sich der Bundesrat nie damit beschäftigen muss­te.24 Zusätzlich hätte das Anliegen auf dem Weg der parlamentarischen Initiative oder der Volksinitiative durchgesetzt werden können. Es ist falsch, der Regierung hier einen Vorwurf zu machen, vielmehr muss man zum Schluss gelangen, dass die politischen Mittel nicht genügend ausgeschöpft wurden. Selbst wenn man davon ausgeht, dass alle politischen Möglichkeiten erfolglos ausgeschöpft wurden, «gibt [dies] Letzteren [den Aktivisten] kein Recht, ihre Anliegen mit strafbaren Methoden zu verfolgen.»25 Wenn radikale Klimaschutzmassnahmen, wie sie die Aktivisten fordern, auf dem Weg der politischen Mitsprache nicht 19 BGE 134 IV 216 E. 6.1. 20 BGE 129 IV 6 E. 3.1. 21 Thommen/Mattmann (Fn. 2), N 32. 22 Urteil des Bezirksgerichts Lausanne PE19.000742/PCL/llb vom 13. Januar 2020, S. 54. 23 Interpellation Thorens (15.3613) «CO2 -Fussabdruck von Investitionen senken (Desinvestition aus fossilen Energieträgern oder ‹Divestment›). Handeln im Hinblick auf die Klimakonferenz in Paris» vom 17. Juni 2015; Interpellation Girod (15.4109) «Klimaschutz und Finanzplatzrisiken. Wie setzt die Schweiz die Empfehlungen um?» vom 8. Dezember 2015; Interpellation Chevalley (17.3120) «Was tut die Schweiz für nachhaltige Finanzen?» vom 15. März 2017; Interpellation Jans (17.3914) «Wann wird der Finanzsektor in die Pflicht genommen?» vom 29. September 2017; Interpellation Jans (17.3915) «Wann kontrolliert die Finma die Klimarisiken?» vom 29. September 2017; Interpellation Thorens (17.3946) «Ein Nachhaltigkeitstest für die künftigen Regulierungen der Finanzmärkte?» vom 29. September 2017; Interpellation Landolt (17.4104) «Zukunftsorientiertes Investieren» vom 13. Dezember 2017. 17 Vgl. Guide on Article 11 of the European Convention on Human Rights, herausgegeben vom Europäischen Gerichtshof für Menschenrechte am 31. Dezember 2020, N 21. 24 Postulat Chevalley (18.3589) «Instrumente zur Überprüfung nachhaltiger Finanzen» vom 14. Juni 2018; Motion Masshardt (18.3918) «Klimastrategie für den Schweizer Finanzmarkt» vom 27. September 2018; Motion Jans (18.3974) «Den grössten Klimaheizern den Geldhahn zudrehen» vom 27. September 2018. 18 Thommen/Mattmann (Fn. 2), N 31. 25 BGE 129 IV 6 E. 3.1. Atila Stocker, Mehrheitsfähige Klimapolitik, sui generis 2021, S. 103 105 9 durchgesetzt werden können, so zeigt dies, dass sie nicht von der Mehrheit der stimm- und wahlberechtigten Bevölkerung unterstützt werden. Dies wiederum lässt darauf schliessen, dass diese Massnahmen nicht sozial erwünscht sind. Die Argumentation der Autoren ist widersprüchlich, wenn sie die Forderungen der Aktivisten einerseits als sozial erwünscht, aber andererseits als demokratisch nicht durchsetzbar, also nicht mehrheitsfähig betrachten. Die Wahrung berechtigter Interessen ist im vorliegenden Fall weder als Rechtfertigungs-, noch als Schuldausschlussgrund anwendbar, da die Aktivisten (1) kein berechtigtes Interesse verfolgen, (2) ihre Aktion nicht Die Argumentation nach Thommen und Mattmann würde darauf hinauslaufen, dass zu illegalen Mitteln gegriffen werden darf, wenn ein Ziel auf politischem Wege nicht erreicht werden kann. Dahinter versteckt sich der Gedanke, dass es erstrebenswerte Ziele gibt, die von der Mehrheit der Bevölkerung nicht erkannt würden. Dies steht dem Konzept der Demokratie entgegen, da es sich in einer Demokratie gerade aus der Mehrheit ergibt, was die erstrebenswerten Ziele und der richtige Weg sind, um diese zu erreichen. Es bleibt unumstritten, dass auch dem Volkswillen Grenzen gesetzt sind, namentlich durch das ius cogens. Eine lasche Klimapolitik steht damit jedoch nicht im Widerspruch. Atila Stocker, Mehrheitsfähige Klimapolitik, sui generis 2021, S. 103 106 IV. Fazit 10 geeignet war, das verfolgte Interesse zu erreichen, und (3) der Grundsatz der Erforderlichkeit nicht erfüllt wurde, da mildere Mittel hätten gewählt werden können. 11
https://openalex.org/W2067093123
https://www.scielo.br/j/csp/a/bHJJCkxfh7pCKmY8rvdq5LJ/?lang=pt&format=pdf
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Os Arquivos Brasileiros de Nutrição: uma revisão sobre produção científica em nutrição no Brasil (1944 a 1968)
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3 0 3 A RTIGO A RT I C L E 3 0 3 A RTIGO A RT I C L E 3 0 3 A RTIGO A RT I C L E Os A rquivos Brasileiros de Nutrição: uma revisão sobre produção científica em nutrição no Brasil (1944 a 1968) Os A rquivos Brasileiros de Nutrição: uma revisão sobre produção científica em nutrição no Brasil (1944 a 1968) A rquivos Brasileiros de Nutrição: a review of scientific re s e a rch on nutrition in Brazil from 1944 to 1968 A rquivos Brasileiros de Nutrição: a review of scientific re s e a rch on nutrition in Brazil from 1944 to 1968 Francisco de Assis Guedes de Vasconcelos 1 Abstract This study re v i ews 209 original articles published in the journal Arq u i vos Bra s i l e i ro s de Nutrição (1944/1968), a periodical edited by Josué de Ca s t ro, p h y s i c i a n , specialist in nutri- t i o n , and founder- d i rector of the Institute of Nutrition at the Un i versity of Brazil (now the Fe d e r- al Un i versity of Rio de Ja n e i ro ) . Our methodology was based on quantitative and qualitative a n a l y s e s , aimed at summarizing both the topic itself and the authors’ b a c k g ro u n d s . Re s u l t s s h owed that 134 of the articles (64%) adopted a biological perspective to nutrition, mostly focus- ing on labora t o ry re s e a rch concerning the chemical composition and nutritional value of Bra z i l- ian foodstuffs. On the other hand, 75 articles (36%) took a social perspective , testifying to the first efforts by Brazilian nutritional ex p e rts to create and improve specific methodological tools for investigating our population’s nutritional conditions, t h e re by helping to consolidate the field of nutrition in the country. 1 De p a rtamento de Nu t r i ç ã o, Ce n t ro de Ciências da Saúde, Un i versidade Fe d e ral de Santa Ca t a r i n a . Campus Un i versitário – Tr i n d a d e , Fl o r i a n ó p o l i s ,S C 8 8 0 4 0 - 9 0 0 , Bra s i l . Francisco de Assis Guedes de Vasconcelos 1 I n t ro d u ç ã o En t re as investigações citadas acima, consi- d e ramos que apenas duas, apesar de suas limi- t a ç õ e s, re a l i z a ram revisões críticas específicas s o b re os Arq u ivos Bra s i l e i ros de Nu t r i ç ã o. O es- tudo de Co i m b ra et al. (1982) sobre a Hi st ór i a da Alimentação Escolar no Bra s i l, uma das pri- m e i ras e mais completas das sistematizações s o b re este tema específico, apresenta impor- tantes informações e comentários a re s p e i t o daquele peri ó d i c o. L’ Abbate (1982, 1988), por sua vez, ao estudar as Pol ít icas de Al i m e n t a ç ã o e Nutrição no Bra s i l, também dedica uma part e de sua atenção à revisão bibliográfica narra t i va do saber científico que foi produzido pelos in- telectuais do campo da nutrição e divulgado a t ravés dos Arq u ivo s. Nos últimos vinte anos, embora muito limita- d a s, foram realizadas importantes inve s t i g a- ções abordando diferentes aspectos acerca do p rocesso de constituição do campo da nutri ç ã o no Brasil, considerada como disciplina, política social e/ou profissão (Ca s t ro, 1977; Co i m b ra et al., 1982; L’ Ab b a t e, 1982, 1988; Natal, 1982; Bo s i , 1988, 1995; Sa n t o s, 1988; Va s c o n c e l o s, 1988; Pra d o, 1993; Lima, 1997; Ma g a l h ã e s, 1997). Nessas distintas inve s t i g a ç õ e s, são sempre o b s e rvadas referências ao periódico Arq u ivo s Bra s i l e i ros de Nu t r i ç ã o, considerado uma das p rincipais fontes documentais da história desse campo específico do saber no Brasil. I n t ro d u ç ã o A publica- ção dessa revista científica, que teve início em maio de 1944, ainda no transcurso do chama- do Estado Novo e da Segunda Gu e r ra Mu n d i a l , constitui uma das inúmeras idealizações da t ra j e t ó ria político-intelectual do eminente mé- dico-nutrólogo pernambucano Josué de Ca s t ro (1908-1973) (Co i m b ra et al., 1982; L’ Ab b a t e, 1988). Por meio de um convênio estabelecido e n t re o Se rviço Técnico da Alimentação Na c i o- nal (STAN) – órgão da Co o rdenação da Mo b i l i- zação Econômica, instituída pelo Pre s i d e n t e Getúlio Va rg a s, em setembro de 1942, em de- corrência da Segunda Gu e r ra Mundial – e a Nu- t r ition Fo u n d a t i o n , de Nova Io rque – órg ã o n o rt e - a m e ricano re s p o n s á vel pela publicação da conceituada Nut r ition Re v i ew s –, Josué de Ca s t ro criou os Arq u ivos Bra s i l e i ros de Nu t r i ç ã o com os seguintes objetivos: a) “contribuir com a difusão de conhecimentos seguros da Ci ê n c i a da Nutrição para a formação deste corpo de nu- t r i c i o n i s t a s , especializados em quaisquer dos d i f e rentes setores da matéria” e b) “dar um co- nhecimento mais efetivo e mais amplo dos re- sultados dos trabalhos e dos estudos re a l i z a d o s no Brasil no campo da Nu t r i ç ã o” ( Ca s t ro, 1944: 5-6). Ainda em agosto de 1944, a publicação dos Arq u ivo s, cuja periodicidade deve ria ser mensal, foi interrompida após a edição do q u a r- to número do seu pri m e i ro vo l u m e, em ra z ã o da extinção do STAN e da Co o rdenação de Mo- bilização Econômica. I n t ro d u ç ã o Em nove m b ro de 1946, os Arq u ivo s vo l t a ram a ser editados sob a re s- ponsabilidade do então Instituto de Nu t ri ç ã o da Un i versidade do Brasil (Inub), atual In s t i t u- to de Nu t rição Josué de Ca s t ro da Un i ve r s i d a d e Fe d e ral do Rio de Ja n e i ro (Arq u i vos Bra s i l e i ro s de Nu t ri ç ã o, 1946; Ca s t ro, 1946). Segundo algu- mas análises, até deze m b ro de 1968, mês de c i rculação do último volume do peri ó d i c o, d e n- t ro e fora deste espaço acadêmico-institucio- nal, os Arq u ivo s c o n s t i t u í ram-se no mais im- p o rtante veículo de difusão do saber científico Assim sendo, o presente estudo tem como o b j e t i vo geral realizar uma reflexão acerca da p rodução científica no campo da nutrição no Brasil, enfocando os artigos originais publica- dos nos Arq u ivos Bra s i l e i ros de Nu t r i ç ã o, n o período de 1944 a 1968. Quanto aos objetivo s e s p e c í f i c o s, a proposta desse trabalho é: a) re a- lizar uma análise quantitativa dos artigos pu- blicados; b) traçar um perfil dos autores dos ar- tigos no que se re f e re a suas identidades pro f i s- s i o n a i s, seus vínculos institucionais e seus campos específicos de conhecimento; c) iden- tificar os conteúdos temáticos abordados pelos a rtigos e d) construir subsídios para uma re f l e- xão sobre a tra j e t ó ria do campo da nutrição em saúde pública no Bra s i l . 3 0 3 A RTIGO A RT I C L E em alimentação produzido no País (Ca s t ro, 1946; Co i m b ra et al., 1982; L’ Ab b a t e, 1988). 3 0 3 A RTIGO A RT I C L E Key words Nut r it i o n ; Hi s t o ry of Nu t r i t i o n ; Serial Pu b l i c a t i o n s ; Pe r i o d i c a l s Resumo Este trabalho consiste em uma revisão de 209 artigos originais publicados pelos A rq u i- vos Bra s i l e i ros de Nu t ri ç ã o ( 1 9 4 4 / 1 9 6 8 ) , um periódico editado pelo médico-nutrólogo Josué de Ca s t ro, d i re t o r-fundador do Instituto de Nutrição da Un i versidade do Brasil (atual Un i ve r s i d a d e Fe d e ral do Rio de Ja n e i ro ) . A metodologia utilizada baseou-se na realização de uma análise q u a n t i - q u a l i t a t i va , buscando sumariar tanto os conteúdos temáticos abord a d o s , como o perf i l dos autores dos art i g o s . Os resultados deste estudo re ve l a m , por um lado, que 134 destes art i g o s (64%) foram inseridos dentro da perspectiva biológica da nutrição e corre s p o n d e m , na maioria, a pesquisas laboratoriais sobre a composição química e o valor nutricional de alimentos nacio- n a i s . Por outro lado, a análise dos 75 artigos (36%) incluídos na perspectiva social atesta o esfor- ço dos primeiros nutrólogos bra s i l e i ros em construir e aperfeiçoar os instrumentos metodológi- cos específicos para investigar as condições nutricionais de nossa população, bem como em con- tribuir para a consolidação do campo da nutrição no Bra s i l . Palavras-chave Nut r iç ã o ; História da Nu t r i ç ã o ; Publicações Se r i a d a s ; Pe r i ó d i c o s Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 3 0 4 VASCONCELOS, F. A. G. em alimentação produzido no País (Ca s t ro, 1946; Co i m b ra et al., 1982; L’ Ab b a t e, 1988). Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 M e t o d o l o g i a De forma semelhante, os a rtigos da perspectiva social foram classifica- dos em outros cinco campos disciplinares ou temáticos da nutrição: avaliação nutricional de populações; política e planejamento em ali- mentação e nutrição; epidemiologia nutri c i o- nal; determinantes do estado nutricional de populações e educação nutricional. Tal classifi- cação corresponde a um constructo metodoló- gico que estabelecemos baseados na atual con- f o rmação desses campos disciplinares ou te- máticos no interior da nutri ç ã o. A opção por investigar apenas a seção Art i- gos Originais dos Arq u ivo s re p resenta, antes de t u d o, um re c o rte metodológico, uma via de a p roximação para melhores aprofundamento e c o m p reensão sobre a produção científica em n u t ri ç ã o, ao longo do extenso período analisa- d o. Cada artigo original (nossa unidade de aná- lise) foi examinado conforme um ro t e i ro de in- vestigação construído para possibilitar tanto a análise quantitativa e de categorização dos ar- tigos por volume editado, como a análise das c a racterísticas dos autores envo l v i d o s. De s s a f o rma, além da elaboração de listagem dos 209 a rt i g o s, conforme normas bibliográficas (nome de autore s, ano, título, nome do peri ó d i c o, vo- l u m e, número, páginas inicial e final e mês), cada artigo foi categorizado de acordo com os seguintes atributos: número de autores; núme- ro de páginas; apresentação de referências bi- bliográficas e outras condições de publicação definidas pelo periódico; vínculo institucional e identidade profissional do(s) autor(es); natu- reza da pesquisa que serviu de base para o art i- go e conteúdo temático abord a d o. Os resultados desse estudo são apre s e n t a- dos sob a forma de tabelas de distribuição de f reqüência dos atributos inve s t i g a d o s. M e t o d o l o g i a a revista pre s t a va homenagem a uma pessoa n o t á vel dentro do cenário universal ou nacio- nal da nutrição; 2a) Ed i t o rial, onde os diri g e n- tes da revista apre s e n t a vam abordagens con- j u n t u rais sobre os mais diversificados temas; 3a) Artigos Ori g i n a i s, que foram objetos de nos- sa reflexão; 4a) Recentes Aquisições da Nu t ro- logia, composta por resenhas dos artigos pu- blicados na Nut r ition Re v i ew s, quase sempre a seção mais extensa do periódico; 5a) Re s e n h a Nacional de Nu t ri ç ã o, composta por re s e n h a s de artigos e de outras modalidades de publica- ções editadas no Brasil; 6a) Tra n s c ri ç õ e s, com- posta por artigos publicados anteri o rmente em o u t ros periódicos; 7a) Cursos e Co n f e r ê n c i a s, onde são apresentadas bre ves inform a ç õ e s, re- s u m o s, pro g ra m a s, agendas e/ou currículos de e ventos científicos, e 8a) Notas e In f o rm a ç õ e s, a p resentação de rel e as e s s o b re uma miscelâ- nea de temas relacionados ao campo da nutri- ç ã o. Tais seções, com exceção das três últimas, m a n t i ve ram-se sem alteração do pri m e i ro ao último volume do peri ó d i c o. Em seguida, na tentativa de garantir inteli- gibilidade à nossa análise, os artigos da pers- p e c t i va biológica foram classificados em qua- t ro campos disciplinares ou temáticos da nu- t rição: nutrição básica e experimental; ciência e tecnologia de alimentos; nutrição clínica ( i n- c l u i n d o f is i ol ogia, patologia e dietoterapia) e n u t rição e dietética. M e t o d o l o g i a O universo do nosso estudo foi constituído por um total de 209 trabalhos publicados na seção de artigos originais dos 24 volumes (76 fascícu- los) dos Arq u ivos Bra s i l e i ros de Nu t r i ç ã o, edita- dos no período de maio de 1944 a deze m b ro de 1968. Cabe ressaltar que, de acordo com as p rincipais condições de publicação dos Arq u i- vo s ( i n s t ruções que não se altera ram ao longo da tra j e t ó ria do periódico), naquela seção se- riam aceitos para publicação trabalhos inédi- tos de autores nacionais sobre nutrologia, que d e ve riam ve r s a r, de modo original, sobre os te- mas a que se pro p u s e ram; deve riam, ainda, a p resentar resumo em português e, se possíve l , em inglês e espanhol, seguindo as normas bi- bliográficas do Ind ex Me d i c u s. De s t a c a m o s, também, que os Arq u ivo s se subdividiam nas seguintes seções seqüenciais: 1a) Grandes Vu l- tos da Nu t rologia, na qual, a cada número, por meio de fotografia e de uma síntese biográfica, Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 0 5 m o d o, os artigos categorizados dentro da pers- p e c t i va biológica originam-se e/ou atendem às finalidades de três das quatro seções org a n i z a- t i vas do então Inub: a Seção de Pesquisas Bi o- l ó g i c a s, a Seção de Patologia da Nu t rição e, de c e rta forma, a Seção de Educação Alimentar. Ig u a l m e n t e, os artigos categorizados dentro da p e r s p e c t i va social originam-se e/ou atendem às finalidades da Seção de Pesquisas Ec o n ô m i- c o - Sociais da re f e rida instituição (Arq u i vo s Bra s i l e i ros de Nu t ri ç ã o, 1946:71-73). M e t o d o l o g i a Assim, p a ra cada um destes atri b u t o s, foi constru í d o um índice descri t i vo (número de artigos por volume; número de artigos por natureza da pesquisa e volume; número de páginas por ar- tigo; número de autores por artigo; apre s e n t a- ção de referências bibliográficas por naturez a da pesquisa; vínculo institucional do(s) au- tor(es) por artigo; identidade profissional do autor principal por artigo etc.), que será anali- sado na seqüência de apresentação dos re s u l- t a d o s. A trajetória dos A rq u ivos Brasileiros de N u t r i ç ã o: volumes, número de art i g o s , periodicidade e dire ç ã o Na seqüência dos procedimentos metodo- lógicos adotados, os artigos foram categori z a- dos dentro de duas modalidades de estudos: i n vestigações de natureza biológica (que cha- m a remos de perspectiva biológica) e inve s t i g a- ções de natureza social (que chamaremos de p e r s p e c t i va social). A ri g o r, tal pro c e d i m e n t o foi adotado pro c u rando-se seguir tanto as fina- l i d a d e s, como a estru t u ra de organização do In u b, idealizadas por Josué de Ca s t ro. De s s e A Tabela 1 apresenta a distribuição dos 209 ar- tigos originais analisados conforme o vo l u m e, bem como a indicação do ano e o cri t é rio de p e riodicidade de circulação da revista. Me- diante a análise do índice número de art i g o s por vo l u m e, observa-se que, ao longo do eixo longitudinal de edição da revista (1944 a 1968), f o ram publicados, em média, 8,7 artigos por Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 VASCONCELOS, F. A. G. 3 0 6 Tabela 1 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o volume (ano/periodicidade) do periódico, 1944-1968. A trajetória dos A rq u ivos Brasileiros de N u t r i ç ã o: volumes, número de art i g o s , periodicidade e dire ç ã o Volume (ano/periodicidade) No de art i g o s % % acumulado 1 (1944/mensal) 8 3 , 8 3 , 8 2 (1946/mensal) 3 1 , 4 5 , 2 3 (1947/mensal) 6 2 , 9 8 , 1 4 (1947/mensal) 1 0 4 , 8 1 2 , 9 5 (1948/bimestral) 6 2 , 9 1 5 , 8 6 (1949/bimestral) 1 4 6 , 7 2 2 , 5 7 (1950/bimestral) 1 1 5 , 3 2 7 , 8 8 (1951/bimestral) 1 0 4 , 8 3 2 , 6 9 (1953/trimestral) 5 2 , 4 3 5 , 0 10 (1954/trimestral) 4 1 , 9 3 6 , 9 11 (1955/semestral) 2 1 , 0 3 7 , 9 12 (1956/semestral) 2 1 , 0 3 8 , 9 13 (1957/semestral) 3 1 , 4 4 0 , 3 14 (1958/semestral) 6 2 , 9 4 3 , 2 15 (1959/semestral) 9 4 , 3 4 7 , 5 16 (1960/semestral) 1 2 5 , 7 5 3 , 2 17 (1961/semestral) 9 4 , 3 5 7 , 5 18 (1962/anual) 1 1 5 , 3 6 2 , 8 19 (1963/semestral) 1 8 8 , 6 7 1 , 4 20 (1964/semestral) 1 0 4 , 8 7 6 , 2 21 (1965/semestral) 1 5 7 , 1 8 3 , 3 22 (1966/semestral) 1 2 5 , 7 8 9 , 0 23 (1967/semestral) 1 3 6 , 2 9 5 , 2 24 (1968/anual) 1 0 4 , 8 1 0 0 , 0 To t a l 2 0 9 1 0 0 , 0 – de publicações, com uma média de 12 art i g o s por vo l u m e, tendo sido publicados 58,3% do total de artigos neste período. Tabela 1 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o volume (ano/periodicidade) do periódico, 1944-1968. Em seu último vo l u m e, re l a t i vo ao ano de 1968, a revista teve como dire t o r- re s p o n s á ve l o médico-nutrólogo Hélio Vecchio Alves Ma u- r í c i o, que, desde 1958, ocupava o cargo de re- d at o r- s e c re t á ri o. Os artigos originais: natureza da pesquisa, tamanho do artigo e normas bibliográficas A Tabela 2 reúne a apresentação de dois dos ín- dices construídos para a análise dos artigos dos Arq u ivo s: o número de artigos por natureza da pesquisa e volume e número de páginas por n a t u reza da pesquisa e vo l u m e. Em relação ao n ú m e ro de artigos por natureza da pesquisa e vo l u m e, observa-se que 134 (64,0%) dos 209 ar- tigos foram incluídos na perspectiva biológica, c o n t ra 75 (36,0%) associados à perspectiva so- cial. Quanto ao número de páginas por nature- za da pesquisa e vo l u m e, os resultados são se- m e l h a n t e s, apontando um total de 61,0% de páginas para a perspectiva biológica, contra 39,0% para a perspectiva social. vo l u m e, entretanto podem ser observados cor- tes tra n s versais que denotam marcantes osci- lações desse índice. No corte inicial (1944 a 1954), corre s p o n- dente à direção Josué de Ca s t ro, a perspectiva social detém apenas 30,0% dos artigos e 27,5% do número de páginas publicadas. Já no cort e final (1955 a 1968), apesar da perspectiva bio- lógica continuar com sua hegemonia quantita- t i va, observa-se um aumento re l a t i vo das pu- blicações da perspectiva social, a qual passou a deter 39,4% dos artigos e 48,3% do número de páginas publicadas. Assim, por exemplo, no período de 1944 a 1954, época em que os Arq u ivo s e ram diri g i d o s por Josué de Ca s t ro, tendo como secre t á ri o s / re d a t o res os médicos nutrólogos Pe d ro Bo rg e s (1946 a 1947) e Rubens de Si q u e i ra (1947 a 1953), foram publicados 36,9% do total de art i- g o s, com uma média de 7,7 artigos por vo l u m e. A trajetória dos A rq u ivos Brasileiros de N u t r i ç ã o: volumes, número de art i g o s , periodicidade e dire ç ã o Neste seu último ano de cir- c u l a ç ã o, assim como em 1962, a sua peri o d i c i- dade foi anual, com dez artigos publicados. A l i á s, é preciso destacar que, em seus últimos vo l u m e s, conforme atestam algumas notas ex- p l i c a t i vas contidas em artigos dos volumes 21, 23 e 24, o periódico circulou com bastante a t ra s o. Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 Os artigos originais: natureza da pesquisa, tamanho do artigo e normas bibliográficas Nesse interva l o, a revista, inicialmente com pe- riodicidade mensal, passou a ter uma circ u l a- ção bimestral (1948 a 1951) e depois tri m e s t ra l (1953 a 1954), enquanto re g i s t rou-se a inter- rupção de sua circulação nos anos de 1944 (as- sociada à extinção do STAN) e em 1952 (justifi- cada por motivo ss up eri ores não explicitados). A análise desses dois índices demonstra q u e, no decorrer de toda a série histórica de c i rculação do peri ó d i c o, a perspectiva biológi- ca esteve à frente da produção e difusão do sa- ber em nutri ç ã o. A perspectiva social, além de vir a reboque ao longo da séri e, não aparece no d e c o r rer de pelo menos cinco volumes da re- vista (3, 5, 12, 13 e 14), com exceção do período 1959-1964, momento em que, quantitativa- m e n t e, supera a perspectiva biológica. Co i n c i- d e n t e m e n t e, este período de auge da perspec- t i va social, conforme discutiremos adiante, c o r re s p o n d e, em termos históri c o - c o n j u n t u- En t re 1955 a 1967, sob a direção de Pe d ro Bo rg e s, que desde 1954 ocupava o cargo de re- d a t o r, passou a ter circulação semestral e, após s u p e rar a crise dos anos 1952-1958 (quando a p resentou os menores índices de publicação de artigos por volume), entrou na fase de auge Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 0 7 Tabela 2 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o número de artigos e o número de páginas por natureza da pesquisa (biológica ou social) e volume (ano) do periódico, 1944-1968. Os artigos originais: natureza da pesquisa, tamanho do artigo e normas bibliográficas Volume (ano) No de art i g o s No de páginas b i o l ó g i c a s o c i a l t o t a l b i o l ó g i c a s o c i a l t o t a l 1 (1944) 5 3 8 4 9 2 8 7 7 2 (1946) 1 2 3 1 6 5 5 7 1 3 (1947) 6 – 6 1 4 9 – 1 4 9 4 (1947) 9 1 1 0 1 4 9 7 1 5 6 5 (1948) 6 – 6 2 1 6 – 2 1 6 6 (1949) 9 5 1 4 1 5 2 6 0 2 1 2 7 (1950) 6 5 1 1 1 2 2 96 2 1 8 8 (1951) 5 5 1 0 1 7 8 1 4 6 3 2 4 9 (1953) 4 1 5 7 8 2 2 1 0 0 10 (1954) 3 1 4 7 4 3 5 1 0 9 11 (1955) 1 1 2 8 3 6 4 4 12 (1956) 2 – 2 4 3 – 4 3 13 (1957) 3 – 3 3 4 – 3 4 14 (1958) 6 – 6 9 4 – 9 4 15 (1959) 4 5 9 3 6 9 9 1 3 5 16 (1960) 5 7 1 2 6 8 1 1 8 1 8 6 17 (1961) 5 4 9 6 3 8 5 1 4 8 18 (1962) 5 6 1 1 6 0 1 2 1 1 8 1 19 (1963) 1 2 6 1 8 1 5 0 1 2 2 2 7 2 20 (1964) 5 5 1 0 5 6 1 1 1 1 6 7 21 (1965) 1 0 5 1 5 1 1 7 6 5 1 8 2 22 (1966) 6 6 1 2 7 5 9 3 1 6 8 23 (1967) 9 4 1 3 1 4 0 4 2 1 8 2 24 (1968) 7 3 1 0 7 2 5 7 1 2 9 To t a l 1 3 4 7 5 2 0 9 2 . 1 9 9 1 . 3 9 8 3 . Os artigos originais: natureza da pesquisa, tamanho do artigo e normas bibliográficas 5 9 7 % ( 6 4 , 0 ) ( 3 6 , 0 ) ( 1 0 0 , 0 ) ( 6 1 , 0 ) ( 3 9 , 0 ) ( 1 0 0 , 0 ) Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o número de artigos e o número de páginas por natureza da pesquisa (biológica ou social) e volume (ano) do periódico, 1944-1968. c), que perf a zem um total de 150 páginas; os de Mo u ra - Campos et al. (1951a e b), que perf a ze m um total de 104 páginas; e os de Orsini ( 1 9 4 7 a , b), com 92 páginas. ra i s, a um momento de grande mobilização so- cial no País (movimento das Ligas Ca m p o n e- sas; movimentos urbanos pelas re f o rmas de ba- s e, incluindo a re f o rma agrária etc.) e que c u l- mina no golpe militar de março de 1964 (Alen- car et al., 1979; Aze v ê d o, 1982; Vi e i ra, 1983). Em relação à distribuição do índice apre- sentação de referências bibliográficas por na- t u reza da pesquisa, observamos que 68 (32,5%) dos 209 artigos publicados não apresentam re- ferências bibliográficas; destes, 40 (58,8%) são da perspectiva social e 28 (41,2%), da perspec- t i va biológica. Além dessas evidências, obser- vamos que muitos dos artigos com re f e r ê n c i a s bibliográficas não as apresentam dentro das n o rmas indicadas pelos Arq u ivo s (Ind ex Me d i- c u s). Os artigos originais: natureza da pesquisa, tamanho do artigo e normas bibliográficas Na seqüência de análise dos aspectos das condições re d a t o riais que contribuem para a d e m o n s t ração do rigor metodológico da re v i s- ta, observamos que os artigos da perspectiva biológica, em sua ampla maioria, encontra m - se dentro da estru t u ra básica tradicional de pu- blicações periódicas (intro d u ç ã o, metodologia, Na Tabela 3, encontra-se a distribuição do índice número de páginas por art i g o, utilizado aqui como um dos demonstra t i vos da inexis- tência de ri g i d ez nos cri t é rios ou condições de publicação do peri ó d i c o. Na distribuição desse í n d i c e, observou-se uma média de cerca de 17, um mínimo de quatro e um máximo de 64 pá- ginas por art i g o. Apesar de a grande maiori a dos artigos estar abaixo de 25 páginas, obser- vamos que o não-estabelecimento de um limi- te máximo de páginas por artigo fez com que os Arq u ivo s publicassem ve rd a d e i ros tra t a d o s de fisiopatologia nutricional ou de ciência e tecnologia de alimentos, a exemplo de alguns a rt i g o s, como os de Si q u e i ra & Vogel (1948a, b, Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 3 0 8 VASCONCELOS, F. A. G. a rtigo (considerada aqui formação básica), to- mamos como referência apenas os 88 autore s p rincipais (excluídos os três autores institucio- nais). Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características Na Tabela 4, apresentamos os dados da distri- buição do índice número de autores por art i g o, onde se observa que 123 (58,9%) dos art i g o s são de autoria individual; 50 (23,9%) são de au- t o ria dupla; 33 (15,8%) são assinados por três ou mais autores e 3 (1,4%), por autores institu- c i o n a i s. Os 209 artigos originais publicados nos Ar- q u ivo s são assinados por um total de 134 dife- rentes autore s, 91 dos quais foram considera- dos autores principais (pri m e i ro nome, no ca- so de mais de um autor) e 43, autores secundá- rios ou colabora d o re s. Pa ra a construção do ín- dice identidade profissional do(s) autor(es) por De acordo com o índice vínculo institucio- nal do(s) autor (es) por art i g o, os resultados ob- s e rvados para os 209 artigos originais foram os seguintes: 95 (45,5%) são assinados por autore s vinculados ao Inub; 38 (18,2%), por autores vin- culados a outras instituições localizadas no Rio de Ja n e i ro, quase sempre com forte art i c u l a ç ã o com o grupo de pesquisadores dos Arq u ivo s ( a exemplo do Se rviço de Alimentação da Pre v i- dência Social – Saps – e da Comissão Na c i o n a l de Alimentação – CNA –, instituições também idealizadas por Josué de Ca s t ro) (Co i m b ra et al., 1982; L’ Ab b a t e, 1988); 45 (21,5%), por autore s vinculados a órgãos da Un i versidade de São Paulo (USP) e de outras instituições deste Esta- do; e 31 (14,8%), por autores vinculados a insti- tuições de outros estados bra s i l e i ros (Ba h i a , Minas Ge ra i s, Alagoas e Pe rnambuco), a insti- tuições internacionais (Organização para a Ali- mentação e Agri c u l t u ra – FAO – e Org a n i z a ç ã o Mundial da Saúde – OMS) e alguns não identifi- c a d o s. Os artigos originais: natureza da pesquisa, tamanho do artigo e normas bibliográficas Apesar da falta de informações acerca da f o rmação básica de boa parcela destes autore s, o que limita bastante a análise desse índice, a d i s t ribuição dos dados obtidos é a seguinte: 39 (44,3%) dos autores são médicos; oito (9,1%) são químicos ou farm a c ê u t i c o - b i o q u í m i c o s ; cinco (5,7%) são nutricionistas; três (3,4%) são e n g e n h e i ros agrônomos; três (3,4%) são médi- cos ve t e ri n á rios; um é assistente social; um é economista; seis são autores estra n g e i ros (con- vidados especiais dos dire t o res da revista) e de 22 (25,0%) não obtivemos inform a ç õ e s. Po d e - se afirm a r, port a n t o, que o perfil do autor dos Arq u ivo s a p resenta as seguintes cara c t e r í s t i c a s : homem, médico-nutrólogo, inserido na pers- p e c t i va biológica da nutri ç ã o. Ca ra c t e r í s t i c a s q u e, apesar da incorporação de novos elemen- t o s, permanecem hegemônicas ao longo da t ra j e t ó ria da re v i s t a . re s u l t a d o s, conclusões, re f e r ê n c i a s, re s u m o s ) , enquanto os artigos da perspectiva social ape- nas em sua minoria seguem essa estru t u ra . Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRAS Tabela 5 Relação dos vinte principais autores dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo f o rmação básica, vínculo institucional, campo disciplinar e número absoluto e percentual de artigos publicados em relação ao total, 1944-1968. No de ord e m / n o m e F o rm a ç ã o I n s t i t u i ç ã o * C a m p o No de artigos % do total 1 Pechnik, E. Q u í m i c a I n u b B i o l ó g i c o 42 2 0 , 2 2 Guimarães, L. R. Q u í m i c o I n u b B i o l ó g i c o 21 1 0 , 1 3 Borges, P. M é d i c o I n u b S o c i a l 17 8 , 2 4 Siqueira, R. M é d i c o I n u b B i o l ó g i c o 15 7 , 2 5 Maurício, H. V. A. M é d i c o I n u b S o c i a l 10 4 , 8 6 Silva, W. M é d i c o I n u b / C N A S o c i a l 9 4 , 3 7 Moura-Campos, F. A. M é d i c o U S P B i o l ó g i c o 9 4 , 3 8 Mucciolo, P. Med. Ve t e r i n á r i a U S P B i o l ó g i c o 7 3 , 4 9 Fioravanti, I. M é d i c a I n u b S o c i a l 7 3 , 4 10 Chaves, J. M. Q u í m i c o I n u b B i o l ó g i c o 7 3 , 4 11 Schneider, I. S. Med. Ve t e r i n á r i a USP B i o l ó g i c o 6 2 , 9 12 Castro, J. Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características M é d i c o Inub S o c i a l 5 2 , 4 13 Dutra-de-Oliveira, J. E. M é d i c o USP B i o l ó g i c o 5 2 , 4 14 Maldonado, G. F. M é d i c o Inub/Saps B i o l ó g i c o 5 2 , 4 15 Ferreira Lima, J. M é d i c o Ascofam/PE S o c i a l 4 1 , 9 16 Luz, H. S. M é d i c o Inub B i o l ó g i c o 4 1 , 9 17 Azevedo, T. M é d i c o DS/Bahia S o c i a l 3 1 , 4 18 Parahim, O. M é d i c o DSP/PE S o c i a l 3 1 , 4 19 Lopes, N. M é d i c o Ufal/AL S o c i a l 3 1 , 4 20 Santos, W. M é d i c o Inub/CNA S o c i a l 3 1 , 4 * Inub: Instituto de Nutrição da Universidade de Brasília; CNA: Comissão Nacional de Alimentação; USP: Universidade de São Paulo; Saps: Serviço de Alimentação da Previdência Social; Ascofam: Associação Mundial de Luta contra a Fome; DS: Departamento de Saúde; DSP: Departamento de Saúde Pública; Ufal: Universidade Federal de Alagoas. * Inub: Instituto de Nutrição da Universidade de Brasília; CNA: Comissão Nacional de Alimentação; USP: Universidade de São Paulo; Saps: Serviço de Alimentação da Previdência Social; Ascofam: Associação Mundial de Luta contra a Fome; DS: Departamento de Saúde; DSP: Departamento de Saúde Pública; Ufal: Universidade Federal de Alagoas. A Tabela 5 apresenta a relação dos vinte p rincipais autores de acordo com o número de a rtigos publicados, constituindo-se em uma síntese do perfil de autoria discutido nesta se- ç ã o. Observa-se que os químicos Emília Pe c h- nik e Luiz Ribeiro Gu i m a r ã e s, técnicos da Se- ção de Pesquisas Biológicas do In u b, lidera m em pri m e i ro e segundo lugare s, re s p e c t i va- m e n t e, a lista de publicações. Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características Os médicos nu- trólogos do Inub Pe d ro Bo rg e s, Rubens de Si- q u e i ra e Hélio Vecchio Alves Ma u r í c i o, que no d e c o r rer do corte longitudinal 1944-1968 re ve- z a ram-se nos cargos de secre t á ri o, redator e di- retor do peri ó d i c o, ocupam os postos de terc e i- ro, quarto e quinto lugare s, re s p e c t i va m e n t e. Os cientistas da USP Franklin A. de Mo u ra Ca m p o s, Paschoal Mu c c i o l o, I. S. Schneider e José Ed u a rdo Du t ra de Ol i ve i ra, todos vincula- dos à perspectiva biológica, assumem os pos- tos de sétimo, oitavo, 11o e 13o l u g a re s, re s p e c- t i va m e n t e. Josué de Ca s t ro, em razão de seu papel na criação e direção da revista e na tra j e- t ó ria de constituição do campo da nutrição no Brasil, neste caso, ostenta apenas a 12a p o s i- ç ã o. De s t a c a m o s, ainda, a presença dos médi- cos nutrólogos nordestinos Jamesson Fe r re i ra Lima (Associação Mundial de Luta Co n t ra a Fo- me – Ascofam/PE), Thales de Aze vedo (De p a r- tamento de Saúde – DS/Bahia), Orlando Pa ra- him (De p a rtamento de Saúde Pública – DSP/ PE) e Nabuco Lopes (Un i versidade Fe d e ral de Alagoas – Ufal/AL), todos vinculados à pers- p e c t i va social. O fato de a cientista Emília Pechnick (na condição de mulher e de química) ocupar, de f o rma isolada, o pri m e i ro lugar da lista de pu- blicações é tão surpreendente e esclare c e d o r, quanto contrário a algumas constatações que temos apontado até aqui. Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características Dessa forma, às características do perf i l do autor dos Arq u ivo s apontadas acima, acre s- centamos: estar vinculado ao Inub e residir no Rio de Ja n e i ro. Ca racterísticas estas que se jus- tificam tanto pelo papel desempenhado pela ci- dade do Rio de Ja n e i ro (Di s t rito Fe d e ral na épo- ca; centro político-intelectual; centro concen- t rador de três das seis escolas de nutrição exis- tentes até então no País e sede das pri n c i p a i s agências estatais da política de alimentação e n u t rição), quanto pelo papel desempenhado pelo grupo de médicos nutrólogos do Inub na- quele período da tra j e t ó ria de consolidação do campo da nutrição no Brasil (Co i m b ra et al., 1982; L’ Ab b a t e, 1982; Bosi, 1988). Tabela 3 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o número de páginas por artigo, 1944-1968. No de páginas n % % acumulado 4 a 10 7 7 3 6 , 8 3 6 , 8 11 a 20 7 9 3 7 , 8 7 4 , 6 21 a 30 2 3 1 1 , 0 8 5 , 6 31 a 40 2 0 9 , 6 9 5 , 2 41 a 50 5 2 , 4 9 7 , 6 51 a 64 5 2 , 4 1 0 0 , 0 To t a l 2 0 9 1 0 0 , 0 – Tabela 4 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o número de autores por artigo, 1944-1968. No de autore s n % 1 1 2 3 5 8 , 9 2 5 0 2 3 , 9 3 2 4 1 1 , 5 4 5 2 , 4 5 3 1 , 4 1 0 1 0 , 5 I n s t i t u c i o n a l 3 1 , 4 To t a l 2 0 9 1 0 0 , 0 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o número de páginas por artigo, 1944-1968. Cad. Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características Por outro lado, reforça as teses da i m p o rtância da química, considerada como uma das disciplinas-matriz na conform a ç ã o deste campo, tanto no cenário universal, co- mo no Brasil ( C h a ve s, 1978; Lima, 1997; Ma g a- l h ã e s, 1997). Além dos vinte autores listados, é pre c i s o f a zer referência a um grupo que emerge ape- nas a partir dos anos 60, denotando uma cert a a b e rt u ra dos Arq u ivo s p a ra a publicação da p rodução científica de outras especialidades que entra vam em jogo na contra d i t ó ria dinâ- mica de competição e interação que se estabe- lecia na tra j e t ó ria de consolidação do campo da nutrição no Brasil. Assim, Enilda Lins da Cruz Go u veia (Saps/RJ) e Ma ria de Lourd e s Mello (Sesi/MG) foram as duas pri m e i ras nu- t ricionistas a publicarem nos Arq u ivo s, cada uma assinando um artigo dentro da perspecti- va social (Go u veia, 1960; Me l l o, 1960). A part i r de então, outras nutricionistas passaram a as- sinar artigos dentro das duas perspectivas que i n ve s t i g a m o s, a exemplo de Lieselotte H. Or- nellas (Escola de En f e rmagem da Un i ve r s i d a d e do Brasil – UB), de Neuza T h e rezinha Reze n d e Ca valcante (Inub), de Mi rtila C. Araújo (Inub) e de Gilda Linhares Mello (Inub). Re f e rimos ain- da a entrada em cena do médico pern a m b u c a- no Nelson Chave s, fundador do Instituto e do Curso de Nu t rição da Un i versidade Fe d e ral de Pernambuco, que, entre 1963 e 1964, assina dois a rtigos naquele peri ó d i c o. Fato que, a nosso ve r, significa mais um reforço à perspectiva bio- lógica, part i c u l a rmente às disciplinas fisiologia e endocrinologia, também consideradas disci- plinas-matriz na conformação do campo da nu- t rição no Brasil (Lima, 1997; Ma g a l h ã e s, 1997). Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características Por um lado, indica um certo rompimento com as teses de que a constituição do campo da nutrição no Bra s i l tenha sido “d em a rc ada por um espaço de luta c o m p e t i t i va pelo monopólio da autoridade c i e n t í f i c a” (Bo u rdieu, 1994:131) entre os gêne- ros masculino e feminino (Sa n t o s, 1988; Pra- d o, 1993; Bosi, 1995; Lima, 1997). Sem dúvida, a existência dos 42 artigos assinados pela quí- mica Emília Pechnik nos volumes 2 ao 24 da revista indica que, apesar de majori t á ria, a p resença do gênero masculino na tra j e t ó ria da Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 VASCONCELOS, F. A. G. 3 1 0 c a d o s. Estes art i g o s, frutos de pesquisas labo- ra t o riais (análises químico-bromatológicas ou ensaios biológico-experimentais), têm como ponto comum a utilização de métodos de de- t e rminação do valor nutricional dos alimentos n a c i o n a i s. Sendo assim, abordam a composi- ção químico-nutricional, o valor calóri c o, o t e o r vitamínico e/ou o teor mineral dos mais dive r- sificados alimentos bra s i l e i ro s, dos mais tra d i- cionais (feijão pre t o, farinha de mandioca, tri- g o, milho, leite, lara n j a s, manga etc.) aos mais exóticos (buriti, tucumã, pupunha, óleo de pa- tauá, pinhão, mucunã, macambira, sururu e t c. ) . Josué de Ca s t ro assina dois artigos do campo temático em questão, um no qual são re ve l a- dos os resultados químico-analíticos de ali- mentos bárbaros do sertão nordestino (Ca s t ro et al., 1947), e outro que apresenta o valor nu- t ri t i vo da mistura de milho com leite (Ca s t ro & Pechnik, 1951). constituição deste campo não é uma condição e xc l u s i va. Os autores dos A rq u iv o s: identidade p rofissional, vínculo institucional e outras características Esse fato re p resenta um dos exemplos que nos ajudam a atestar que não existiam fro n t e i- ras rígidas entre as duas perspectivas inve s t i- g a d a s, nem tampouco entre campos temáticos ou disciplinares dentro de uma mesma pers- p e c t i va. V á rios autores dos Arq u ivo s c i rc u l a m e n t re as duas perspectivas e entre os distintos campos temáticos existentes no interior de ca- da uma delas, ocorre n d o, inclusive, tentativa s c o n c retas de conexão entre o biológico e o so- cial, a exemplo de artigo incluído nessa temáti- ca (Pechnik et al., 1950), no qual os autore s a p resentam os resultados de uma inve s t i g a ç ã o s o b re a castanha-do-pará, investigação esta composta por um detalhado estudo econômi- co-social da produção e por um ensaio bioló- g ic o - e x p e rimental do valor nutricional deste p ro d u t o. A temática tecnologia dos alimentos apare- ce em segundo lugar dentro da perspectiva biológica, em 35 (26,1%) dos artigos publica- d o s. Trata-se da única temática alheia aos pes- q u i s a d o res do In u b, uma vez que quase a tota- lidade destes artigos são oriundos de pesquisa- d o res do Estado de São Pa u l o. Paschoal Mu c- ciolo e I. S. Schneider, da Faculdade de Me d i c i- na Ve t e ri n á ria da USP, são os dois pri n c i p a i s a u t o res dessa linha, cujos artigos abordam os mais diversificados temas ligados à industri a l i- zação de alimentos (métodos de pro c e s s a m e n- t o, de enri q u e c i m e n t o, de conserva ç ã o, de em- balagem, de controle de qualidade etc.). Em- b o ra apareça, de forma bastante tímida, desde 1947, esta temática só teve uma melhor expre s- são numérica a partir de 1954. Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 A perspectiva biológica: dos métodos de determinação do valor nutricional aos métodos de industrialização dos alimentos A temática nutrição e dietética aparece em último lugar, em 13 (9,7%) dos artigos publica- d o s. Os artigos desta linha abordam temas re- lacionados a aspectos do planejamento e ad- m i n i s t ração de serviços de alimentação, da téc- nica dietética e culinária e da dietética pro p ri a- mente dita. Quase a totalidade destes art i g o s (92,0%) foram publicados entre 1959 a 1968 e versam, em sua maioria, sobre o planejamento da alimentação de grupos militares (exérc i t o, m a rinha e aeronáutica). En t re os seus autore s, destacamos os médicos nutrólogos Pe d ro Bo r- g e s, Hélio Vecchio A. Maurício e Irma Fi o ra va n- ti (Seção de Pesquisas Ec o n ô m i c o - Sociais do Inub); Ge raldo Francisco Maldonado (Saps) e a n u t ricionista Lieselotte H. Ornellas (Escola de En f e rmagem da UB). Em síntese, através dos conhecimentos ob- j e t i vos produzidos por estas pesquisas labora- t o riais e ensaios biotecnológicos sobre a com- posição e o metabolismo dos alimentos; sobre os métodos de industrialização e enri q u e c i- mento dos alimentos e sobre os modern o s p rincípios da dietética e da dietotera p i a , o s médicos nutrólogos vinculados aos Arq u ivo s c o n s t ru í ram uma importante base cognitiva p a ra a legitimidade da tese, atribuída a Jo s u é de Ca s t ro, de que “a partir de uma alimentação ra c i o n a l , seria possível valorizar a ra ç a ,c o n s- truir o homem bra s i l e i ro e forjar a nação” (Li- ma, 1997; Ma g a l h ã e s, 1997:79). No campo temático Pol ít ica e Pl a n e j a m e n- to em Alimentação e Nu t r i ç ã o, foram incluídos 20 (26,7%) dos art i g o s. A perspectiva biológica: dos métodos de determinação do valor nutricional aos métodos de industrialização dos alimentos Tais art i g o s, publicados do pri m e i ro ao último volume da revista, demonstram que, após o inquérito pioneiro: As condições de vida das classes operárias no Re c i f e, realizado por Jo- sué de Ca s t ro em 1933 (Ca s t ro, 1935), esta mo- dalidade de inve s t i g a ç ã o, além de apri m o ra r- se enquanto instrumental básico de pesquisa d e n t ro da perspectiva social da nutri ç ã o, ga- nhou adeptos em todo o país, a exemplo das pesquisas realizadas pelos médicos nutrólogos Orlando Pa rahim e Jamesson Fe r re i ra Lima, em Pe rnambuco; Thales de Aze ve d o, na Bahia; Os- waldo Costa e Rubem de Si q u e i ra, no Rio de Ja- n e i ro; Domingos Lopes e Benedito Ph i l a d é l p h o Si q u e i ra, em Minas Ge rais; Nabuco Lopes, em A l a g o a s, e Walter Si l va, no Rio Grande do No rt e. • Oito artigos sobre avaliação nutricional ba- seada em métodos antro p o m é t ri c o s, clínicos e s ó c i o - e c o n ô m i c o s. Ressalte-se que tais ava l i a- ções clínico-antro p o m é t ricas tive ram como di- re t ri zes comuns: a população em idade esco- l a r; algum cri t é rio de estratificação sócio-eco- nômica desta população; a utilização de pro c e- dimentos estatísticos na amostragem e análise dos dados e resultados apontando melhore s p e rfis antro p o m é t ri c o - n u t ricionais entre os es- c o l a res com maior poder aquisitivo. Estes art i- g o s, publicados entre 1949 a 1968, são assina- dos pelos médicos nutrólogos Josué de Ca s t ro, Walter Si l va, Pe d ro Bo rg e s, Thales de Aze ve d o, Irma Fi o ra vanti, Nabuco Lopes e Ge raldo Fra n- cisco Ma l d o n a d o. A perspectiva biológica: dos métodos de determinação do valor nutricional aos métodos de industrialização dos alimentos Esta temática emerg e em setembro de 1947, a partir de um artigo de Walter Si l va que apresenta uma abord a g e m t e ó rica sobre os três grupos de fatores a sere m c o n s i d e rados na formulação de uma política alimentar para o Brasil (disponibilidade de ali- m e n t o s, poder aquisitivo e capacidade de sele- ção de alimentos). A articulação destes três g rupos de fatores apontados por Si l va (1947), no nosso entender, caminhará para a form u l a- ção da matriz teórico-conceitual sobre a pro- blemática alimentar e nutricional bra s i l e i ra , que unificará os intelectuais do campo da nu- t rição em torno do seu cri a d o r, Josué de Ca s t ro (Lima, 1997; Ma g a l h ã e s, 1997). Ainda, no nosso e n t e n d e r, implicitamente, através do re f e ri d o a rtigo (Si l va, 1947), os médicos nutrólogos vin- A perspectiva biológica: dos métodos de determinação do valor nutricional aos métodos de industrialização dos alimentos uso de métodos antro p o m é t ri c o s, clínicos, la- b o ra t o ri a i s, dietéticos, demográficos e sócio- econômicos para a realização de diagnósticos n u t ricionais de comunidades), foram incluídos 21 (28,0%) dos art i g o s, destacando-se: • Nove artigos sobre consumo alimentar, uti- lizando distintas modalidades de inquéri t o s d i e t é t i c o s. Tais art i g o s, publicados do pri m e i ro ao último volume da revista, demonstram que, após o inquérito pioneiro: As condições de vida das classes operárias no Re c i f e, realizado por Jo- sué de Ca s t ro em 1933 (Ca s t ro, 1935), esta mo- dalidade de inve s t i g a ç ã o, além de apri m o ra r- se enquanto instrumental básico de pesquisa d e n t ro da perspectiva social da nutri ç ã o, ga- nhou adeptos em todo o país, a exemplo das pesquisas realizadas pelos médicos nutrólogos Orlando Pa rahim e Jamesson Fe r re i ra Lima, em Pe rnambuco; Thales de Aze ve d o, na Bahia; Os- waldo Costa e Rubem de Si q u e i ra, no Rio de Ja- n e i ro; Domingos Lopes e Benedito Ph i l a d é l p h o Si q u e i ra, em Minas Ge rais; Nabuco Lopes, em A l a g o a s, e Walter Si l va, no Rio Grande do No rt e. • Nove artigos sobre consumo alimentar, uti- lizando distintas modalidades de inquéri t o s d i e t é t i c o s. A perspectiva biológica: dos métodos de determinação do valor nutricional aos métodos de industrialização dos alimentos Nesta seção, passaremos à análise do conteúdo temático dos 134 artigos incluídos na perspec- t i va biológica da nutri ç ã o, dentro dos quatro campos temáticos ou disciplinares em que es- tes foram categori z a d o s. A temática nutrição básica e experi m e n t a l , l i d e rada pelos químicos Emília Pechnik, Lu i z R i b e i ro Guimarães e José Ma ria Chave s, da Se- ção de Pesquisas Biológicas do In u b, e pelo médico Franklin A. de Mo u ra Ca m p o s, do De- p a rtamento de Fisiologia da Faculdade de Me- dicina da USP, detém a hegemonia dos tra b a- lhos desta linha, com 61 (45,5%) artigos publi- A temática nutrição clínica aparece em ter- c e i ro lugar, com 25 (18,7%) artigos publicados. Os médicos Rubens de Si q u e i ra, Hélio de So u- Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 1 1 za Luz, Clementino Fraga Filho (da Seção de Patologia da Nu t rição do Inub), José Ed u a rd o Du t ra de Ol i ve i ra e Demosthenes Orsini (da Fa- culdade de Medicina da USP) são os pri n c i p a i s a u t o res dessa linha, cujo auge de produção ve- rificou-se no período de 1947 a 1953, com 16 (64,0%) art i g o s. Os artigos desta área abord a m aspectos de fisiologia, patologia e dietotera p i a ; o ri g i n a ram-se de pesquisas do tipo experi m e n- tal (ensaios biológicos) e estudos de casos (en- saios clínicos), tratando dos mais diversos te- m a s, tais como semiótica das hipov i t a m i n o s e s ; metabolismo basal; dietoterapia em diabetes, disfunções hepáticas e gastrectomias e padro- nização de dietas hospitalare s. A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome No campo temático Ed uc ação Nu t r i c i o n a l, f o ram classificados sete (9,3%) dos art i g o s, en- t retanto apenas três são especificamente fiéis a este campo disciplinar: uma extensa re v i s ã o bibliográfica sobre a temática educação nutri- cional no Brasil (à época denominada Ed u c a- ção Al i m e n t a r), publicada em 1951 pelo oficial médico do exército e nutrólogo Walter José dos Santos; um relato da experiência da nutri c i o- nista Ma ria de Lourdes Mello (Sesi/MG) em educação alimentar para menores tra b a l h a d o- res e um esboço de projeto de pesquisa sobre tabus e hábitos alimentares no Brasil, de auto- ria do economista Souza Ba r ros (Se s i / A s c o- fam). Os quatro demais estão relacionados à f o rmação de recursos humanos (nutri c i o n i s- t a s, médicos, tecnólogos de alimentos e ve t e ri- n á rios), sendo incluídos neste grupo por ser esta a temática mais semelhante. Nesse aspec- t o, é importante registar que esses tra b a l h o s a c e rca da formação de recursos humanos, pu- blicados a partir de 1963, parecem datar o acir- ramento da discussão em torno da demarc a ç ã o dos campos de saber, de competência e de po- der entre os profissionais (especialidades) do campo da nutri ç ã o. Nesse sentido, em 1963, foi publicado um artigo sobre tecnologia de ali- mentos na formação do ve t e ri n á rio (Mu c c i o l o, 1963); a revista re l a t i va ao pri m e i ro semestre de 1966 publicou um artigo sobre o ensino de n u t rição para estudantes de Medicina (Du t ra - d e - Ol i ve i ra, 1966) e, ainda no segundo semes- t re de 1966 e em 1967, foram publicados dois a rtigos sobre a formação do profissional nutri- cionista (Ca va l c a n t e, 1966, 1967). A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome De s t a c a- mos a publicação, no período 1961-1963, de cinco artigos apresentando propostas de pro- d u ç ã o, abastecimento de alimentos e de re f o r- ma agrária (Ma u r í c i o, 1961; Fe r re i ra-Lima & Ba n c ov s k y, 1962 e 1963; Fe r re i ra Lima et al., 1962; Salomão & Gomes da Si l va, 1963), quan- do o Brasil inteiro, part i c u l a rmente a re g i ã o No rd e s t e, vive n c i a va uma intensa mobilização dos tra b a l h a d o res ru rais com a criação das Li- gas Camponesas e dos Sindicatos Ru rais (Aze- v ê d o, 1982). De s t a c a m o s, ainda, no período 1951-1968, a publicação de quatro artigos a b o r- dando a questão da mere n d a e sc ol a r; a nosso ve r, foram importantes veículos de difusão das idéias em torno da u t i l i d a d e da política nacio- nal de alimentação escolar, implantada no Bra- sil, no decorrer dos anos 50, a partir dos pro g ra- mas internacionais de ajuda alimentar (Co i m- b ra et al., 1982). E, finalmente, os artigos que p re n u n c i a vam o futuro envolvimento que teri a a indústria de alimentos na formulação e con- dução da política de alimentação e nutrição do País no pós-1964. A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome Os 75 artigos incluídos na perspectiva social da n u t rição foram categorizados dentro dos cinco campos temáticos ou disciplinares que discuti- remos a seguir. No campo temático Aval i ação Nu t r i c i o n a l de Po p u l a ç õ e s (que compreendemos como o Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 VASCONCELOS, F. A. G. 3 1 2 3 1 2 VASCONCELOS, F. A. G. t rição pro t é i c o - c a l ó rica (DPC), de autoria dos médicos Irma Fi o ra vanti e Nelson Chave s, que t ratam, re s p e c t i va m e n t e, de um estudo de pre- valência sobre DPC hospitalar e de uma pre l e- ção conferida pelo pro f e s s o r, abordando as- pectos epidemiológicos desta carência nutri- cional no No rdeste; c) um artigo sobre hipov i- taminose A, de autoria de Thales de Aze ve d o ; t rata-se de uma curta revisão bibliográfica, a p resentando alguns dados sobre a pre va l ê n- cia desta carência nutricional na Ba h i a . culados aos Arq u ivo s b u s c a vam gerar consen- so e legitimidade para a regulamentação da Comissão Nacional de Alimentação (CNA), agência criada em 1945, mas que só passaria a o p e ra r, efetiva m e n t e, a partir de 1951, com o re t o rno de Getúlio Va rgas à Presidência da Re- pública (L’ Ab b a t e, 1982). Aliás, a maioria des- tes artigos são de natureza teóri c o - c o n c e i t u a l , a p resentando enfoques e propostas políticas s o b re temas emergentes em determinadas c o n- j u n t u ra s. A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome Como exemplo, podemos apontar o a rtigo Plano de política alimentar na Amazônia ( Ca s t ro, 1951), um re l a t ó rio técnico, no qual Jo- sué de Ca s t ro, dentro da abordagem geográfi- co-ecológica que cara c t e rizará a sua obra (Ma- g a l h ã e s, 1997; Lima, 1997), já àquela época, su- g e ria a implantação de medidas de interve n ç ã o a l i m e n t a r- n u t ricional visando ao desenvo l v i- m e n t o e c on ôm ic o s u st e nt ável para aquela re- gião bra s i l e i ra, que estava em evidência desde o início da Segunda Gu e r ra Mundial. De s t a c a- mos a publicação, no período 1961-1963, de cinco artigos apresentando propostas de pro- d u ç ã o, abastecimento de alimentos e de re f o r- ma agrária (Ma u r í c i o, 1961; Fe r re i ra-Lima & Ba n c ov s k y, 1962 e 1963; Fe r re i ra Lima et al., 1962; Salomão & Gomes da Si l va, 1963), quan- do o Brasil inteiro, part i c u l a rmente a re g i ã o No rd e s t e, vive n c i a va uma intensa mobilização dos tra b a l h a d o res ru rais com a criação das Li- gas Camponesas e dos Sindicatos Ru rais (Aze- v ê d o, 1982). De s t a c a m o s, ainda, no período 1951-1968, a publicação de quatro artigos a b o r- dando a questão da mere n d a e sc ol a r; a nosso ve r, foram importantes veículos de difusão das idéias em torno da u t i l i d a d e da política nacio- nal de alimentação escolar, implantada no Bra- sil, no decorrer dos anos 50, a partir dos pro g ra- mas internacionais de ajuda alimentar (Co i m- b ra et al., 1982). A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome E, finalmente, os artigos que p re n u n c i a vam o futuro envolvimento que teri a a indústria de alimentos na formulação e con- dução da política de alimentação e nutrição do País no pós-1964. culados aos Arq u ivo s b u s c a vam gerar consen so e legitimidade para a regulamentação da Comissão Nacional de Alimentação (CNA), agência criada em 1945, mas que só passaria a o p e ra r, efetiva m e n t e, a partir de 1951, com o re t o rno de Getúlio Va rgas à Presidência da Re- pública (L’ Ab b a t e, 1982). Aliás, a maioria des- tes artigos são de natureza teóri c o - c o n c e i t u a l , a p resentando enfoques e propostas políticas s o b re temas emergentes em determinadas c o n- j u n t u ra s. Como exemplo, podemos apontar o a rtigo Plano de política alimentar na Amazônia ( Ca s t ro, 1951), um re l a t ó rio técnico, no qual Jo- sué de Ca s t ro, dentro da abordagem geográfi- co-ecológica que cara c t e rizará a sua obra (Ma- g a l h ã e s, 1997; Lima, 1997), já àquela época, su- g e ria a implantação de medidas de interve n ç ã o a l i m e n t a r- n u t ricional visando ao desenvo l v i- m e n t o e c on ôm ic o s u st e nt ável para aquela re- gião bra s i l e i ra, que estava em evidência desde o início da Segunda Gu e r ra Mundial. Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome Além disso, é preciso re g i s t rar que: a) a par- tir de 1959, com a adesão de novos médicos m i l i t a res à nutrologia (Jair de Matos Mo n t e- d ôn i o, José João Barbosa e Ge raldo Fra n c i s c o Maldonado), começaram a ser publicados art i- gos sobre nutrição nas forças armadas e b) em 1961 foi publicado um re l a t ó rio técnico sobre a a t u a ç ã o, no Brasil, do pro g rama Al im e ntos pa- ra a Pa z, desenvolvido pelo gove rno nort e - a m e- ri c a n o, prenunciando o assistencialismo ali- mentar que vigora ria no País no pós-64 (Co i m- b ra et al., 1982; L’ Ab b a t e, 1982). Por fim, entre 1964 e 1968, a discussão cen- t ral girou em torno dos temas desenvo l v i m e n- to econômico, população, alimentação, tecno- logia e, pri n c i p a l m e n t e, sobre o papel da in- d ú s t ria alimentar no combate à fome (Orn e l- l a s, 1965). p q Nesse sentido, é preciso atentar para a e m e rgência de temas específicos em determ i- nados cortes tra n s versais da tra j e t ó ria da re v i s- ta. En t re 1944 a 1955, permanecia em evidên- cia a tese da ignorância alimentar do povo bra- s i l e i ro, presente em todas as camadas sociais, e m b o ra com certas distinções de classe social ( Pa rahim, 1944; Si q u e i ra, 1950). Po rt a n t o, tor- n a va-se fundamental a sedutora proposta da “a l im e nt ação ra c i o n a l , como fundamento da vida e condição precípua de saúde, eugenia e p ro g re s s o” (Pa rahim, 1944:23), legitimadora da constituição do campo da nutrição e da educa- ção alimentar como um dos seus instru m e n- tais básicos (Lima, 1997). A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome s é rie histórica 1944-1968, a nosso ve r, pode ser a t ribuída à articulação de distintos elementos na tra j e t ó ria dos intelectuais vinculados aos Arq u ivo s, tais como a maior liberdade de ex- p ressão que, gra d a t i va m e n t e, foi sendo am- pliada no País a partir da queda do Estado No- vo; a incorporação de novas concepções cientí- ficas e filosóficas, a exemplo, entre outra s, da t e o ria do círculo vicioso da pobreza e da doen- ça no início dos anos 50 (Braga & Paula, 1981); das teorias do subdesenvo l v i m e n t o - d e s e n vo l- vimento a partir de meados dos anos 50 (Ma n- tega, 1985) e do discurso pre ventivista dos anos 60 (Arouca, 1975) e, finalmente, a maior auto- nomia que o campo da nutrição foi conquis- tando no interior do espaço acadêmico (nas u n i versidades e em outras instituições de ensi- no e pesquisa), do espaço político-institucio- nal (nas agências e instituições estatais vincu- ladas à política s ocial de alimentação e nutri- ção) e no seio da sociedade civil (Co i m b ra et al., 1982; L’ Ab b a t e, 1982; Natal, 1982). Assim como nos demais, este campo temático tam- bém reflete aspectos das conjunturas específi- cas em que os artigos foram produzidos e di- fundidos pelos Arq u ivo s. ( ) En t re 1959 a 1963, a discussão assumiu no- vos contorn o s, ampliando-se o debate em tor- no dos determinantes demográficos, econômi- co-sociais e estru t u ra i s, part i c u l a rmente sobre re f o rma agrária, produção e abastecimento de a l i m e n t o s, conforme apontamos anteri o rm e n- t e. A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome No campo temático Ep id em i ol ogia Nu t r i- c i o n a l (que compreendemos como o estudo das doenças nutricionais em uma determ i n a d a população), foram identificados apenas sete (9,3%) dos art i g o s, sendo: a) quatro sobre bó- cio endêmico, publicados entre 1944 a 1959, o riginados das atividades desenvolvidas pelo então De p a rtamento Nacional de Sa ú d e, que relatam estudos de pre valência entre escola- res ou apresentam revisões sobre pre va l ê n c i a , á reas e n d ê m i c a s, profilaxia e tratamento da doença. Seus autores são os médicos Álva ro Lobo Leite Pe re i ra, Amílcar Ba rca Pellon, Wa l- ter Si l va e Pe d ro Bo rges; b) dois sobre desnu- No campo temático Det e rm in a ntes do Es t a- do Nutricional de Po p u l a ç õ e s, foram incluídos 20 (26,7%) dos art i g o s. Apesar de implícito nos a rtigos da perspectiva social desde 1944, este campo temático só ganhou especificidade a p a rtir de 1950 e expressão numérica nos anos 60. Os artigos nele incluídos, de natureza teóri- co-conceitual, tratam das concepções ou pon- tos de vista de distintos autores acerca da gê- n e s e, re p rodução e conseqüências da pro b l e- mática alimentar e nutricional no Brasil. Esta politização observada nos artigos ao longo da Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 1 3 p ro m ovam atividades que visem aumentar as disponibilidades de alimentos de alto valor nu- t r i t i vo” (Costa & Si l va, 1946:16-17). A perspectiva social: da eugenia através da alimentação racional à indústria alimentar no combate à fome Observa-se que a te- se do mal de fome e não de raça, formulada por Josué de Ca s t ro nos anos 30 – sua forma de in- terlocução com os cientistas de outros campos d i s c i p l i n a res que, à época, pro c u ra vam desfo- car da questão biológica para a questão sócio- c u l t u ral o preconceito de clima (meio) e de ra- ça que se tinha sobre o povo bra s i l e i ro (Lima, 1997; Ma g a l h ã e s, 1997) –, continuava, nos anos 44-55, ecoando entre os intelectuais do campo da nutri ç ã o. En t re t a n t o, nesse mesmo contex- t o, outras teses foram sobrepondo-se a essa an- t e ri o r, a exemplo da linha que concluía que a questão alimentar estava “mais na dependên- cia do poder aquisitivo que de outro fator qual- quer e, na impossibilidade de agir sobre o salá- r i o, impõe-se que os serviços de saúde pública, ao lado das atividades de educação alimentar, Considerações finais Fase esta liderada por um grupo de médicos nutrólogos que, ao mesmo tempo em que pro- c u ra ram estabelecer as conexões entre o bioló- gico e o social, com base na interd i s c i p l i n a ri- dade e na complexidade inerentes ao novo c a m- po do saber que se constituía, contra d i t o ri a- m e n t e, cri a ram a demarcação de outras fro n- t e i ra s, definidoras dos limites de competência, de autonomia e de poder – na concepção teóri- ca de Bo u rdieu (1987 e 1994) –, entre as espe- cialidades do campo da nutri ç ã o, part i c u l a r- mente entre médicos nutrólogos e nutri c i o n i s- t a s. Assim, em 1968, formou-se um hiato para a (re ) c o n s t rução de uma nova escola bra s i l e i ra de nutri ç ã o. Mas isso é uma outra históri a . q u ivos de Ga s t ro e n t e ro l o g i a, os Arq u ivos de Bro m a t o l o g i a e t c.) não nos autoriza a genera l i- zar as conclusões deste estudo. Sendo assim, deixamos acesa a chama para o trilhar de no- vas investigações neste campo. A análise dos 209 artigos originais publica- dos pelos Arq u ivo s nos possibilitou a identifi- cação não apenas do elenco de intelectuais que part i c i p a ram do processo de constituição do campo da nutri ç ã o, mas também de como as temáticas por estes abordadas foram sendo t ra n s f o rmadas ao longo dos 24 anos de circ u l a- ção da revista. Considerações finais A opção metodológica adotada neste estudo – a realização de uma investigação sistemati- zada baseada em princípios da meta-análise ( Greenland, 1987; Jenicek, 1989; Riegelman & Hirsch, 1992; Jo n e s, 1993; T h a c k e r, 1993; Esco- b a r, 1994), buscando sumariar aspectos quan- t i - q u a l i t a t i vos dos artigos originais publicados nos Arq u ivos Bra s i l e i ros de Nutrição –, apesar de demonstra r-se eficiente na consecução dos o b j e t i vos a que nos pro p u s e m o s, apre s e n t a também as suas limitações. Em pri m e i ro lugar, é preciso destacar que o nosso olhar debru ç o u - se apenas sobre uma das seções da estru t u ra i n t e rna de organização do periódico (a seção de artigos originais), o que não nos autoriza a t i rar conclusões sobre a tra j e t ó ria desta publi- cação científica como um todo. Em segundo l u g a r, é preciso re g i s t rar que, embora acre d i t e- mos que os artigos publicados nos Arq u ivo s s e- jam re p re s e n t a t i vos do que foi produzido e di- fundido no campo da nutrição ao longo da sé- rie histórica 1944-1968, a inexistência de estu- dos acerca da produção difundida em outra s publicações científicas contemporâneas ao pe- riódico (os Arq u ivos Bra s i l e i ros de Me d i c i n a, a Revista Bra s i l e i ra de Me d i c i n a, os Arq u ivos Bra- s i l e i ros de Endocrinologia e Me t a b o l o g i a, os Ar- Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 3 1 4 VASCONCELOS, F. A. G. tos idealizados por seu dire t o r-fundador de c o n t ribuir com a difusão dos modernos conhe- cimentos científicos sobre nutrição para a for- mação de novas gerações de nutrólogos, nutri- cionistas e outros especialistas deste campo ( Ca s t ro, 1944). Considerações finais Além desse mérito técnico-cien- t í f i c o, é preciso reconhecer a importância dos Arq u ivo s como instrumento político-ideológi- co a serviço de interesses específicos dos cien- tistas que os conduziram. A análise da séri e h i s t ó rica 1944-1968 nos faz concluir que, mes- mo com o afastamento oficial de Josué de Ca s- t ro da direção da revista, quando o mesmo pas- sou a exercer sua militância política no perío- do de 1955-1964 (ocupando os cargos de depu- tado federal pelo estado de Pe rnambuco e de embaixador bra s i l e i ro junto à Organização das Nações Unidas – ONU) (Co i m b ra et al., 1982; L’ Ab b a t e, 1982), sob a direção dos seus discípu- los mais diletos (Pe d ro Bo rges e Hélio Ve c c h i o A. Maurício), os Arq u ivo s m a n t i ve ram a mesma d i re t riz e até dinamizaram quantitativa m e n t e o seu número de publicações. Após o golpe mi- litar de 1964, com o exílio de seu dire t o r- f u n- d a d o r, os Arq u ivo s c o n s e g u i ram circular até 1968, encerra n d o, assim, uma importante fase da nossa história, quando “d e nt ro das pare d e s fechadas dos labora t ó r i o s , no campo aberto da pesquisa social, na cátedra e nos livro s , se criou a escola bra s i l e i ra de Nu t r i ç ã o” (Ca s t ro, 1 9 4 4 : 3 ) . Considerações finais Grosso modo, podemos dize r q u e, no período 1944-1954, correspondente à d i reção Josué de Ca s t ro, os estudos sobre a composição química e o valor nutricional de alimentos nacionais, desenvolvidos pelos inte- lectuais vinculados à perspectiva biológica, aliados aos estudos sobre consumo alimentar e estado nutricional da população bra s i l e i ra, de- s e n volvidos pelos intelectuais vinculados à p e r s p e c t i va social, constituíram a base cogniti- va gera d o ra da especificidade do campo da nu- t rição no Brasil, considerada como disciplina, política social e/ou pro f i s s ã o. A matriz teóri c a que implícita ou explicitamente unificou tais i n t e l e c t u a i s, “a tese do valor eugênico da ali- mentação na construção da nação e do homem brasileiro” (Lima, 1997; Magalhães, 1997), a nos- so ve r, sintetiza a ressonância das concepções de cunho nacionalista que cara c t e ri z a ram os anos 30-40, no campo da nutri ç ã o, no Brasil (Mi- celi, 1979; Pecaut, 1990; Go m e s, 1996). Por ou- t ro lado, no período 1955-1968, corre s p o n d e n- te à direção Pe d ro Bo rges/Hélio Vecchio Ma u- r í c i o, a maior politização dos intelectuais vin- culados aos Arq u ivo s em suas concepções acer- ca da gênese, re p rodução e enfrentamento da f o m e / s u b n u t rição da população bra s i l e i ra, si- naliza, em última instância, a comunhão destes intelectuais com as teorias do nacional desen- volvimentismo dos anos 50-60 (Mantega, 1985). Fi li d di í ó Fi n a l i z a n d o, diríamos que, para nós, ao longo dos seus 24 anos de circ u l a ç ã o, os Arq u i- vo s p a recem ter cumprido os objetivos explíci- Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 R e f e r ê n c i a s ALENCAR, F.; CARPI, L. & RIBEIRO, M. V., 1979. Hi s- tória da Sociedade Bra s i l e i ra. Rio de Ja n e i ro: Ao L i v ro T é c n i c o. C H AVES, N., 1978. Nutrição Básica e Ap l i c a d a. Rio de Ja n e i ro: Gu a n a b a ra - Ko o g a n . DUTRA-DE-OLIVEIRA, J. E., 1966. Ensino de nutri ç ã o p a ra estudantes de Medicina. Arq u i vos Bra s i l e i ro s de Nu t r i ç ã o, 22:21-36. A ROUCA, A. S. S., 1975. O Dilema Pre ve n t i v i s t a :C o n- tribuição para a Compreensão e Crítica da Me d i- cina Pre ve n t i va. Tese de Doutora d o, Ca m p i n a s : Faculdade de Ciências Médicas, Un i versidade Es- tadual de Ca m p i n a s. E S COBAR, A. L., 1994. Malária no Sudeste da Am a z ô- n i a : Uma Me t a - An á l i s e. Di s s e rtação de Me s t ra d o, Rio de Ja n e i ro: Escola Nacional de Saúde Pública, Fundação Oswaldo Cru z . A RQU I VOS BRASILEIROS DE NUTRIÇÃO, 1946. In s t i- tuto de Nutrição (Notas e In f o r m a ç õ e s ), 2:71-73. FERREIRA-LIMA, J.; CO S TA, F. & MELLO, F. B., 1962. Algumas medidas para a melhoria do abasteci- mento regional. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 1 8 : 9 5 - 1 2 0 . A Z EV Ê D O, F. A., 1982. As Ligas Ca m p o n e s a s. Rio de Ja n e i ro: Paz e Te r ra . BOSI, M. L. M., 1988. A Face Oculta da Nu t r i ç ã o : Ci ê n- cia e Id e o l o g i a. R e f e r ê n c i a s Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 8: 1 9 5 - 2 0 4 . L’ A B B ATE, S., 1988. As políticas de alimentação e nu- t rição no Brasil. I. Período de 1940 a 1964. Re v i s t a de Nutrição da PUCCAMP, 1:87-138. LIMA, E. S., 1997. Gênese e Constituição da Ed u c a ç ã o Al i m e n t a r : A In s t a u ração da No r m a. Tese de Dou- t o ra d o, São Paulo: Pontifícia Un i versidade Ca t ó l i- ca de São Pa u l o. C A S T RO, J.; PECHNIK, E.; PARAHIM, O.; MATTO S O, I. V. & CHAVES, J. M., 1947. Os “alimentos bárbaro s” dos Se rtões do No rd e s t e. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 3:5-29. M AGALHÃES, R., 1997. Fo m e : Uma (Re ) l e i t u ra de Jo- sué de Ca s t ro. Rio de Ja n e i ro: Ed i t o ra Fi o c ru z . C A S T RO, J. & PECHNIK, E., 1951. Valor nutri t i vo da m i s t u ra de milho com leite. Arq u i vos Bra s i l e i ro s de Nu t r i ç ã o, 8:13-30. MANTEGA, G., 1985. A Economia Política Bra s i l e i ra. Petrópolis: Ed i t o ra Voze s / Po l i s. C AVA LCANTE, N. T. R., 1966. O profissional em nu- t rição: form a ç ã o, atividades e perspectivas fu- t u ra s. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 22:51-64. M AU R Í C I O, H. V. A., 1961. Alimentação e re f o rm a a g r á ria, problemas sociais. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 17:17-27. C AVA LCANTE, N. T. R., 1967. R e f e r ê n c i a s Ep i d e m i o- logic Re v i ew s, 9:1-30. C A S T RO, A. M., 1977. Nutrição e De s e n vo l v i m e n t o : Análise de uma Po l í t i c a. Tese de Livre - D o c ê n c i a em Sociologia, Rio de Ja n e i ro: Instituto de Nu- t ri ç ã o, Ce n t ro de Ciências da Sa ú d e, Un i ve r s i d a- de Fe d e ral do Rio de Ja n e i ro. JENICEK, M., 1989. Meta-analysis in Medicine – w h e re we are and where we want to go? Journal of Clinical Ep i d e m i o l o gy, 42:35-44. C A S T RO, J., 1935. As Condições de Vida das Classes Operárias no Re c i f e. Rio de Ja n e i ro: Mi n i s t é rio do Tra b a l h o. JONES, D. R., 1993. El metanálisis en los estudios epi- demiológicos observa c i o n a l e s. Boletín de la Ofi- cina Sanitaria Pa n a m e r i c a n a, 115:438-445. L’ A B B ATE, S., 1982. Fome e De s n u t r i ç ã o : Os De s c a m- inhos da Política Social. Di s s e rtação de Me s t ra d o, São Paulo: De p a rtamento de Ciências Sociais da Faculdade de Filosofia, Letras e Ciências Hu m a- n a s, Un i versidade de São Pa u l o. C A S T RO, J., 1944. O “Se rviço Técnico da Alimentação Na c i o n a l” e os “A rq u i vos Bra s i l e i ros de Nu t ri ç ã o”. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 1:3-6. C A S T RO, J., 1946. Ed i t o rial. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 2:3-5. C A S T RO, J., 1951. Plano de política alimentar na Amazônia. R e f e r ê n c i a s Rio de Ja n e i ro: Espaço e Te m p o / Ed i t o ra da Un i versidade Fe d e ral do Rio de Ja- n e i ro. FERREIRA-LIMA, J. & BANCOV S K Y, J., 1962. O dra m a da fome no No rd e s t e. Arq u i vos Bra s i l e i ros de Nu- t r i ç ã o, 18:155-192. BOSI, M. L. M., 1995. Definindo Fro n t e i ra s : O Nu t r i- cionista e sua Pro f i s s i o n a l i z a ç ã o. Tese de Douto- ra d o, Rio de Ja n e i ro: Escola Nacional de Sa ú d e Pública, Fundação Oswaldo Cru z . FERREIRA-LIMA, J. & BANCOV S K Y, J., 1963. Plano de alimentação racional. Arq u i vos Bra s i l e i ros de Nu- t r i ç ã o, 19:85-115. GOMES, A. C., 1996. História e Hi s t o r i a d o re s : A Po l í t i- ca Cu l t u ral do Estado Novo. Rio de Ja n e i ro: Ed i t o- ra da Fundação Getúlio Va rg a s. BOURDIEU, P., 1987. A Economia das Trocas Si m b ó l i- c a s. São Paulo: Pe r s p e c t i va . BOURDIEU, P., 1994. El campo científico. Revista de Estudios Sociales de la Ciencia (Re d e s ), 1:131-160. GOUVEIA, E. L. C., 1960. O papel dos inquéritos ali- m e n t a res nos pro g ramas educacionais e assisten- ciais de coletividades. Arq u i vos Bra s i l e i ros de Nu- t r i ç ã o, 16:69-78. B R AGA, J. C. S. & PAU LA, S. G., 1981. Saúde e Pre v i- d ê n c i a : Estudos de Política Social. São Pa u l o : Ce b e s / Hu c i t e c. G R E E N LA N D, S., 1987. Qu a n t i t a t i ve methods in the review of epidemiological litera t u re. A g r a d e c i m e n t o s Aos pro f e s s o res Ricardo Ve n t u ra Santos e Carlos E. A. Co i m b ra Jr. (Escola Nacional de Saúde Pública, Fu n- dação Oswaldo Cruz), pela prestimosa orientação e l e i t u ra crítica do texto ori g i n a l . PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 1 5 R e f e r ê n c i a s Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 3:6- 3 6 . S I QUEIRA, R. & VOGEL, J., 1948b. Aspectos semióti- cos das carências vitamínicas no homem e nos animais domésticos (continuação). Arq u i vos Bra- s i l e i ros de Nu t r i ç ã o, 5:5-58. ORSINI, D., 1947b. O metabolismo de base de jove n s b ra s i l e i ras (continuação). Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 3:6-66. PARAHIM, O., 1944. O problema alimentar no Se rt ã o No rdestino: a eugenia através da alimentação ra- cional. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 1:23-36. S I QUEIRA, R. & VOGEL, J., 1948c. Aspectos semióti- cos das carências vitamínicas no homem e nos animais domésticos (conclusão). Arq u i vos Bra- s i l e i ros de Nu t r i ç ã o, 5:5-58. PE C AU T, D., 1990. Os Intelectuais e a Política no Bra s i l : En t re o Povo e a Na ç ã o. São Paulo: Ed i t o ra Á t i c a . T H ACKER, S. B., 1993. Metanálisis: un enfoque cuan- t i t a t i vo para la integración de inve s t i g a c i o n e s. Boletín de la Oficina Sanitaria Pa n a m e r i c a n a, 1 1 5 : 3 2 8 - 3 3 9 . PECHNIK, E.; BORGES, P. & SIQUEIRA, R., 1950. Estu- do sobre a Castanha do Pará. Arq u i vos Bra s i l e i ro s de Nu t r i ç ã o, 7:7-42. VA S CO N C E LOS, F. A. G., 1988. A Política Social de Al i- mentação e Nutrição no Bra s i l : Acumulação de Capital e Re p rodução da Fo m e / Desnutrição (do Estado Novo à Nova Re p ú b l i c a ). R e f e r ê n c i a s Pre p a ração do nutri- cionista face o desenvo l v i m e n t o. Arq u i vos Bra s i- l e i ros de Nu t r i ç ã o, 23:77-88. M E L LO, M. L., 1960. 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Self-organization of swimmers drives long-range fluid transport in bacterial colonies
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ARTICLE Self-organization of swimmers drives long-range fluid transport in bacterial colonies Haoran Xu1, Justas Dauparas2, Debasish Das2, Eric Lauga 2 & Yilin Wu 1 Motile subpopulations in microbial communities are believed to be important for dispersal, quest for food, and material transport. Here, we show that motile cells in sessile colonies of peritrichously flagellated bacteria can self-organize into two adjacent, centimeter-scale motile rings surrounding the entire colony. The motile rings arise from spontaneous segregation of a homogeneous swimmer suspension that mimics a phase separation; the process is mediated by intercellular interactions and shear-induced depletion. As a result of this self-organization, cells drive fluid flows that circulate around the colony at a constant peak speed of ~30 µm s−1, providing a stable and high-speed avenue for directed material transport at the macroscopic scale. Our findings present a unique form of bacterial self-organization that influences population structure and material distribution in colonies. 1 Department of Physics and Shenzhen Research Institute, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People’s Republic of China. 2 Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge CB3 0WA, UK. Correspondence and requests for materials should be addressed to Y.W. (email: ylwu@cuhk.edu.hk) NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 1 TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/ ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 M M icrobes commonly live in structured communities that play important roles in human health and ecology1–5, such as biofilm aggregates during chronic infections6, commensal microbiome in animal guts, microbial mats in river beds or ocean floors, and bacterial colonies in soils or food pro- ducts5. The multicellular lifestyle is key to the prospering of microbes in diverse habitats as it confers high resistance to var- ious environmental stresses7–11. Understanding the physiology of these structured microbial communities is not only essential to the treatment of chronic infections, but also important to industrial applications, such as bioremediation, anti-biofouling and food hygiene. In addition, as a type of active matter, microbial communities have provided model systems for the research of biological self-organization and complex fluids12–16. colony-scale motile rings is present in colonies of Proteus mir- abilis, Escherichia coli, and Bacillus subtilis, suggesting that the phenomenon is conserved among bacteria species with peri- trichous flagella. Results S lf 1e, Supplementary Fig. 1; Supplementary Movie 2). None- theless, the motion of cells in the inner ring shows a weak CCW bias [defined as NCCWVCCW/NCWVCW, where NCCW (or NCW) and VCCW (or VCW) denote the number and the mean speed of CCW (or CW) moving cells, respectively] ranging from ~1.1 to ~1.2, and the inner motile ring collectively circles CCW around the colony at a mean speed of ~1 µm s−1 (Fig. 1d). Our phe- nomenon is distinct from the self-organized vortex reported in microscale circular confinement31,32, which did not display chirality bias and nematic order in cells’ moving directions. Note that hereinafter in the paper CW and CCW refer to the sense of chirality with respect to the entire colony viewed from above, unless otherwise indicated. Colony mode of bacterial growth on solid substrates (e.g., food products and solidified nutrient agar) is a common experimental approach to study structured microbial communities11 and is closely related to biofilm development23,24. Cells in a bacterial colony move in confined space surrounded by boundaries between gas, liquid, and solid phases. Interfaces and spatial confinement are known to affect motile behavior of bacteria in more artificial settings such as microfluidic systems. Bacteria swimming near liquid–solid interfaces have the tendency moving in parallel to the wall due to hydrodynamic trapping25–30, but it was less clear how swimming bacteria behave at the gas–liquid–solid three phase interface, such as the edge of bac- terial colonies. Also it was reported that confined bacterial sus- pension droplets of size less than ~100 micron31,32 can self- organize into vortices, in which two concentric, counter-rotating regions of bacteria are present near the edge of the droplet. This type of bacterial self-organization has not yet been observed in the context of naturally developed colonies. Understanding how bacterial motility adapts to the physical environment in colonies may therefore have implications for active matter self- organization and for the engineering of self-assembled micro- fluidic systems that perform fluid pumping and cargo trans- port33–38. Here we sought to examine the behavior of motile cell popu- lations in bacterial colonies and to explore their potential phy- siological functions. In routinely cultured bacterial colonies where most cells have transitioned into a sessile state, we discover that motile cells can self-organize into two adjacent, centimeter-scale motile rings that surround the entire colony. Results S lf Heterogeneous populations, including motile and non-motile populations, often coexist in bacteria communities17. Motile subpopulations in microbial communities are believed to be important to dispersal18,19, quest for food20, and material trans- port21. However, except in circumstances where motile cells drive colony expansion (e.g., bacterial swarming8,10,22), the physiolo- gical functions of motile subpopulations in bacterial communities are largely unclear. For example, in many occasions bacterial colonies are sessile and their expansion is driven by growth rather than by cell motility, but these sessile colonies normally preserve a subpopulation of motile cells for reasons that are not well understood17. Self-organization of colony-scale motile rings. Our organism of choice was P. mirabilis, a Gram-negative rod-shaped bacterium well known for its swarming behavior on hard agar surfaces39,40. P. mirabilis is widely distributed in the natural environment and as a human pathogen with clinical importance, it is often asso- ciated with urinary tract infections41. When grown on soft agar plates in humid environment (~85% relative humidity) for ~24 h (Methods; Supplementary Table 1), P. mirabilis colonies have not initiated swarming yet and most cells have become sessile, although a sub-population of motile planktonic cells (ranging from 2 to 10 µm in length) are present. Such sessile colonies of P. mirabilis provide a model system for studying the behavior and physiological function of motile sub-populations. We discovered that, surprisingly, motile cells in such colonies self-organize into two adjacent motile rings that surround the entire colony (Fig. 1a–d; Supplementary Movie 1). The outer motile ring is located at the outmost rim of the colony; it measures ~10 µm in width and ~1 µm in height. Cells in this ring are well aligned with each other, with the average orientation making an angle of ~30° with colony edge, presumably due to steric repulsion between densely packed cells42; they circle exclusively clockwise (CW) around the colony (viewed from above; observed in >100 P. mirabilis colonies on tens of agar plates) at a uniform, constant speed (mean: 27.8 µm s−1, S.D.: 2.7 µm s−1, N = 10), i.e., their collective motion is polarly ordered. On the other hand, the inner motile ring is ~20–40 µm in width and ~2–3 µm in height; it is bounded by the outer ring and the sessile part of the colony, and cells in this ring tend to swim in parallel to colony edge bi- directionally with nematic order in cells’ moving directions (Fig. Self-organization of swimmers drives long-range fluid transport in bacterial colonies As a result of this self-organization, cells in the outer motile ring drive fluid flows in the inner motile ring to circulate counterclockwise (CCW) around the colony at a con- stant peak speed of ~30 µm s−1, providing a stable and high- speed avenue for directed material transport at the macroscopic scale. Our findings reveal a unique form of colony-scale self- organization and active transport in bacterial colonies, which may shape the population structure and material distribution of bac- terial communities in widespread environments. NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunicatio NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 2 and Supplementary Movie 3), albeit less robust against environmental perturbations. B. subtilis displays vigorous swarming due to the secretion of surfactin, a lipoprotein having potent surfactant activities43. Nonetheless, B. subtilis colonies prior to the onset to swarm expansion (~3-h post inoculation; “Methods”) displayed self-organization of motile rings as well (Supplementary Fig. 2 and Supplementary Movie 4). These findings suggest that the phenomenon is conserved among bacteria species with peritrichous flagella. We note that smooth- inoculation, at which time a highly organized outer motile ring very rapidly emerges at the colony edge. The full development is completed within 10 min, as shown by the sharp transition of mean tangential speed at T = 0 min in Fig. 1f. Since the motility and the density of cells at colony edge is similar before and after this transition point, we suggest that the physicochemical conditions of the colony, presumably water content (Supplemen- tary Table 1) and surface tension, may just reached an appropriate state at the transition point permitting the formation of a thin wetting film ~1–2 µm in thickness at the colony edge that can support 2D motion of cells. Once emerged, the motile rings remain stable for many hours until the initiation of swarming. Environmental humidity is key to the formation of motile rings of Proteus mirabilis. Motile rings cannot be observed if the relative humidity is below 47% and they appear earlier on plates with higher agar concentrations (Supplementary Table 1). Motile ring self-organization arises from physical interactions. Next we sought to understand the self-organization process of motile rings. A recent study reported that concentrated swim- ming bacteria confined in 1D microfluidic channels can display unidirectional collective motion due to flagella-driven fluid flows generated by cells at channel edge44. The geometry in our phe- nomenon is similar to a 1-D channel, since the two motile rings are bounded by colony edge and the sessile part of the colony. In contrast to the phenomenon reported earlier44, cells in the inner motile ring only display weakly directed collective motion (Fig. 1), suggesting a different mechanism at work. Nonetheless, we decided to examine whether channel-like confinement is necessary for motile ring self-organization. We collected motile cells from swarming colonies of P. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 Time = 0 min is chosen at the onset of collective motion with high polar order and it corresponds to ~20 h colony growth 1e–1.0 1e–0.0 c 1e+1.5 1e+0.5 1e+1.0 1e+0.0 (µm/s) a b X Y b X Y c a b f d 0 20 40 60 80 –4 0 4 8 12 16 Probability 0 20 40 0 0.1 0.2 0  0 0.05 e 2 60 f X (µm) V ( / ) Vring (µm/s) Vcell (µm/s) Probability 0 20 40 0 0.1 0.2 0  0 0.05 e 2 60 Vcell (µm/s) −300 −200 −100 0 100 0 4 8 12 f Time (min) Vring (µm/s) f e Fig. 1 Self-organization of two adjacent colony-scale motile rings at the edge of a P. mirabilis colony. a Phase contrast image of P. mirabilis colony. Scale bar, 0.5 mm. b Enlarged view of the area enclosed by magenta box in panel A. Scale bar 10 μm. Also see Supplementary Movie 1. c Time-averaged collective velocity field of cells in the region of panel b computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude in log scale (with the color bar provided to the right, in μm s−1). d The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from colony edge. Positive value of speed indicates motion along CW direction, i.e., along + Y axis of the coordinate system specified in panel b, and X = 0 is set at the position of colony edge. e Speed distribution of individual bacteria at the inner motile ring moving in CCW direction (gray) and in CW direction (brown). Cell speed was computed by tracking fluorescent cells seeded into the colony (“Methods”) and located near the center of inner motile ring where the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel d. Inset: Probability distribution of velocity direction, with 3/2π and 1/2π corresponding to −Y and +Y directions, respectively. See Supplementary Fig. 1 and Supplementary Movie 2. f Dynamics of motile ring emergence during colony growth. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 1e–1.0 1e–0.0 a b d X Y c 0 20 40 60 80 –4 0 4 8 12 16 Probability 0 20 40 0 0.1 0.2 0  0 0.05 e 2 60 −300 −200 −100 0 100 0 4 8 12 f Time (min) 1e+1.5 1e+0.5 1e+1.0 1e+0.0 (µm/s) X (µm) Vring (µm/s) Vring (µm/s) Vcell (µm/s) Fig. 1 Self-organization of two adjacent colony-scale motile rings at the edge of a P. mirabilis colony. a Phase contrast image of P. mirabilis colony. Scale bar, 0.5 mm. b Enlarged view of the area enclosed by magenta box in panel A. Scale bar 10 μm. Also see Supplementary Movie 1. c Time-averaged collective velocity field of cells in the region of panel b computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude in log scale (with the color bar provided to the right, in μm s−1). d The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from colony edge. Positive value of speed indicates motion along CW direction, i.e., along + Y axis of the coordinate system specified in panel b, and X = 0 is set at the position of colony edge. e Speed distribution of individual bacteria at the inner motile ring moving in CCW direction (gray) and in CW direction (brown). Cell speed was computed by tracking fluorescent cells seeded into the colony (“Methods”) and located near the center of inner motile ring where the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel d. Inset: Probability distribution of velocity direction, with 3/2π and 1/2π corresponding to −Y and +Y directions, respectively. See Supplementary Fig. 1 and Supplementary Movie 2. f Dynamics of motile ring emergence during colony growth. The mean tangential speed of bacteria (computed by optical flow analysis with phase contrast images) in the 10-μm-wide outmost rim of colonies (i.e., the region of outer motile ring) is plotted against time. Results S lf Located at the outmost rim of the colony, the outer motile ring measures about ten microns in width; cells in this ring circle clockwise around the colony (viewing from above) with high polar order. On the other hand, the inner motile ring is several tens of microns in width; cells in this ring tend to swim in parallel to colony edge bi- directionally with nematic ordering in cells’ moving directions. We demonstrate that the motile rings arise from spontaneous segregation of a homogeneous swimmer suspension that mimics a phase separation; the process is mediated by intercellular inter- actions and by a shear-induced depletion that concentrates bac- teria in sheared regions. The remarkable self-organization of To characterize the dynamics of motile ring development, we measured the collective speed of bacteria at colony edge over a time course of 7 h starting from ~16 h after colony inoculation. At the early stage of colony development, cells adapt to the surface environment, extract water from the substrate, and become able to move on agar surface. At this stage the motion of cells displayed certain degree of ordering (as shown by the non-zero mean tangential speed of ~4 µm s−1 prior to time = 0 min in Fig. 1f). This stage lasts for several hours until ~20 h after colony 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 The mean tangential speed of bacteria (computed by optical flow analysis with phase contrast images) in the 10-μm-wide outmost rim of colonies (i.e., the region of outer motile ring) is plotted against time. Time = 0 min is chosen at the onset of collective motion with high polar order and it corresponds to ~20 h colony growth swimming mutants of E. coli and B. subtilis do not display the self- organization of motile rings, at least under our experimental conditions. Smooth-swimming cells are not able to switch flagellar rotation direction autonomously. Consequently they tend to get stuck to each other during collisions and form jammed clusters. Indeed, jammed clusters frequently formed at the edge of such colonies (Supplementary Movie 5). Due to these jammed clusters, the orientation of cells at the colony edge remained disordered (Supplementary Fig. 3, panel A), and unidirectional motion could not develop there, despite a weak CW bias in the average speed of cells (Supplementary Fig. 3, panel B). inoculation, at which time a highly organized outer motile ring very rapidly emerges at the colony edge. The full development is completed within 10 min, as shown by the sharp transition of mean tangential speed at T = 0 min in Fig. 1f. Since the motility and the density of cells at colony edge is similar before and after this transition point, we suggest that the physicochemical conditions of the colony, presumably water content (Supplemen- tary Table 1) and surface tension, may just reached an appropriate state at the transition point permitting the formation of a thin wetting film ~1–2 µm in thickness at the colony edge that can support 2D motion of cells. Once emerged, the motile rings remain stable for many hours until the initiation of swarming. Environmental humidity is key to the formation of motile rings of Proteus mirabilis. Motile rings cannot be observed if the relative humidity is below 47% and they appear earlier on plates with higher agar concentrations (Supplementary Table 1). To see whether this remarkable self-organization phenomenon was due to any physiological properties specific to P. mirabilis, we examined colonies of another two model flagellated bacteria, E. coli and B. subtilis. E. coli colonies were grown on LB agar plates that do not support swarm expansion (“Methods”), and we observed the same self-organization of motile rings described above (Supplementary Fig. ARTICLE Assuming that the flagella bundle is rotating about cell body long axis with angular speed Ω, then the torque balance at low Reynolds number gives: Gdrive ¼ D1Ω; ð3Þ Gdrag ¼ D2ω; ð4Þ ω ¼ D1 D2 Ω; ð5Þ Gdrive ¼ D1Ω; ð3Þ Gdrag ¼ D2ω; ð4Þ ω ¼ D1 D2 Ω; ð5Þ ð3Þ ð4Þ ω ¼ D1 D2 Ω; ð5Þ ð5Þ where D1 is the constant reorientation coefficient relating the driving torque to the rotation rate Ω of the flagella bundle, and the constant coefficient D2 relates the drag torque to the angular speed ω of cell body rotation about the cell pole in contact with drop edge. We derived D1 and D2 based on slender body theory47,48 and resistive force theory49, respectively (“Methods”), which allowed us to compute ω as a function of the flagellar bundle distance to the bottom surface h. Our simplified model is more appropriate for E. coli than for P. mirabilis, since P. mirabilis has 2–3 fold higher flagellar density on cell surface than E. coli and its flagellar filaments may not form a single bundle as modeled here12. Therefore we chose to verify our model with E. coli. Using the reorientation angular speed ω ≈2.1 rad s−1 measured in our experiment as well as other known parameters of E. coli, we found the flagellar bundle distance to substrate as h ≈0.4 μm (“Methods” and Supplementary Fig. 4), i.e., approxi- mately half of cell body width. This result is reasonable, since cells are supposed to sit just above the substrate surface at the edge of liquid drop. CW bias is a result of cell-substrate hydrodynamic interaction. To understand the origin of CW bias of collective motion in the outer motile ring, we examined the motion pattern of individual cells at low densities. P. mirabilis colony edge was diluted by adding external liquid, creating a liquid drop dispersed with isolated cells that mimicked the fluid environment of colonies (“Methods”). We found that cells colliding with the edge of such a diluted colony tended to turn CW with respect to the contact point about an axis normal to the underlying substrate and subsequently move to the right along the edge (Fig. 3; Supple- mentary Movie 8). In fact, cells moved exclusively CW after collision with the diluted colony edge for colliding angles between 0 and 1.75 rad (Fig. 3e). As expected, isolated cells of both E. ARTICLE We varied cell speed by tuning environmental temperature (“Meth- ods”) and found that the width of inner motile ring increases linearly with cell speed at the outer motile ring (Fig. 2e). This result suggests that physical interaction between cells, mediated by either steric repulsion or hydrodynamic forces or both, controls the formation of inner motile ring. We also tuned the cell density in suspension drops and found that the order of cellular motion in the 10-μm-wide outmost rim of the drop depends on cell density (Fig. 2f): At low densities, cellular motion in this rim does not display polar order, despite a weak CW bias (mean tangential speed ~2 μm s−1); beyond a critical cell density (cell occupation ratio ~0.4), cellular motion experiences a sharp transition to maximal polar order (mean tangential speed ~12 μm s−1), and a highly ordered CW motile ring emerges. This result demonstrates that the emergence of a highly ordered outer motile ring is mediated by intercellular interactions. The results presented in Fig. 1f and Fig. 2f reveals the two essential requirements of forming a highly ordered outer motile ring: (1) Appropriate physicochemical conditions of the colony edge that support 2D motion of cells; (2) Sufficiently high cell density that allows for intensive cell-cell interactions. As suggested earlier, the first requirement was not satisfied in naturally developed P. mirabilis colonies prior to T = 0 min in Fig. 1f, although the second requirement had already been met. By contrast, the first requirement was always satisfied in suspension drops of P. mirabilis with the help of exogenously supplied surfactant. We concluded that the self-organization of motile rings near the edge of the colony is a collective effect arising from physical interactions between swimmers under 2D confinement. X i Gi ¼ 0 ð1Þ Gdrive ¼ Gdrag ð2Þ ð1Þ Gdrive ¼ Gdrag ð2Þ ð2Þ In our model, the driving torque Gdrive arises in the system because the bacterium rotates its flagella bundle CCW around flagellar axis (when looked from behind the cell body) next to the edge of the drop, where the bottom surface (agar) induces more friction than the top air-liquid interface46. Gdrive depends on the height of flagellar bundle above bottom surface. As the body with flagellar bundle reorients, the driving torque balances a resisting drag torque Gdrag due to rotation of the whole cell in the viscous fluid. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 ARTICLE three regions with different types of order, mimicking a phase separation: a dense and polarly ordered layer at the edge undergoing CW collective motion (~10 µm in width), a dense layer with nematic order in cells’ moving directions just adjacent to the polar-order layer (~30 µm in width), and a low-density disordered phase in the bulk where cells swim randomly. The two densely-packed ordered layers resemble the colony-scale motile rings observed in naturally grown colonies (Fig. 2a–c, Supple- mentary Fig. 1; Supplementary Movies 6, 7). Our results suggest that channel-like confinement is not required for motile ring self- organization in colonies. The results also exclude the possibility that the sessile part of the colony may have orchestrated the collective motion in both motile rings through chemical signaling. We note that the speed distribution of cells in the nematic layer is Gaussian like (Fig. 2d), which is different from the speed dis- tribution in the inner motile ring of colonies (Fig. 1e); this is because cells in the inner motile ring of colonies occasionally collide with sessile cells lying underneath and get stuck tran- siently, thus contributing to a large number of low-speed traces. contact point (Supplementary Movie 9, 10). Interestingly, by imaging fluorescently labeled flagellar filaments of GFP-tagged E. coli cells45, we found that the flagellar bundle of cells colliding with the edge of a suspension drop did not change its orientation with respect to the cell body throughout the turning process (Supplementary Movie 9; “Methods”). Presumably the CW bias of cell reorientation upon contact with colony edge gives rise to the exclusively CW bias of the collective motion in the outer motile ring of colonies. g To understand the mechanism of CW turning around contact point upon collision, we measured the time dependence of cell orientation and found that cells rotated at a constant angular speed ω during CW turning around contact point (for P. mirabilis, ω = 8.3 (3.3) rad s−1, mean (SD), N = 56; for E. coli, ω = 2.1 (0.9) rad s−1, mean (SD), N = 15). This constant angular speed of reorientation suggests the balance of torque about the cell pole in contact with drop edge. We build a simplified swimmer model to understand this torque balance, as illustrated in Fig. 3f: We further used suspension drops of P. mirabilis to characterize the dynamics of motile ring self-organization. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 mirabilis, re-suspended them in a medium containing surfactant Tween 20, and deposited them at appropriate densities on agar surface, forming a non-spreading liquid drop with motile cells alone that resembles a colony (referred to as an artificial colony; “Methods”). We found that motile cells in the artificial colony spontaneously separated into To see whether this remarkable self-organization phenomenon was due to any physiological properties specific to P. mirabilis, we examined colonies of another two model flagellated bacteria, E. coli and B. subtilis. E. coli colonies were grown on LB agar plates that do not support swarm expansion (“Methods”), and we observed the same self-organization of motile rings described above (Supplementary Fig. 2 and Supplementary Movie 3), albeit less robust against environmental perturbations. B. subtilis displays vigorous swarming due to the secretion of surfactin, a lipoprotein having potent surfactant activities43. Nonetheless, B. subtilis colonies prior to the onset to swarm expansion (~3-h post inoculation; “Methods”) displayed self-organization of motile rings as well (Supplementary Fig. 2 and Supplementary Movie 4). These findings suggest that the phenomenon is conserved among bacteria species with peritrichous flagella. We note that smooth- TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 8 a b 0 0 –2 2 4 6 20 40 60 80 X (µm) c X Y d 0 2 4 6 8 10 (µm/s) Probability 2 0  0 5 10 15 20 0 0.02 0.04 0.06 0.08 4 8 12 16 20 20 40 60 80 Width (µm) e f 0.03 0.06 0 0 0 Cell occupation ratio 0 0.2 0.4 0.6 0.8 1.0 0 4 8 12 16 Vring (µm/s) Vcell (µm/s) Vcell (µm/s) Vcell (µm/s) Fig. 2 Self-organization of two adjacent motile rings at the edge of a P. mirabilis suspension drop (artificial colony). a Fluorescent image of the drop edge (“Methods”). Scale bar, 10 μm. See Supplementary Movie 6 (phase contrast) and Supplementary Movie 7 (fluorescence). b Time-averaged collective velocity field of cells in the region of panel a computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude (with the color bar provided to the right, in μm s−1). c The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from suspension-drop edge. Following the coordinate system specified in panel a, positive value of speed indicates motion along +Y axis, i.e., along CW direction viewed from above the suspension drop, and X = 0 is set at the position of drop edge. d Speed distribution of individual bacteria obtained by single-cell tracking at the inner motile ring where the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel c. Brown and orange columns represent statistics for cells moving in CCW and CW direction, respectively. Inset: Distribution of velocity direction of individual bacteria; 3/2π and 1/2π correspond to −Y and +Y directions, respectively. Also see Supplementary Fig. 1. e The width of inner motile ring plotted against the mean tangential speed of collective motion at the outer motile ring. Dashed line is a linear fit with R2 = 0.84. Error bars indicate standard error of the mean (n = 1200 measurements in three biologically independent samples). f The mean tangential speed of cells plotted against cell occupation ratio in the 10-μm-wide outmost rim of the suspension drop (corresponding to the region of outer motile ring in naturally developed colonies). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 Horizontal error bars indicate standard error of the mean (n = 300 measurements in three biologically independent samples); vertical error bars indicate standard deviation (n = 10 cells) | p g b 0 2 4 6 8 10 (µm/s) a X Y b a 8 0 0 –2 2 4 6 20 40 60 80 X (µm) c Vring (µm/s) d Probability 2 0  0 5 10 15 20 0 0.02 0.04 0.06 0.08 0.03 0.06 0 Vcell (µm/s) d c 4 8 12 16 20 20 40 60 80 Width (µm) e 0 0 Vcell (µm/s) f Cell occupation ratio 0 0.2 0.4 0.6 0.8 1.0 0 4 8 12 16 Vcell (µm/s) f f e Fig. 2 Self-organization of two adjacent motile rings at the edge of a P. mirabilis suspension drop (artificial colony). a Fluorescent image of the drop edge (“Methods”). Scale bar, 10 μm. See Supplementary Movie 6 (phase contrast) and Supplementary Movie 7 (fluorescence). b Time-averaged collective velocity field of cells in the region of panel a computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude (with the color bar provided to the right, in μm s−1). c The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from suspension-drop edge. Following the coordinate system specified in panel a, positive value of speed indicates motion along +Y axis, i.e., along CW direction viewed from above the suspension drop, and X = 0 is set at the position of drop edge. d Speed distribution of individual bacteria obtained by single-cell tracking at the inner motile ring where the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel c. Brown and orange columns represent statistics for cells moving in CCW and CW direction, respectively. Inset: Distribution of velocity direction of individual bacteria; 3/2π and 1/2π correspond to −Y and +Y directions, respectively. Also see Supplementary Fig. 1. e The width of inner motile ring plotted against the mean tangential speed of collective motion at the outer motile ring. Dashed line is a linear fit with R2 = 0.84. Error bars indicate standard error of the mean (n = 1200 measurements in three biologically independent samples). ARTICLE coli and B. subtilis displayed similar CW bias of reorientation around Colony-scale directed flows are present in the inner motile ring. Fluid pumping due to flagellar rotation of cells in the outer motile ring may generate strong unidirectional fluid flows, as suggested by previous studies50,51. To examine this idea, we NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 f The mean tangential speed of cells plotted against cell occupation ratio in the 10-μm-wide outmost rim of the suspension drop (corresponding to the region of outer motile ring in naturally developed colonies). Horizontal error bars indicate standard error of the mean (n = 300 measurements in three biologically independent samples); vertical error bars indicate standard deviation (n = 10 cells) visualized fluid flows in P. mirabilis colonies by adding 0.1-μm fluorescent microspheres (Life Technologies, Cat. No. F8820) as tracers. We found that tracers were transported in a CCW manner around the entire colony at a peak constant speed of ~30 µm s−1 (Fig. 4a–d; Supplementary Movie 11), along a narrow channel of width ~20–30 µm that coincides with the inner motile ring. A similar stream of fluid flows was found with a much smaller tracer, fluorescently labelled dextran of a size ~10 nm (FITC–Dextran, mol. wt. 2000 kDa; Sigma, FD2000S) that can move freely in between cells (Supplementary Fig. 5). The peak speed and the spatial range of fluid flows we uncovered are comparable to those of the flagella-driven flows measured near individual cells whose motion were restricted in quasi-2D liquid films50,51. The CCW directed flows in the inner motile ring cause a shear rate up to ~10 s−1 in the plane perpendicular to substrate surface; this shear rate falls within the range of intermediate shear rates (~9–12 s−1) for slight downstream motion bias of flagellated bacteria52, which may account for the weak CCW bias (i.e., downstream bias) of collective motion in the inner motile ring. Our flow measurement revealed a stable, high-speed avenue for directed material transport in bacterial colonies at the macro- scopic scale. In addition, we also found that microspheres and TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 0.00 s 0.30 s 0.24 s 0.18 s 0.06 s 0.12 s −50 0 50 50 0 Y coordinate (µm) X (µm) −1 0 1 2 −20 0 20 40 Y (µm) Time (s) a b c  e Y X d CW bias CCW bias 0  0 0.2 0.4 0.6 0.8 1 1 0.8 0.6 0.4 0.2 0 Interaction angle  (rad) /2 /4 3/4 f CCW CW CCW CW Drop edge Gdrag=D2 Gdrive=D1 x Lb Lf  y y′ x′ Fig. 3 Single-cell motion pattern of P. mirabilis near diluted colony edge. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 a A sequence of phase contrast images recording the CW turning of an isolated cell colliding with the diluted colony edge (“Methods”). The dark area in upper portion of images is virgin agar. Scale bar, 5 μm. b Trajectory of the cell in panel a generated by overlaying a series of phase contrast images. Magenta arrows by the side of trajectory (white dotted line) indicate instantaneous moving direction of the cell. See Supplementary Movie 8. θ denotes interaction angle, defined as the angle between the diluted colony edge and the cell orientation just before collision. Scale bar, 20 μm. c Representative trajectories (N = 31) of cells that collided with diluted colony edge. Following the coordinate system specified in panel b, X = 0 μm is set at the edge of liquid drops. The starting point of all trajectories are aligned and set as Y = 0 μm and X = 55 μm. Blue (or magenta) lines represent the trajectory of cells that turned CW (or CCW) around the contact point during collision. d Tangential position of cells (whose trajectories were shown in panel e) plotted against time. The collision positions of all trajectories are aligned and set as Y = 0 μm, and T = 0 is set at the time of collision. Positive slope at T > 0 indicates that the cell moved in CW sense around the colony after collision. e Probability distribution of cells’ motion bias after collision with diluted colony edge plotted against interaction angle (Blue column: CW bias; Magenta column: CCW bias). f Model of a swimming bacterium in contact with liquid drop edge (top view). The torque driving reorentation Gdrive and the drag torque Gdrag balance, which leads to rotation at a constant angular speed ω about the x = 0; y = 0 point (green dot). The flagellar bundle is rotating at an angular speed Ω. The length of cell body and flagellar bundle is Lb and Lf, respectively. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 Also see “Methods” 0.00 s 0.30 s 0.24 s 0.18 s 0.06 s 0.12 s a b a  Y X −1 0 1 2 −20 0 20 40 Y (µm) Time (s) d CCW CW −50 0 50 50 0 Y coordinate (µm) X (µm) c CCW CW d c e CW bias CCW bias 0  0 0.2 0.4 0.6 0.8 1 1 0.8 0.6 0.4 0.2 0 Interaction angle  (rad) /2 /4 3/4 f ( ) f Drop edge Gdrag=D2 Gdrive=D1 x Lb Lf  y y′ x′ e Fig. 3 Single-cell motion pattern of P. mirabilis near diluted colony edge. a A sequence of phase contrast images recording the CW turning of an isolated cell colliding with the diluted colony edge (“Methods”). The dark area in upper portion of images is virgin agar. Scale bar, 5 μm. b Trajectory of the cell in panel a generated by overlaying a series of phase contrast images. Magenta arrows by the side of trajectory (white dotted line) indicate instantaneous moving direction of the cell. See Supplementary Movie 8. θ denotes interaction angle, defined as the angle between the diluted colony edge and the cell orientation just before collision. Scale bar, 20 μm. c Representative trajectories (N = 31) of cells that collided with diluted colony edge. Following the coordinate system specified in panel b, X = 0 μm is set at the edge of liquid drops. The starting point of all trajectories are aligned and set as Y = 0 μm and X = 55 μm. Blue (or magenta) lines represent the trajectory of cells that turned CW (or CCW) around the contact point during collision. d Tangential position of cells (whose trajectories were shown in panel e) plotted against time. The collision positions of all trajectories are aligned and set as Y = 0 μm, and T = 0 is set at the time of collision. Positive slope at T > 0 indicates that the cell moved in CW sense around the colony after collision. e Probability distribution of cells’ motion bias after collision with diluted colony edge plotted against interaction angle (Blue column: CW bias; Magenta column: CCW bias). f Model of a swimming bacterium in contact with liquid drop edge (top view). Discussion of supply from upstream, microspheres became barely visible in the inner motile ring downstream of the motility-deactivation region (Fig. 5f). Flagellated bacteria grown on solid substrates often develop into structured communities, in which most cells have transitioned into a non-motile state while a small population remains motile. In this paper, we investigated the behavior of motile cell popu- lation in sessile colonies of flagellated bacteria. Our major dis- covery is that motile cells in routinely cultured bacterial colonies can self-organize into two adjacent motile rings surrounding the entire colony via spontaneous segregation that mimics a phase separation. Cells in the outer motile ring circle CW around the colony (viewing from above) with high polar order, while cells in the inner ring swim in parallel to colony edge bi-directionally with nematic order in cells’ moving directions. The self- organization of motile cells is mediated by intercellular interac- tions and shear-induced depletion. Flagellar rotation of cells in the outer motile ring generates directed fluid flows in the inner motile ring that circulate CCW around the colony at a constant speed of ~30 µm s−1. Overall, our findings reveal that motile sub- populations in sessile colonies of flagellated bacteria can self- organize in a remarkable and heretofore unnoticed manner. Shear-induced depletion facilitates inner motile ring forma- tion. Here we introduce a conceptual model to explain the for- mation of inner motile ring. The results presented above provide strong evidence that the CCW directed flows in inner motile ring are driven by flagellar motility of cells in the outer motile ring. The directed fluid flows can cause shear-induced depletion to aid the formation of inner motile ring; shear-induced depletion is a phenomenon recently reported in bacterial suspensions that leads to the accumulation of swimmers in high-shear regions55. To examine this idea, we used fluorescent microspheres as tracers to map the flow speed profile generated by the CW outer motile ring of P. mirabilis suspension drops in the absence of the inner motile ring (thus avoiding the influence of cells in the inner motile ring on tracer motion, as microspheres tend to stick to cells) (Sup- plementary Fig. 8). We found that the region with horizontal shear rate coincided with the expected range of inner motile ring (ranging from ~10 to ~40 μm in X-axis of Supplementary Fig. 8). We further measured the radial profile of bacterial density in suspension drops (Supplementary Fig. 9). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 The torque driving reorentation Gdrive and the drag torque Gdrag balance, which leads to rotation at a constant angular speed ω about the x = 0; y = 0 point (green dot). The flagellar bundle is rotating at an angular speed Ω. The length of cell body and flagellar bundle is Lb and Lf, respectively. Also see “Methods” motile ring, we used patterned illumination modulated by a digital mirror device (Andor Mosiac; “Methods”) to optically deactivate the flagellar motility of cells in outer motile ring, while measuring how the transport of fluorescent microspheres in inner motile ring was affected. We took advantage of the photosensitiz- ing effect of membrane stain FM 4–64 (Life Technologies, Cat. No. T13320; “Methods”) for motility deactivation; upon excita- tion, FM4–64 disrupts flagellar motility, most likely due to the release of reactive oxygen species53,54. When cell motility was deactivated in a selected area of outer motile ring, we found rapid accumulation of microspheres in the region of inner motile ring just adjacent to the deactivation area (Fig. 5a–g) and the disappearance of CCW bias of collective motion in this region (Fig. 5h–i), indicating that CCW directed fluid flows were substantially halted in this region. Due to the substantial decrease FITC–Dextran get transported into the sessile, interior part of the colony along some crack-like conduits (Fig. 4e–h; Supplementary Movie 12; Supplementary Fig. 5). The conduits typically formed at ~15–20 h after inoculation and became fully developed within 1 h (Supplementary Fig 6, panel A); they remained stable for many hours until the colony started to swarm, and then they gradually disappeared within ~3 h (Supplementary Fig. 6, panel B). The conduits have a mean radial extension of ~65 µm, with the longest ones up to ~200 µm (Supplementary Fig. 7). The inwards moving flows along the conduits are presumably driven by flagellar rotation of motile cells confined along the boundary of these conduits, and the flows may increase nutrient availability for the interior part of the colony. y To further verify that the observed CCW directed flows in the inner motile ring are driven by flagellar rotation of cells in outer NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 0 s 200 s 100 s 300 s e g h f a 0 s c 120 s b 60 s d 180 s Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 4 Long-range, colony-scale directed transport in a P. mirabilis colony. a–d Image sequence showing the directed transport of 0.1-μm fluorescent microspheres in the inner motile ring around colony edge (“Methods”). Fluorescent images of microspheres were overlaid with phase contrast images of the colony (at low light intensity). Bright blob in the upper part of the images is where the fluorescent microspheres were deposited. Arrows indicate the position of microspheres transported for the longest distance at indicated times; these foremost microspheres are only visible when enlarging the figure or when viewing the associated Supplementary Movie 11. Scale bar, 500 μm. e–h Image sequence showing the directed transport of 0.1-μm fluorescent microspheres inwards along crack-like conduits present near colony edge. The conduits are inside the rectangular area showed in the images. Also see Supplementary Movie 12. Scale bar, 50 μm 100 s f 0 s e 300 s h 200 s g g 200 s g 0 s h Fig. 4 Long-range, colony-scale directed transport in a P. mirabilis colony. a–d Image sequence showing the directed transport of 0.1-μm fluorescent microspheres in the inner motile ring around colony edge (“Methods”). Fluorescent images of microspheres were overlaid with phase contrast images of the colony (at low light intensity). Bright blob in the upper part of the images is where the fluorescent microspheres were deposited. Arrows indicate the position of microspheres transported for the longest distance at indicated times; these foremost microspheres are only visible when enlarging the figure or when viewing the associated Supplementary Movie 11. Scale bar, 500 μm. e–h Image sequence showing the directed transport of 0.1-μm fluorescent microspheres inwards along crack-like conduits present near colony edge. The conduits are inside the rectangular area showed in the images. Also see Supplementary Movie 12. Scale bar, 50 μm Discussion CCW bias of cellular motion changed from ~1.19 to ~1.04 in this experiment b 20 s a 16 s b 24 s c a c d 28 s e 32 s f 84 f 84 s d f e 0.10 0.15 0.20 Probability h 10 20 40 30 50 0 0.05 Vcell (µm/s) Vcell (µm/s) 0.10 0.15 0.20 i 0 0.05 10 20 40 30 50 0 10 20 40 30 0 0.2 0.4 0.6 0.8 1.0 Time (s) Fluorescence intensity (a.u.) g 0.10 0.15 0.20 Probability h 10 20 40 30 50 0 0.05 Vcell (µm/s) Vcell (µm/s) 0.10 0.15 0.20 i 0 0.05 10 20 40 30 50 g 0 i i h Fig. 5 Optical deactivation of flagellar motility in the outer motile ring. A region of the outer motile ring (enclosed by solid white rectangles in panels a–f was chosen for motility deactivation with intense light (“Methods”). a–e Fluorescent image sequence of 0.1-μm microspheres in the same field of view before (a–b) and after (c–e) the deactivation of flagella motility. Motility-deactivation light at the selected region was turned on at time = 24 s (panel c). Scale bars, 50 μm. f Fluorescent image of 0.1-μm microspheres overlaid with the phase-contrast image of colony edge at time = 84 s. Microspheres were barely visible in the inner motile ring downstream of the motility-deactivation region. Scale bar, 50 μm. g Mean fluorescence intensity of microspheres averaged over a selected area of the inner motile ring (enclosed by blue rectangles in panels a–f) plotted against time. The shaded area starting at time = 24 s indicates the duration of motility-deactivation. Before motility deactivation (from 0 to 24 s), the slope of fluorescence intensity given by linear fit is 0.013 ± 0.002 a.u. s−1 (±sign indicates fitting error; R2 = 0.94); the slope increased to 0.041 ± 0.002 a.u. s−1 (R2 = 0.95) after motility deactivation. h–i Speed distribution of individual cells in the inner motile ring before (panel h) and after (panel i) motility deactivation, respectively. The speed distribution of cells moving in CCW or CW direction were represented by columns filled in magenta or yellow color, respectively, and both types of columns were plotted together in the same histogram with the overlapping portion filled with white color. Discussion Near the transition region between the dilute phase and the inner motile ring (at ~40 micron from the edge), the surface cell density experienced a sharp increase when moving towards the edge. This increase of surface cell density cannot be attributed to the variation of the height of liquid film, since the fluid height in the dilute phase is comparable or greater than that in the inner motile ring; indeed, the increase of surface cell density reflects the increase of volume cell density and proves that cells accumulate towards the edge from the dilute phase. The region with cell accumulation (~10–40 micron from the edge) corresponds to the region of inner motile ring (Fig. 2a), and it coincides with the region with horizontal shear shown in Supplementary Fig. 8. These results (Supple- mentary Figs. 8, 9), together with the fact that the width of inner motile ring increases linearly with cell speed (Fig. 2e), supports the idea that shear-induced depletion helps attract cells to colony edge and contributes to the formation of inner motile ring in colonies. g The mechanism underlying motile-ring self-organization deserves further study in the context of flagellar hydrodynamics and active matter self-organization. Shear-induced depletion may account for the accumulation of cells that leads to the formation of inner motile ring. As the inner motile ring is densely packed with swimming cells, would the modification of flow field by these cells affect the width or the stability of the motile rings? We note that these cells are moving bi-directionally and therefore the flows they generated would cancel one another out; consequently the flows generated by the outer motile ring would be largely pre- served, as demonstrated by the strong CCW flows present in the inner motile ring in colonies (Fig. 4). Nonetheless, the weak CCW bias of collective motion in the inner motile ring would compli- cate the issue. Moreover, hydrodynamic attraction between cells, similar to that responsible for cohesive swimming of cells in 2D confinement56, may help to reduce the orientational noise and thereby stabilize the motile rings. On the other hand, accumu- lation of cells via the shear-depletion mechanism is not sufficient to account for the nematic order in cells’ moving directions in the inner motile ring. Discussion CCW bias of cellular motion changed from ~1.19 to ~1.04 in this experiment The unique mechanism of bacterial self-organization we uncovered here may be important to the physiology and stress response of bacterial communities in widespread environments. Self-segregation of motile subpopulations to the colony edge apparently reshapes the population structure in bacterial com- munities, which could facilitate the initiation of dispersal or range expansion (e.g., via swarming process). In addition, the self- organization of swimmers generates highly directed fluid flows, providing a stable high-speed avenue for long-range directed material transport in bacterial communities. Diffusion has been assumed to dominate material transport within bacterial com- munities58–60, and only a few examples of directed transport have been found so far51,61–63. It was reported that B. subtilis colonies can make use of evaporation-driven fluid flows to aid colony- scale material transport;62 however, the process is not driven by stabilize unidirectional collective motion (in which cells align their orientations nematically but move unidirectionally)32, and nematically ordered collective motion (in which cells move bi- directionally in parallel with each other as seen in our phenom- enon) was not reported. In a recent study, computational mod- eling predicted that self-propelled polar particles interacting locally by nematic alignment could self-segregate into high- density bands with nematic order in particles’ moving directions in the midst of low-density disordered regions57. It will be intriguing to examine the relevance of these model results to our findings. However, the model did not include hydrodynamic interaction between cells. The dynamics of motile rings may be better understood by modeling dense suspensions of swimmer that takes into account both hydrodynamic and steric interactions. stabilize unidirectional collective motion (in which cells align their orientations nematically but move unidirectionally)32, and nematically ordered collective motion (in which cells move bi- directionally in parallel with each other as seen in our phenom- enon) was not reported. In a recent study, computational mod- eling predicted that self-propelled polar particles interacting locally by nematic alignment could self-segregate into high- density bands with nematic order in particles’ moving directions in the midst of low-density disordered regions57. It will be intriguing to examine the relevance of these model results to our findings. However, the model did not include hydrodynamic interaction between cells. The dynamics of motile rings may be better understood by modeling dense suspensions of swimmer that takes into account both hydrodynamic and steric interactions. Discussion Advection due to fluid flows generated by flagellated bacteria at confinement boundary was shown to 7 TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 24 s c b a f e d 0 10 20 40 30 0 0.2 0.4 0.6 0.8 1.0 Time (s) Fluorescence intensity (a.u.) g 16 s 20 s 28 s 32 s 84 s 0.10 0.15 0.20 Probability h 10 20 40 30 50 0 0.05 Vcell (µm/s) Vcell (µm/s) 0.10 0.15 0.20 i 0 0.05 10 20 40 30 50 Fig. 5 Optical deactivation of flagellar motility in the outer motile ring. A region of the outer motile ring (enclosed by solid white rectangles in panels a–f was chosen for motility deactivation with intense light (“Methods”). a–e Fluorescent image sequence of 0.1-μm microspheres in the same field of view before (a–b) and after (c–e) the deactivation of flagella motility. Motility-deactivation light at the selected region was turned on at time = 24 s (panel c). Scale bars, 50 μm. f Fluorescent image of 0.1-μm microspheres overlaid with the phase-contrast image of colony edge at time = 84 s. Microspheres were barely visible in the inner motile ring downstream of the motility-deactivation region. Scale bar, 50 μm. g Mean fluorescence intensity of microspheres averaged over a selected area of the inner motile ring (enclosed by blue rectangles in panels a–f) plotted against time. The shaded area starting at time = 24 s indicates the duration of motility-deactivation. Before motility deactivation (from 0 to 24 s), the slope of fluorescence intensity given by linear fit is 0.013 ± 0.002 a.u. s−1 (±sign indicates fitting error; R2 = 0.94); the slope increased to 0.041 ± 0.002 a.u. s−1 (R2 = 0.95) after motility deactivation. h–i Speed distribution of individual cells in the inner motile ring before (panel h) and after (panel i) motility deactivation, respectively. The speed distribution of cells moving in CCW or CW direction were represented by columns filled in magenta or yellow color, respectively, and both types of columns were plotted together in the same histogram with the overlapping portion filled with white color. Methods B t i l t Bacterial strains. The following strains were used: wildtype P. mirabilis BB2000, and a fluorescent P. mirabilis KAG108 (BB2000 background with constitutive expression of Green Fluorescent Protein (GFP);69 from Karine Gibbs, Harvard University, Cambridge, MA); E. coli HCB1737 (a derivative of E.coli AW405 with wildtype flagellar motility but with FliC S219C mutation for fluorescent labeling; from Howard Berg, Harvard University, Cambridge, MA); E. coli HCB1737 GFP (HCB1737 transformed with pAM06-tet plasmid with constitutive GFP expres- sion70 from Arnab Mukherjee and Charles M. Schroeder, University of Illinois at Urbana-Champaign); wildtype B. subtilis 3610 and a smooth-swimming mutant B. subtilis DK2178, which is a cheB-deleted derivative of B. subtilis 3610 (from Daniel B. Kearns, Indiana University at Bloomington). Single-colony isolates were grown overnight (~13–14 h) with shaking in LB medium (1% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl) at 30 °C to stationary phase. For E. coli HCB1737 GFP, kanamycin (50 μg per mL) was added to the growth medium for maintaining the plasmid. Single-cell motion pattern at the edge of liquid drops. For P. mirabilis BB2000, 1 μL motility buffer was deposited near the edge of a P. mirabilis colony. The diluted colony edge formed a liquid drop dispersed with isolated cells. The motion pattern of isolated cells near the diluted colony edge was observed in phase contrast with the 20× dark phase objective and was recorded at 30 fps for 10 min (Fig. 3). For B. subtilis, overnight culture was diluted with motility buffer 10−4, and 1-μL drops of the diluted suspension were deposited onto 2.0% LB agar plates. The motion pattern of isolated B. subtilis cells near the edge of the suspension drop was observed in phase contrast with the 20× dark phase objective and was recorded at 30 fps for 10 min (Supplementary Movie 10). For E. coli (HCB1737 GFP), flagellar filaments were fluorescently labeled prior to observation of single-cell motion pattern, following procedures established in ref. 45 and modified in ref. 56. Briefly, 0.1 μL overnight culture of E. coli (HCB1737 GFP) was diluted 10−4 to a volume of 1 mL in a glass test tube. Labeling dye stock solution (Alexa Flour 546 C5- Preparation of agar plates for colony growth. LB agar (Difco Bacto agar at specified concentrations infused with 1% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl) was autoclaved and stored at room temperature. ARTICLE mirabilis occurs under our experimental conditions following the emergence of motile rings (>24 h after inoculation, with large variability of the exact initiation time of swarming). Observations were made in phase contrast with the 40× dark phase objective (Nikon CFI Super Plan Fluor ELWD ADM 40XC, N.A. 0.60, W.D. 3.6–2.8 mm) and were recorded at 30fps. For single-cell tracking at the edge of P. mirabilis colonies, overnight culture of the GFP-tagged P. mirabilis KAG108 was mixed with wildtype P. mirabilis at 1:2000 before inoculating the agar plates. P. mirabilis KAG108 cells were imaged with an FITC single band filter set (excitation 482/35 nm, emission 536/40 nm, dichroic: 506 nm; FITC-3540C-000, Semrock Inc.) and were recorded at 30 fps for 2 min. To monitor the development of motile rings and crack-like conduits during colony growth, the edge of P. mirabilis colonies was recorded at 30 fps for 10 s duration every 20 min with the 20× dark phase objective (Nikon CFI Super Plan Fluor ELWD ADM 20XC, N.A. 0.45, W.D. 8.2–6.9 mm). Bacterial self-organization at the edge of cell suspension drops. To observe self-organization at the edge of cell suspension drops (artificial colonies) (Fig. 2), overnight culture of the GFP-tagged P. mirabilis KAG108 was either used alone (Fig. 2a, e, f, Supplementary Movies 6, 7) or mixed with wildtype P. mirabilis BB2000 at 1:50 (Fig. 2b–d), and 1-μL drops of the pure culture or the mixture were inoculated on 0.6% LB agar plates. The plates were incubated at 30 °C and ~85% relative humidity for 36 h; at this time the colony had started to expand. Cells were collected from the leading edge of the colony with motility buffer (0.01 M potas- sium phosphate, 0.067 M NaCl, 10−4 M EDTA, pH 7.0). The collected suspension was washed by mobility buffer for three times and resuspended in motility buffer to reach appropriate cell densities. Tween 20 (Sigma-Aldrich, Cat. No. P7949) was then added to the cell suspension at a final concentration of 0.002% (wt/wt). 1-μL drops of this suspension were deposited onto 2.0% LB agar plates supplemented with 0.002% (wt/wt) Tween 20. Bacterial self-organization at the edge of such suspension drops was observed in phase contrast and in fluorescence (via the FITC single band filter set described above) with the 40× dark phase objective. Images were recorded at 30 fps. ARTICLE On the other hand, we wonder whether the long-range directed flows could be exploited to perturb the behavior of bacterial communities; for example, drugs and chemical effectors loaded in vesicles could be supplied locally and transported by the flows to manipulate distant parts of a bacterial colony. plates were dried for another 10 min without a lid inside the Plexiglas box. The plates were then covered and incubated at 30 °C and ~85% relative humidity in an incubator with water tray for specified durations of time (see below). To vary the environmental humidity for colony growth, the plates were incubated at 30 °C in a custom-built incubator in which different kinds of saturated salt solutions (NaBr: 47.4%RH; KI: 55.0%RH; NaCl: 60.4%RH) or DI water (~97% RH) were supplemented, or incubated in a regular incubator without water tray (~42% RH) at 30 °C. Phase contrast and fluorescence imaging. Imaging was performed on a motorized inverted microscope (Nikon TI-E). The following objectives were used in different experiments for both phase contrast and fluorescence imaging: Nikon CFI Plan Fluor DL4X, N.A. 0.13, W.D. 16.4 mm; Nikon CFI Achromat DL 10×, N. A. 0.25, W.D. 7.0 mm; Nikon CFI Super Plan Fluor ELWD ADM 20XC, N.A. 0.45, W.D. 8.2–6.9 mm; and Nikon CFI Super Plan Fluor ELWD ADM 40XC, N.A. 0.60, W.D. 3.6–2.8 mm. Fluorescence imaging was performed in epifluorescence using filter sets specified below, with the excitation light provided by a mercury pre- centered fiber illuminator (Nikon Intensilight). All recordings were made with a sCMOS camera (Andor ZYLA 4.2 PLUS USB 3.0) at full frame size (2048 × 2048 pixels) using the software NIS-Elements AR (Nikon). In all experiments the petri dishes were covered with lid to prevent evaporation and air convection, and the sample temperature was maintained at 30 °C using a custom-built temperature control system installed on microscope stage, unless otherwise stated. Bacterial self-organization at the edge of sessile colonies. To observe self- organization at the edge of sessile colonies (Fig. 1 and Supplementary Fig. 2), P. mirabilis BB2000 or E. coli HCB1737 colonies were grown on 0.6% LB agar plates at ~85% relative humidity (as described above) for 24 h, and B.subtilis colonies were grown on 2.0% LB agar plates ~85% relative humidity for 2 h. Swarming of P. ARTICLE To study the velocity dependence of the width of inner motile ring, we made use of the fact that the swimming speed of flagellated bacteria depends on temperature71, and we varied cell speed in P. mirabilis suspension drops by changing environmental temperature from 15 to 37 °C with a temperature-controlled water bath. ARTICLE On the other hand, we wonder whether the long-range directed flows could be exploited to perturb the behavior of bacterial communities; for example, drugs and chemical effectors loaded in vesicles could be supplied locally and transported by the flows to manipulate distant parts of a bacterial colony. autonomous cellular activities. Expanding swarms of flagellated bacteria can generate CW directed flows at colony edge at a similar magnitude (~10 µm s−1)51, but it is not stable due to constant swarm expansion (lasting for <1 min) and material transport can only persist for up to ~500 µm; these time and length scales are two orders of magnitude smaller than that associated with the CCW flows in inner motile ring, which last for hours and persist for centimeters. Moreover, the long-range active transport we report here is enabled by bacterial self-organization, whereas cells at the edge of bacterial swarms do not display stable ordering of orientations or moving directions despite the pre- sence of transient jets and vortices lasting for a fraction of a second. The unique means of long-range active transport in bacterial colonies we uncovered here is reminiscent of mucus flow driven by motile cilia of epithelial cells in various organ systems, such as the respiratory tract and oviduct64. We speculate that this long-range active transport may have profound effect on the physiology of bacterial communities. Bacteria communities in natural and clinical settings normally grow in anisotropic, structured environments with heterogeneous nutrient or chemical distribution. In such environments, bacterial communities do not have rotational symmetry and the self-organization of motile subpopulation may occur in various locations not limited to the colony edge; thus the long-range directed flows enabled by motile-cell self-organization may efficiently redistribute slowly- diffusing substances or molecules with high binding affinity to cell surface within bacterial communities. For example, high- molecular-weight polymeric metabolites65 and membrane vesicles that encapsulate nutrients or signaling molecules66 could travel for 1 cm in ~5 min via the long-range directed flows, which is much more efficient than diffusion process alone (either passive diffusion driven by thermal energy or active diffusion driven by active forces in a bath of motile cells61,67,68). In fact, the 0.1-μm fluorescent microspheres we used to track the flows are com- parable to the size of membrane vesicles secreted by many bac- terial species, and thus the motion of microspheres should closely resemble the transport of membrane vesicles in situ in colonies. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 autonomous cellular activities. Expanding swarms of flagellated bacteria can generate CW directed flows at colony edge at a similar magnitude (~10 µm s−1)51, but it is not stable due to constant swarm expansion (lasting for <1 min) and material transport can only persist for up to ~500 µm; these time and length scales are two orders of magnitude smaller than that associated with the CCW flows in inner motile ring, which last for hours and persist for centimeters. Moreover, the long-range active transport we report here is enabled by bacterial self-organization, whereas cells at the edge of bacterial swarms do not display stable ordering of orientations or moving directions despite the pre- sence of transient jets and vortices lasting for a fraction of a second. The unique means of long-range active transport in bacterial colonies we uncovered here is reminiscent of mucus flow driven by motile cilia of epithelial cells in various organ systems, such as the respiratory tract and oviduct64. We speculate that this long-range active transport may have profound effect on the physiology of bacterial communities. Bacteria communities in natural and clinical settings normally grow in anisotropic, structured environments with heterogeneous nutrient or chemical distribution. In such environments, bacterial communities do not have rotational symmetry and the self-organization of motile subpopulation may occur in various locations not limited to the colony edge; thus the long-range directed flows enabled by motile-cell self-organization may efficiently redistribute slowly- diffusing substances or molecules with high binding affinity to cell surface within bacterial communities. For example, high- molecular-weight polymeric metabolites65 and membrane vesicles that encapsulate nutrients or signaling molecules66 could travel for 1 cm in ~5 min via the long-range directed flows, which is much more efficient than diffusion process alone (either passive diffusion driven by thermal energy or active diffusion driven by active forces in a bath of motile cells61,67,68). In fact, the 0.1-μm fluorescent microspheres we used to track the flows are com- parable to the size of membrane vesicles secreted by many bac- terial species, and thus the motion of microspheres should closely resemble the transport of membrane vesicles in situ in colonies. Discussion NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunicatio ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 maleimide, 5 mg per ml in DMSO; Life Technologies) was added to the cell sus- pension in test tube at a final concentration of 25 μg per ml. Labeling was allowed to proceed for 7 min in a shaker (30 °C and 180 rpm). When labeling was com- pleted, the cell suspension was transferred to 1.5 ml Eppendorf tubes, brought to a volume of 10 mL with mobility buffer, and washed free of unreacted dyes by centrifugation and resuspension for three times (10,000 × g for 4 min in the first wash, 2000 × g for 10 min in the second and third washes). After washing, cells were resuspended in mobility buffer to 1 mL, and 1-µl drops of this cell suspension were deposited onto 2.0% LB agar plates supplemented with 0.002% (wt/wt) Tween 20. The motion pattern of cell body and flagellar filaments of isolated E. coli cells near the edge of the suspension drop was imaged in fluorescence with the 20× dark phase objective, via an EGFP/mCherry dual band filter set (excitation: 471/38 nm and 571/34 nm, emission: 522/42 nm and 634/62 nm, dichroic: 522/52 nm and 636/ 84 nm; Part No. 59022, Chroma), and was recorded at 30 fps (Supplementary Movie 9). different ways for the outer motile ring and for the inner motile ring, respectively. In the outer motile ring, cells move at an almost uniform speed, so we randomly chose 10 cells there and computed their mean speeds over ~10 s. The speed of cells in the inner motile ring has a broad distribution, so we tracked ~1000 cells in ~5- min movies and divided their trajectories into ~10,000 segments, each segment with a duration of 1 s. The mean speed of these 1-s segments were computed to yield the velocity statistics of cells. The velocity statistics of microspheres were obtained in a similar way. To study the cell density dependence of collective motion in outer motile ring, the cell occupation ratio at the 10-μm-wide outmost rim of suspension drops was defined as the area occupied by cells divided by total area of the rim. To determine the width of inner motile ring in P. mirabilis suspension drops, GFP-tagged P. Model of cell reorientation upon collision with colony edge. The driving ll d h l d d h Model of cell reorientation upon collision with colony edge. The driving reorientation torque Gdrive is numerically estimated using the Slender Body Theory Model of cell reorientation upon collision with colony edge. The driving reorientation torque Gdrive is numerically estimated using the Slender Body Theory (SBT)47,48 for the helix rotating above the no-slip wall ignoring the top air/fluid Interface. The SBT model takes into account full hydrodynamic interactions with the surface (except for lubrication when the helix is all but touching the surface). Firstly the force distribution f on the stationary helix rotating next to the no-slip wall is calculated. From this force distribution we calculate the instantaneous reorientation torque about the tip of the cell (x = 0; y = 0 point in Fig. 3f): Visualization of fluid flows in colonies and suspension drops. P. mirabilis BB2000 colonies were grown on 0.6% LB agar plates as described above for 24 h. To visualize fluid flows (Fig. 4, Supplementary Figs. 5 and 8), fluorescent microspheres (Fluo-Spheres, carboxylate-modified, 0.1 μm diameter; Life Technologies Cat. No. F8820) or fluorescently labeled dextran (FITC–Dextran, mol. wt. 2000 kDa; Sigma, FD2000S) were deposited near the colony edge. Fluorescence of microspheres was excited via a Cy3/TRITC single band filter set (excitation 535/50 nm, emission 610/ 75 nm, dichroic: 565 nm; Part No. 41007, Chroma), and FITC–Dextran was excited via the FITC single band filter set. Phase contrast images of colony edge (low- intensity illumination from above the sample provided by the built-in halogen lamp of the microscope) and fluorescent images of microspheres were taken with the 4× phase-contrast objective (Nikon CFI Plan Fluor DL4X, N.A. 0.13, W.D. 16.4 mm) and recorded simultaneously at 5 fps (Supplementary Movie 11). The directed transport of fluorescent microspheres inwards along crack-like conduits near col- ony edge was observed with the 10× objective (Nikon CFI Achromat DL 10X, N.A. 0.25, W.D. 7.0 mm), and was recorded at 5 fps simultaneously in phase contrast (illumination from above the sample provided by the built-in halogen lamp of the microscope) and in fluorescence (illumination provided by Nikon Intensilight via the Cy3/TRITC single band filter set) (Supplementary Movie 12). The transport of FITC–Dextran near colony edge was observed with the 10× objective and recorded at 5 fps in fluorescence (illumination provided by Nikon Intensilight via the FITC single band filter set). To measure the flow speed profile generated by CW outer motile ring (Supplementary Fig. Model of cell reorientation upon collision with colony edge. The driving ll d h l d d h 8), P. mirabilis BB2000 suspension drops were deposited onto 2.0% LB agar as described above, and fluorescent microspheres were deposited near the suspension drop edge. Fluorescence of microspheres was excited via the Cy3/TRITC single band filter set and recorded with the 20× objective at 30 fps. GdriveðtÞ ¼ ZLf 0 Lb þ x′ ð ÞfY′ðtÞsec Ψdx′; tan Ψ ¼ 2πR P ð6Þ ð6Þ where fy′ (t, h, r, x′) is the force density in the y′ direction which is driving the reorientation (Fig. 3f), h the height above the wall of the helical axis, r the radius of flagellar bundle, Lf the length of the flagellum along its axis, Lb the length of the cell body, x′ the length parameter x′ ∈[0, Lf], R the radius of flagellar helix, and P the pitch of flagellar helix. The contribution due to the force component fx will be small because of the geometry. The instantaneous torque is averaged over one period T of the flagellar rotation: 0.25, W.D. 7.0 mm), and was recorded at 5 fps simultaneously in phase contrast (illumination from above the sample provided by the built-in halogen lamp of the microscope) and in fluorescence (illumination provided by Nikon Intensilight via the Cy3/TRITC single band filter set) (Supplementary Movie 12). The transport of FITC–Dextran near colony edge was observed with the 10× objective and recorded at 5 fps in fluorescence (illumination provided by Nikon Intensilight via the FITC single band filter set). To measure the flow speed profile generated by CW outer motile ring (Supplementary Fig. 8), P. mirabilis BB2000 suspension drops were deposited onto 2.0% LB agar as described above, and fluorescent microspheres were deposited near the suspension drop edge. Fluorescence of microspheres was excited via the Cy3/TRITC single band filter set and recorded with the 20× objective at 30 fps. Gdrive ¼ 1=T ZT 0 GdriveðtÞdt ð7Þ ð7Þ The parameters for the helix are: the pitch P = 2.3 μm73, radius R = 0.3 μm73, the axial length Lf = 7.3 ± 2.4 μm73, the length of the cell body Lb = 1.9 μm74, radius of the bundle r = 24 nm (double radius of a single flagellum)75, frequency of rotation 1/T = Ω/2π = 110 Hz73. The driving torque as a function of height is computed as shown in Supplementary Fig. 4. Model of cell reorientation upon collision with colony edge. The driving ll d h l d d h The closer the flagellar bundle is to the bottom wall, the larger reorientation torque is created. Optical deactivation of motility via patterned illumination. P. mirabilis BB2000 colonies were grown on 0.6% LB agar plates supplemented with FM 4–64 (final concentration 1.0 μg per mL) as described above for 24 h. Fluorescent microspheres were deposited near the colony edge (described above) and got transported along the colony edge. A selected region of the outer motile ring (length: 150 μm; width: 20 μm) was chosen for motility deactivation. A beam of intense green-orange light provided by an LED illuminator (Thorlabs 530 nm Mounted High-Power LED L3, item No. M530L3, installed on X-Cite XLED1; Excelitas Technologies Corp) was modulated by a digital mirror device (Andor Mosiac) to form a spatially defined illumination pattern, and the modulated light passed through the 40× objective via the Cy3/TRITC filter set (described above) to excite FM4–64 in the selected region for motility deactivation. Meanwhile, the entire field of view was illuminated at low light intensity for imaging in phase contrast (illumination from above the sample provided by the built-in halogen lamp of the microscope) and/or in fluorescence (illumination provided by Nikon Intensilight via the Cy3/TRITC filter set). Recordings were made at 30 fps. We use the Resitive Force Theory (RFT)49 to estimate the drag torque Gdrag due to the rotation of the body with flagellar bundle around the tip of the cell. The majority of the drag torque comes from the flagellar bundle because it is much longer than the body and the flagellar bundle is further away from the rotation point. Therefore we approximate the cell-flagella system as a slender rod of length L = Lb + Lf (which is a sum of the body length and flagellum length) and of radius r (i.e., the radius of flagellar bundle). The force per unit length f on a slender straight filament moving with velocity u is given by: f ¼ ðξ?nn þ ξkttÞ  u ð8Þ ð8Þ where t is the vector along the axis of filament and n perpendicular to it. In this case the body-flagella system is rotating about the tip of the cell. The point which is l distance away from the tip is moving at the linear speed ωl. Methods B t i l t Before use, the agar was melted in a microwave oven, cooled to ~60 °C, and pipetted in 10-mL aliquots into 90-mm polystyrene petri plates. For optical deactivation of flagellar motility, the membrane stain FM 4–64 (Life Technologies, Cat. No. T13320) was dissolved in deionized water and added at a final concentration of 1.0 μg per mL to the liquefied agar before pipetting. The plates were swirled gently to ensure surface flatness, and then cooled for 10 min without a lid inside a large Plexiglas box. Drops of 1 μL overnight bacterial cultures were inoculated onto the LB agar plates. 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Code availability 31. Wioland, H., Woodhouse, F. G., Dunkel, J., Kessler, J. O. & Goldstein, R. E. Confinement Stabilizes a Bacterial Suspension into a Spiral Vortex. Phys. Rev. Lett. 110, 268102 (2013). The custom codes used in this study are available from the corresponding author upon request. 32. Lushi, E., Wioland, H. & Goldstein, R. E. Fluid flows created by swimming bacteria drive self-organization in confined suspensions. Proc. Natl Acad. Sci. USA 111, 9733–9738 (2014). Received: 6 June 2018 Accepted: 28 March 2019 Model of cell reorientation upon collision with colony edge. The driving ll d h l d d h The drag torque is given by:76 Gdrag ¼  ZL 0 f  n ð Þldl ¼ ZL 0 ξ?ωl ð Þldl ¼ ξ?ω L3 3 Image processing and data analysis. Images were processed using the open source Fiji (ImageJ) software (http://fiji.sc/Fiji) and custom-written programs in MATLAB R2016b (The MathWorks; Natick, Massachusetts, United States). The velocity field of cells’ collective motion was obtained by performing optical flow analysis based on microscopy movies using the built-in functions of MATLAB. Prior to computing the optical flow fields, the images were first smoothed to reduce noise by convolution with a Gaussian kernel of standard deviation 1. The optical flow field for any two consecutive video frames was computed using the Horn- Schunck algorithm72 (maximum iteration number, 128; smoothness parameter, 1) and then smoothed by local averaging. The grid size of the optical flow field was 1 pixel × 1 pixel and the initial value of optical flow vectors was set to zero. The obtained optical flow fields were used to compute the collective speed profile in Figs. 1d and 2c. To visualize the collective velocity field (Figs. 1c and 2b), the optical flow fields were coarsened to a grid size of 5.2 μm × 5.2 μm. The results were insensitive to different parameters of smoothing. For single-cell and single- microsphere tracking, bacteria and microsphere trajectories were obtained by a custom-written particle-tracking program in Matlab (The MathWorks, Inc) based on phase-contrast or fluorescence movies. Single-cell speed calculation was done in ξ? ¼ 4πμ 1=2 þ lnð0:18Psec Ψ=rÞ ; tan Ψ ¼ 2πR P ð9Þ ð9Þ which means that the reorientation angular speed is which means that the reorientation angular speed is ω ¼ 3Gdrive ξ?L3 ð10Þ ð10Þ We then use the result from the SBT for the driving torque Gdrive to calculate the reorientation angular speed ω as a function of h, as shown in Supplementary Fig. 4. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 10 Competing interests: The authors declare no competing interests. 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The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 75. Yonekura, K., Maki-Yonekura, S. & Namba, K. Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 424, 643–650 (2003). 76. Cox, R. G. The motion of long slender bodies in a viscous fluid Part 1. Competing interests: The authors declare no competing interests. General theory. J. Fluid Mech. 44, 791–810 (1970). ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 Grants 4053230&4053310; to Y.W.). This project has also received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement 682754 to EL). 63. Petroff, A. & Libchaber, A. Hydrodynamics and collective behavior of the tethered bacteriumThiovulum majus. Proc. Natl Acad. Sci. USA 111, E537–E545 (2014). 64. Huang, B. K. & Choma, M. A. Microscale imaging of cilia-driven fluid flow. Cell. Mol. Life Sci. 72, 1095–1113 (2015). Author contributions 65. Hartley, J. L., Adams, G. A. & Tornabene, T. G. Chemical and physical properties of lipopolysaccharide of Yersinia pestis. J. Bacteriol. 118, 848–854 (1974). H.X. performed the experiments, analyzed and interpreted the data. J.D., D.D., and E.L. development the mathematical model. Y.W. discovered the phenomena, designed the study, performed the initial experiments, analyzed and interpreted the data. Y.W. wrote the paper, with input from other authors. 66. Mashburn, L. M. & Whiteley, M. 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Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-09818-2. Competing interests: The authors declare no competing interests. © The Author(s) 2019 Acknowledgements We thank Howard C. Berg, Karine Gibbs, Daniel B. Kearns, Arnab Mukherjee, and Charles M. Schroeder for their kind gifts of bacterial strains, and Howard C. Berg for helpful comments. The work of H.X. and Y.W. was supported by the National Natural Science Foundation of China (NSFC 21473152; to Y.W.) and by the Research Grants Council of Hong Kong SAR (RGC Ref. No. GRF 14322316&14301915, CUHK Direct © The Author(s) 2019 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunicatio
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Abstract Spatial information on the varying composition of coral reefs is beneficial for the management and preservation of natural resources in coastal areas. Its availability is inseparable from environmental management goals; however, it can also be used as a means of supporting tourism activities and predicting the emergence of certain living species. A satellite image is one of the effective and efficient data sources that provide spatial information on coral reef variations. This study aimed to evaluate the classification scheme of coral reef life-form using images with different spatial resolutions on Parang Island, Karimunjawa Islands, Central Java. These images were from PlanetScope (3m), PlanetScope resampling (6m), and Sentinel-2A MSI (10m), whose spatial resolutions functioned as the base for building the 3m, 6m, and 10m classification schemes producing 12, 11, and 9 classes, respectively. As for the classification method, it integrated both object-based and pixel-based approaches. The results showed that the highest overall accuracy (60%) was obtained using Sentinel-2A MSI image (10m), followed by PlanetScope (3m) with 48% accuracy, and PlanetScope resampling (6m) with 40% accuracy. This finding indicates that multiresolution images can be used to produce complex coral reef life-form maps with different levels of information details. Keywords: Coral reef; Life-form; Planetscope; Spatial resolution; Classification scheme Assessment of Coral Reef Life-Form Classification Scheme using Multiresolution Images on Parang Island, Indonesia Wahyu Lazuardi1, Pramaditya Wicaksono2,* 1Geospatial Information Agency, Bogor, 16911, Indonesia 2Department of Geographic Information Science, Faculty of Geography, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia Wahyu Lazuardi1, Pramaditya Wicaksono2,* 1Geospatial Information Agency, Bogor, 16911, Indonesia 2Department of Geographic Information Science, Faculty of Geography, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia Received 26 October 2021/Revised 14 December 2021/Accepted 21 December 2021/Published 30 December 2021 Received 26 October 2021/Revised 14 December 2021/Accepted 21 December 2021/Published 30 December 2021 *Corresponding author. Email address : prama.wicaksono@ugm.ac.id (Pramaditya Wicaksono) Geosfera Indonesia Geosfera Indonesia Vol. 6 No. 3, December 2021, 377-397 p-ISSN 2598-9723, e-ISSN 2614-8528 https://jurnal.unej.ac.id/index.php/GEOSI DOI : 10.19184/geosi.v6i3.27592 377 *Corresponding author. Email address : prama.wicaksono@ugm.ac.id (Pramaditya Wicaksono) *Corresponding author. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 management and monitoring of coral reefs are paramount to improve the sustainability and usefulness of these shallow-water ecosystems, especially when under a significant amount of environmental stress. In this situation, spatial information on the characteristics of benthic compositions and coral substrates becomes necessary (Hoegh-Guldberg et al., 2007). When available, this information is not only beneficial for detecting and monitoring the impact of environmental management and changes in the living patterns of marine biota due to altered environmental conditions, but it can also be used as support facilities in tourism (Dudgeon et al., 2010). Most importantly, spatial information on coral reef life-forms is acquired for its close relationship with coastal environment dynamics, especially with several coastal environment aspects. Complex geomorphological characteristics and diverse life-form compositions can be used in biodiversity assessment. Certain life-forms are also known to affect the growth and development of organisms, particularly the abundance of fish (Madduppa et al., 2019), and some are highly sensitive to the coastal environment dynamics. Therefore, their spatial information can be the basis for monitoring, explaining the potential of and threats to coastal resources, and ultimately creating opportunities to build and grow the blue economy (Voyer et al., 2018). Large-scale mapping and monitoring are a stepin coral reefs management and conservation (Green et al., 2000). To meet these requirements means, among others, to utilize multispectral remote sensing technology, that is, satellite imagery that can provide an effective and efficient alternative source of data for coral reef mapping (Hedley et al., 2005). Since multispectral satellite images with visible to near-infrared (VNIR) channels can be used to remotely characterize benthic habitats, such as coral reefs, seagrass beds, and macroalgae (Goodman et al., 2013), they have been widely used to map ones in optically shallow waters (Phinn et al., 2012; Wicaksono et al. 2019a). The specifications of multispectral satellite imagery continue to evolve, including increased spatial and spectral resolution and higher data acquisition capacity. Straightforward integration and access to multispectral satellite imagery enable its further development, in particular, applications for mapping, monitoring, and modeling for coral reef studies. Such advancement can contribute to increasing the accuracy of benthic habitats classification (Roelfsema et al., 2013). 1. Introduction Coral reefs are one of the natural resources in coastal areas that play fundamental ecological roles for the life of marine flora and fauna (Larkum et al., 2006). If managed, preserved, and utilized optimally, coral reefs can provide many benefits for coastal communities. Their exceptional socio-economic and cultural values and interests spread beyond their function as fish habitat, including abundant sources of seafood, coastal protection, and marine tourism (Giyanto et al., 2017). Therefore, effective and efficient 377 *Corresponding author. Email address : prama.wicaksono@ugm.ac.id (Pramaditya Wicaksono) 377 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 used (Roelfsema et al., 2013). Therefore, each remote sensing image needs to be assessed for its capability to produce spatial information on a particular complexity of benthic habitats. used (Roelfsema et al., 2013). Therefore, each remote sensing image needs to be assessed for its capability to produce spatial information on a particular complexity of benthic habitats. Several studies related to coral reefs use a classification scheme at the life-form level. For example, Wicaksono (2016) mapped 28 classes of coral life-forms around Kemujan Island, Zhang et al. (2013) used two classification schemes—namely group level (3 classes) and code level (12 classes), Phinn et al. (2012) mapped benthic habitat communities with 15 class schemes, and Roelfsema et al. (2013) also mapped coral reefs with a 20-class scheme. In some of these studies, the classification results do not cover all existing classes of coral reef life-forms, indicating limitations in the ability of the used images to discriminate between them. In addition, each coastal environment has different characteristics of coral reefs, meaning that any classification scheme and model developments must be first adjusted to them. Mapping coral reefs at the life-form level is one of the challenges several researchers remain to experience, mainly because the model designed for a coastal environment depends on the complexity of the coral reef composition and, thus, may very well be inapplicable to other coasts. Therefore, it is imperative that the role of multiresolution imagery in producing spatial information of coral reefs with various levels of detail be examined. Therefore, this study aimed to evaluate the classification scheme of coral reef life- form using images with different spatial resolutions on Parang Island, Karimunjawa Islands, Central Java. The multispectral satellite images used in this study are Planet Scope (3m), PlanetScope resulted from the resampling process (6m), and Sentinel-2A MSI (10m). Despite their easy access and fitting spatial resolution for mapping the individual variation of a class of coral reefs, these images are still rarely used in detailed benthic habitat studies, underlying their uses in this research. The study was designed to assess the classification scheme for coral reef life-form mapping using satellite images with different spatial resolutions on Parang Island, Karimunjawa Islands, Central Java Province (Figure 1). Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Using different images can produce detailed information regarding the existence, diversity, and type of coral reefs, the composition of the geomorphological zone of coral reefs, benthic habitats, and coral life-form composition (Goodman et al., 2013). The level of detail of coral life-form information offers depends on the spatial resolution of the image 378 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Parang is one of many islands in Indonesia that has highly diverse coastal ecosystems and is part of the Karimunjawa National Park Region. Compared with the other regions in Karimunjawa Islands, Parang is the least visited destination because it is located somewhat far from the main island of Karimunjawa. Low tourism activity has little to no effect on the condition of shallow water ecosystems on Parang Island. Accordingly, the coral reef cover on the island remains widely diverse and preserved. 379 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Figure 1. The location of Parang Island Figure 1. The location of Parang Island 2.1 Image Preprocessing The multispectral images used in the research were PlanetScope (3m spatial resolution), PlanetScope Resampling (6m) recorded on April 30, 2018, and Sentinel-2A MSI (10m) on April 17, 2018. These images were corrected to minimize any biases caused by sensory and atmospheric conditions. In this study, the Sentinel-2A MSI image (10m) had the level 1C, and its Digital Number (DN) was converted to Top-Of-Atmosphere Reflectance (RTOA). Furthermore, this image was subjected to atmospheric, sunglint, and water column corrections. The PlanetScope image was the SR Product that had been corrected to Bottom- of-Atmosphere Reflectance (RBOA) and subjected to water column correction. However, sunglint correction was not applied to the PlanetScope image because of a missed coregistration between visible and NIR bands, entirely removing the possibility of performing this correction.The specifications of both images can be seen in Table 1. Table 1. Specification of the PlanetScope and Sentinel-2A MSI Images (Suhet, 2014; PlanetLabs, 2017) Image Spesification PlanetScope Sentinel-2A MSI Spatial Resolution (m) 3 10 Radiometric Resolution 12-bit 12-bit Temporal Resolution 1 day 15‒30 days Spectral Resolution (µm) - - Blue Band 0.42‒0.53 0.45‒0.52 Green Band 0.50‒0.59 0.54‒0.58 Red Band 0.61‒0.70 0.65‒0.68 near-infrared Band 0.77‒0.90 0.78‒0.90 Note : For the Sentinel-2A MSI image, only the bands used in the study are presented, including Visible – NIR bands Specification of the PlanetScope and Sentinel-2A MSI Images (Suhet, 2014; s, 2017) 380 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 2.3 Sunglint Correction Sunglint is a mirror-like specular reflection on the water surface that can produce noise in remote sensing applications for mapping in the optically shallow and optically deepwater regions. This study used a sunglint correction method by Hedley et al. (2005). Kay et al. (2009) compared various sunglint correction methods and showed that the Hedley et al. (2005) and Lyzenga et al. (2006) has the most constant results compared to other methods. Kay et al. (2009) also explained that the use of the Hedley et al. (2005) is more suitable for mapping benthic habitats, whereas for the correction method Lyzenga et al. (2006) is more suitable for use in bathymetry mapping. 2.2 Atmospheric Correction Dark-Object Subtraction (DOS) was applied in this research because although it is one of the simplest atmospheric correction methods (Chavez, 1996 in Nazeer et al., 2014), it produces better results than other methods (Nazeer et al., 2014). DOS was carried out by analyzing the reflectance of dark objects in the image, and then the reflectance value was used to remove the atmospheric offset in the Sentinel-2A MSI image at RTOA level (Chavez, 1996 in Nazeer et al., 2014). 2.5 Field Data Collection The field data were collected by the photo-transect method, which is very efficient in terms of cost, time, and energy (Roelfsema et al., 2006). Photos of benthic habitat were captured using an underwater camera by the surveyors while snorkeling in the optically shallow water. A GPS 78s was attached to the surveyor and set to the tracking mode to record coordinates at an interval of two seconds, with a UTM coordinate system in the 49M zone and the datum WGS84. The time indicators on both GPS and underwater cameras were synchronized (up to seconds) to geotag the taken benthic habitat photos with GPS coordinates accurately (Phinn et al., 2012). The compositions of benthic habitat and coral reef life-forms were obtained by interpreting the photo samples taken during the photo-transect survey in Coral Point Count Excel (CPCe) software. 2.4 Water Column Correction The water column correction of sunglint-free images aimed to minimize changes in the spectral response of benthic habitats due to depth variations and the attenuation of electromagnetic energy in the water column (Maritorena, 1996). Lyzenga (1978) developed one of the most widely used algorithms for water column correction (Zhang et al., 2013) and is one of the simplest methods of water column correction (Zoffoli et al., 2014). This water column correction method utilizes the water column attenuation coefficient or ratio at two different wavelengths to eliminate the need for depth information and the water column attenuation coefficient at each wavelength (Lyzenga, 1978). The attenuation coefficient value for each band pairis presented in Table 2. Table 2. The ratio of water column attenuation coefficient value for each band pair Image Attenuation Coefficient (ki/kj) DII 12 DII 13 DII 23 Planet (3 m) 0.822 0.600 0.733 Planet Resampling (6 m) 0.854 0.601 0.710 Sentinel 2 MSI (10 m) 0.7195 0.518 0.722 Table 2. The ratio of water column attenuation coefficient value for each band pair 381 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 2.6 Image Classification Images that had been subjected to water column correction functioned as the input to the two-level classification process. In the first level, the major classifier was types of benthic habitats, namely coral reefs, seagrass, macroalgae, and bare substratum. In the second level, the minor classifier was the coral life-form, which was built according to the spatial resolution and segments of the used satellite images and based on the dominant life-form samples in each segment. In this level, the cross-classification of coral life-forms was carried out in each spatial resolution to assess whether the classification results were consistently accurate. After applying all classification schemes, the most suitable one for each satellite image was determined based on the highest accuracy produced (Table 4). As an attempt to improve mapping accuracy, this study integrated two different approaches in the classification scheme, namely object-based and pixel-based (Ma et al., 2017). Several studies have proven that using the hybrid OBIA-Supervised classification method can improve mapping accuracy (Ma et al., 2017). The object-based approach adopted in this study was limited to the segmentation process in IDRISI Selva 17. Meanwhile, the pixel-based supervised classification involved the use of algorithms, such as Support Vector Machine (SVM) and Classification Tree Analysis (CTA), both of which possess the capacity to provide accurate classification of remote sensing images (Zambon et al., 2006; Wicaksono et al., 2019a). Compared with other algorithms in supervised classification, SVM can produce higher mapping accuracy than Maximum Likelihood (ML) and Artificial Neural Network (ANN) (Pal & Mather, 2005; Wahidin et al., 2015). CTA can analyze large amounts of data with complex structures (Cappelli et al., 2002 in Zambon et al., 2006). In terms of 382 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 classification, CTA algorithm has been reported to increase classification accuracy in several previous studies (Wicaksono et al., 2019b) and provide better results than ML. In addition, the Object-Based Image Analysis (OBIA) relies on object characters or aspects to create classification, namely shape, texture, and relationship between objects (Mafanya et al., 2017). Although it can provide good mapping results, this study did not solely use OBIA because it lacks quantitative verification (Ma et al., 2017) and is highly dependent on the developed rules when applied to remote sensing data. 2.6 Image Classification Furthermore, designing and generating the rules requires adequate knowledge and experience, and even then, it is not easy to ascertain the error or truth of said rules. In addition, the development of OBIA rules strongly depends on the number of classes to be used in the classification. For instance, a high class diversity requires the developed rules to pay attention to each class, which is a lengthy process where repeated experiments are conducted to obtain a good rule for each step. 2.7 Accuracy Assessment Accuracy assessment is an approach used to determine the extent to which a study result can be trusted. In the case of benthic habitat classification, its accuracy was assessed using a confusion matrix analysis. Samples for validation were used as reference data. Confusion matrix analysis offers information about user's accuracy (UA), producer's accuracy (PA), overall accuracy (OA), and kappa coefficient (Congalton & Green, 2009). We determined the most appropriate classification scheme for each image based on the confusion matrix results. 3.1 Field Data Collection The field survey of benthic habitat was carried out on April 17-19, 2018, and the location of the transects for data collection is presented in Figure 2. During this field survey, 1,658 samples were obtained. Based on the life-form variations at the study location, the dominant coral reef life-forms obtained by analyzing the photo samples were branching, stag horn, massive, pillar, foliage, encrusting, tabular, and dead coral; thereby, only these classes were used as the training areas for object classification. However, not all life forms can be used as a single class for training areas in the classification process, except for the frequently encountered and homogeneous ones. In some locations, the life-forms of coral reefs were not always homogeneous but consisted of multiple types. 383 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Figure 2. Field data collection using the photo-transect method Figure 2. Field data collection using the photo-transect method 3.2 Coral Reef Life-Forms Classification Scheme The classification scheme of coral reef life-forms was obtained through the dominance analysis of each segment in the three satellite images (Phinn et al., 2012). However, should the existing sample consist of different classes and has the same percentage, it is classified based on the number of classes in the image segment (mixed class). Based on Table 3, the classification schemes of coral reef life-forms, built from each image, have different numbers of classes. 384 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Table 3. The classification schemes of benthic habitats and coral reef life-forms at each spatial resolution Benthic Habitat Classification Scheme Classes Classification Validation Bare Substratum 105 119 Macroalgae 69 55 Seagrass 219 194 Coral Reef 412 437 Coral Life-formClassification Scheme PlanetScope (3m) Classification Validation PlanetScope Resampling (6m) Classification Validation Sentinel-2A MSI (10m) Classification Validation Branching 55 65 Branching 45 57 Branching 78 95 Branching Staghorn 15 10 Branching Staghorn 33 21 Branching Tabular 62 51 Branching Tabular 79 74 Branching Tabular 79 85 Foliage Staghorn 23 16 Foliage 19 9 Foliage 22 15 Massive 54 68 Massive 54 64 Massive 24 30 Massive Branching 72 65 Massive Branching 36 41 Massive Branching 85 80 Massive Staghorn 47 41 Massive Foliage 11 6 Massive Branching Tabular 29 22 Massive Tabular 24 30 Massive Staghorn 50 69 Massive Staghorn 19 26 Staghorn 29 38 Massive Tabular 45 36 Massive Tabular 46 41 Tabular 48 32 Staghorn 27 16 Staghorn 31 39 Staghorn Tabular 14 8 Tabular 24 20 Tabular 32 38 e classification schemes of benthic habitats and coral reef life-forms at each i 3. The classification schemes of benthic habitats and coral reef life-forms at each resolution 3.3 Benthic Habitat Mapping Benthic habitat mapping sought to obtain the best spatial distribution of coral reef (coral reef mask image), which was used as a basis for the classification of coral reef life- forms. In the classification process, the benthic habitat was a major classifier that defined four types of habitat, namely coral reef, seagrass, macroalgae, and bare substratum (Figure 3). The classification scheme of benthic habitat has an OA of 57-63% (Table 4). PlanetScope Resampling (6m) images produced the highest accuracy, with an OA of 63.10% and a kappa coefficient of 0.38, which was obtained using the CTA classification algorithm and was higher than using the SVM algorithm. The accuracies of benthic habitat mapping using the CTA and SVM algorithms differed by approximately 5%. The overall accuracies of benthic habitat classification from different algorithms and images are compared in Table 4. Table 4. Summary of the assessment of benthic habitat mapping accuracy Image CTA SVM OA (%) Kappa OA (%) Kappa PlanetScope (3 m) 57.26 0.49 61.86 0.32 PlanetScope Resampling (6 m) 63.10* 0.38 58.75 0.22 Sentinel-2A MSI (10 m) 57.26 0.16 57.14 0.08 Note : *indicates the highest overall accuracy Table 4. Summary of the assessment of benthic habitat mapping accuracy Judging from the overall classification schemes of benthic habitat, CTA algorithm produced a spatial distribution of benthic habitats more consistently—in which all types of 385 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 habitats were classified—and with a higher OA for each satellite image than SVM algorithm, even though the accuracy difference is merely 5%. SVM algorithmprovided less accurate results because several types of benthic habitats, such as macroalgae and sand were not applicable for classification. Another reason was inter-class misclassification; for example, sand was misclassified into seagrass and macroalgae. Sand has a spectral response that is similar to low seagrass and macroalgae due to the contribution of sand reflectance as the substrate of seagrass and macroalgae (Wicaksono, 2016). Also, coral reefs were misclassified into macroalgae because both objects have comparable spectral response due to similar pigmentation (Hochberg & Atkinson, 2000; Wicaksono, 2016). Figure 3. A comparison of benthic habitat classification resulted from different classification algorithms and satellite images Figure 3. 3.3 Benthic Habitat Mapping A comparison of benthic habitat classification resulted from different classification algorithms and satellite images Figure 3. A comparison of benthic habitat classification resulted from different classification algorithms and satellite images able 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of coral asses Table 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of co classes Table 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of coral reef classes Table 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of coral reef classes Image CTA-based SVM-based PA (%) UA (%) Resulted Accuracy (%) PA (%) UA (%) Resulted Accuracy (%) PlanetScope (3 m) 80.55 73.79 59.43 89.93 59.10 53.14 PlanetScope resampling (6m) 83.07 73.19 60.79 87.87 61.84 54.33 Sentinel-2A MSI (10 m) 85.35 68.69 58.62 99.77 56.26 56.13 386 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Based on the overall results of the benthic habitat classification, the individual accuracy (UA and PA) of the coral reef life-forms classification ranged from 56 - 99% (Table 5). The CTA-based classification from PlanetScope (3m) was used in the masking of coral reef images or to exclude non-coral reef pixels in the mapping. Although the OA of the CTA- based classification from PlanetScope (3m) image was slightly lower than PlanetScope Resampling (6m), the spatial resolution was higher than PlanetScope Resampling (6m) and Sentinel-2A MSI (10m), and producing more precise coral reef masking delineation. 3.4 Coral Reef Life-Form Mapping The coral reef life-forms were mapped by applying the classification schemes built on each satellite image (see Table 3). The coral reef life-form classification (Figure 4) with the highest OA (60.78%) was obtained from Sentinel-2A MSI image using CTA algorithm and the 10m scheme, and then followed by PlanetScope (48.39%), and PlanetScope Resampling (40.83%) (Table 6). Although the CTA algorithm could consistently produce maps of coral reef life-forms with higher OA than SVM, the generated classification was relatively low, in particular, because of a large number of misclassifications as a result of similar statistics between the classes of coral reef life-forms. For this reason, both CTA and SVM classification algorithms could not separate one life-form from another straightforwardly. The coral reef life-form classifications built from the three satellite images showed different spatial distribution (Figure 4). PlanetScope (3m) and Sentinel-2A MSI (10m) images presented similar distributions of life-forms in some locations, and different spread patterns are found in some others. This similarity indicates consistency in the classification results produced using different images. The spatial distribution of several coral reef life- forms corresponds to the theory proposed by English et al. (1994), that is, coral reef life- forms spread following the locations of optimal environmental conditions for their growth. The opposite is true for the classification scheme built from the PlanetScope Resampling (6m), which only has one class, i.e., the tabular branching that dominated the study area. It is caused by the effects of the image pixel resampling process from 3m to 6m. 387 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Figure 4. Comparison of coral reef life-form maps with the highest OA Figure 4. Comparison of coral reef life-form maps with the highest OA Table 6. Comparison of the overall accuracies of the resulted coral reef life-form classifications Images 3m Scheme 6m Scheme 10m Scheme CTA (%) SVM (%) CTA (%) SVM (%) CTA (%) SVM (%) PlanetScope (3m) 38.76 30.05 35.32 27.52 48.39 39.45 PlanetScope resampling (6m) 40.83 31.65 39.91 27.52 36.01 40.60 Sentinel-2A MSI (10m) 39.91 26.38 46.56 30.28 60.78 42.20 The resampling process generalizes pixel values by considering the surrounding pixels (i.e., mean values). Therefore, variations in the pixel values of the analyzed objects canbe very similar, and as a result, pixels that should be classified as different objects are categorized as the same object. 3.4 Coral Reef Life-Form Mapping Accordingly, the image resampling process is not recommended for addressing issues of unavailable images with a particular spatial resolution due to its poor results. Different distributions of coral reef life-forms obtained from the multiresolution images were potentially caused by the size of the spatial resolution itself. As evidence, different spatial resolutions have different spectral responses from the object composition. Accordingly, the distributions of coral reef life-forms are different from one satelliteimage to another (Figure 4 and Table 7). The classification results of the satellite image with 6m spatial resolution were not included in the comparison because the 388 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 generalization ofthe spatial resolution was believed to produce less representative distribution. Table 7. The classification schemes of coral reef life-forms 3m Scheme 6m Scheme 10m Scheme CORAL REEF LIFE-FORM CLASSES Branching Branching Branching Branching Staghorn Branching Staghorn Branching Tabular Foliage Foliage Foliage Staghorn Branching Tabular Branching Tabular Massive Massive Massive Massive Branching Massive Branching Massive Branching Tabular Massive Staghorn Massive Foliage Massive Branching Massive Tabular Massive Staghorn Massive Staghorn Staghorn Massive Tabular Massive Tabular Tabular Staghorn Staghorn Staghorn Tabular Tabular Tabular Table 7. The classification schemes of coral reef life-forms Based on the highest accuracy of the maps produced from each satellite image and classification scheme (Table 6), the coral reef life-forms captured in PlanetScope (3m) and Sentinel-2A MSI (10m) were best mapped using the 10m scheme. Unlike in the two satellite images, the coral reef life-forms featured in PlanetScope Resampling (6m) were best mapped using the 3m scheme. However, not all life-forms were applicable for classification. Eight classes of coral reef life-forms were consistently classified in all images and schemes, namely branching, tabular branching, massive, massive branching, massive staghorn, massive tabular, staghorn, and tabular. As seen in Tables 8, 9, and 10, each life-form was properly classified for each image with high UA and PA. Nevertheless, overestimate and underestimate could occur. Therefore, the classes above can be used in the classification schemes of coral reef life-forms in various images (Table 7). Table 8. Confusion matrix of PlanetScope (3m) using the 10m scheme based on the CTA- based classification result 389 Table 8. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA- based classification result Classes References 1 2 3 4 5 6 7 8 9 Total UA % 7 0 0 3 0 0 0 15 0 6 24 62.50 8 0 0 1 0 0 0 0 3 0 4 75.00 9 1 8 0 0 2 0 0 0 4 15 26.67 Total 95 51 16 68 65 41 30 38 32 436 PA (%) 65.26 72.55 0.00 63.24 66.15 9.76 50.00 7.89 12.50 OA (%) 48.39 Legend: 1 :Branching 4 :Massive 7: Massive Tabular 2: Branching Tabular 5 :Massive Branching 8 :Staghorn 3: Foliage Staghorn 6: Massive Staghorn 9: Tabular Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA- based classification result Classes References 1 2 3 4 5 6 7 8 9 Total UA % 7 0 0 3 0 0 0 15 0 6 24 62.50 8 0 0 1 0 0 0 0 3 0 4 75.00 9 1 8 0 0 2 0 0 0 4 15 26.67 Total 95 51 16 68 65 41 30 38 32 436 PA (%) 65.26 72.55 0.00 63.24 66.15 9.76 50.00 7.89 12.50 OA (%) 48.39 Legend: 1 :Branching 4 :Massive 7: Massive Tabular 2: Branching Tabular 5 :Massive Branching 8 :Staghorn 3: Foliage Staghorn 6: Massive Staghorn 9: Tabular Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA- based classification result Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA- based classification result Table 9. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA- based classification result Class References 1 2 3 4 5 6 7 8 9 10 11 12 Total UA (%) 1 46 5 6 0 14 19 0 23 0 0 8 4 125 36.80 2 0 0 0 2 0 0 0 0 0 0 0 0 2 0.00 3 16 1 48 1 20 1 6 13 6 9 0 11 132 36.36 5 0 0 1 1 16 0 0 0 1 3 0 0 22 72.73 6 0 4 0 5 3 21 0 3 0 0 0 0 36 58.33 8 2 0 14 0 4 0 0 25 26 0 0 8 79 31.65 9 0 0 0 0 0 0 0 0 3 0 0 0 3 100 10 0 0 0 0 0 0 0 0 0 4 0 0 4 100 12 1 0 5 0 7 0 0 5 0 0 0 15 33 45.45 Total 65 10 74 9 64 41 6 69 36 16 8 38 436 PA (%) 70.77 0 64.86 0 25.00 51.22 0 36.23 8.33 25.00 0 39.47 OA (%) 41.00 Legend: 1 :Branching 4 :Foliage 7: Massive Foliage 10 :Staghorn 2: Branching Staghorn 5 :Massive 8 :Massive Staghorn 11 :Staghorn Tabular 3: Branching Tabular 6: Massive Branching 9: Massive Tabular 12 :Tabular Table 10. Confusion matrix of Sentinel-2A MSI (10m) using the 10m scheme based on the CTA-based classification result Class References 1 2 3 4 5 6 7 8 9 Total UA % 1 83 0 5 28 17 20 0 3 0 156 53.21 2 7 46 0 12 2 0 0 0 14 81 56.79 3 0 0 11 0 0 0 0 0 0 11 100 4 0 5 0 28 8 0 0 5 3 49 57.14 5 2 0 0 0 38 6 17 0 5 68 55.88 6 0 0 0 0 0 10 0 0 0 10 100 7 0 0 0 0 0 5 13 0 0 18 72.22 8 3 0 0 0 0 0 0 30 4 37 81.08 9 0 0 0 0 0 0 0 0 6 6 100 Total 95 51 16 68 65 41 30 38 32 436 PA (%) 87.36 90.19 68.75 41.17 58.46 24.39 43.33 78.94 18.75 OA (%) 60.78 Table 10. 3.4 Coral Reef Life-Form Mapping Confusion matrix of PlanetScope (3m) using the 10m scheme based on the CTA- based classification result Classes References 1 2 3 4 5 6 7 8 9 Total UA % 1 62 0 7 22 13 12 0 2 0 118 52.54 2 3 37 2 3 6 0 7 6 10 74 50.00 3 0 0 0 0 0 1 0 0 0 1 0.00 4 18 4 3 43 1 7 7 19 1 103 41.75 5 11 0 0 0 43 17 1 8 11 91 47.25 6 0 2 0 0 0 4 0 0 0 6 66.67 389 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Confusion matrix of Sentinel-2A MSI (10m) using the 10m scheme based on the CTA-based classification result 390 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Legend: 1: Branching 4: Massive 7: Massive Tabular 2: Branching Tabular 5: Massive Branching 8: Staghorn 3: Foliage Staghorn 6: Massive Staghorn 9: Tabular The classification schemes of coral reef life-forms are constructed from three satellite images with different spatial resolutions. Three schemes of classification with varying numbers of classes have been built in this study (Table 3). Some classes in these schemes depict similar types of coral reef life-forms, such as branching, tabular branching, massive, massive branching, massive staghorn, massive tabular, staghorn, and tabular. Nevertheless, some classes are only found in a particular scheme because their generation depends on the uniquely formed image segments (Phinn et al., 2012). Each satellite images of which indeed produces classification schemes with different suitability for coral reef life-form mapping. Here, suitability is defined by the highest OA in each image. Table 6 shows that PlanetScope (3m) and Sentinel-2A MSI (10m) images are suitable using the 10m scheme, whereas PlanetScope Resampling (6m) image using the 3m scheme. PlanetScope (3m) is not suitable using the 3m and 6m schemes because the statistical value of each pixel in its segment is too precise. Consequently, the same objects have varying pixel values, causing too many misclassifications (Purkis, 2018). Meanwhile, the 10m scheme produces higher accuracy than the 3m and 6m schemes because the classification wasbuilt from images with a lower spatial resolution (general information) (Zhang et al., 2013). As a result, variations of pixel values in a segment are considered one life-form class so that the said segment can be classified according to the input sample (Phinn et al., 2012). PlanetScope Resampling (6m) is more suitable using the 3m scheme because the generalization during the spatial resolution resampling results in similarities. In this case, the 10m scheme is not suitable for use because the effect of generalization increases the homogeneity of image statistics to PlanetScope (3m) instead of the Sentinel-2A MSI (10m), even though the level of precision is different. Several studies have shown that the higher the number of classes used, the lower the accuracy (Wicaksono, 2016; Wicaksono et al., 2019a). However, the contrary is true for this study, where the findings correspond to Zhang et al. (2013) instead. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Sentinel-2A MSI (10m) is more suitable using the 10m scheme because it produces classes that are more general than the 3m and 6m schemes. The classified types of coral reef life-forms in the Sentinel-2A MSI image will not be optimal if the used classification scheme is built from images with more detailed spatial resolutions. For example, if two different classes from the 3m or 6m scheme occupy a segment, one that appears dominantly will be selected over the other, creating low accuracy. On the contrary, if the used classification scheme is built from the same image, each sample of each class undoubtedly corresponds to the previously formed segments. Consequently, each segment can be classified more accordingly to the input sample, leading to a higher OA than the other schemes. The selected satellite images can map the life-forms of coral reefs with overall accuracies of 48.39% (PlanetScope 3m, CTA, 10m scheme), 40.83% (PlanetScope Resampling 6m, CTA, 3m scheme), and 60.78% (Sentinel-2A MSI 10 m, CTA, 10m scheme). Multiresolution images produce classifications with multilevel accuracy (Mumby & Edward, 2002), and satellite imagery with a more detailed spatial resolution can yield higher accuracy (Goodman et al., 2013). However, the highest accuracy in this study is shown by images with a lower spatial resolution (10m) because the more detailed ones, i.e., 3m and 6m, cannot represent a community or a dominant class of coral reef life-forms. In addition, Sentinel 2 MSI also has a fairly consistent image quality (Hedley et al., 2018; Wicaksono et al., 2021) compared with PlanetScope, whose pixels between scenes are inconsistent and SNR is of poor quality (Wicaksono & Lazuardi, 2018). Based on the percentage, the coral reef life-forms are covered with diverse objects, meaning that the resulted spectral response is a function of various classes. Sentinel-2A MSI (10m) has successfully produced the highest accuracy, followed by PlanetScope (3m) and PlanetScope Resampling (6m). The resampling process generalizes the spectral response value of the image, and the results are less suitable for representing a variety of objects because the spectral response is the mean value of the surrounding pixels. In other terms, the spectral response produced in the resampling process shows similarities among different objects. These numerous cases of misclassifications contribute to the overall accuracy of PlanetScope Resampling (6m), which is lower than that of PlanetScope (3m). Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 They used two classification schemes consisting of group level (3 classes) and code level (12 classes) and found that the latter produces a higher accuracy (86.7%) than the former (84.3%). 391 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 When the accuracies are compared, the CTA algorithm evidently produces the best and most consistent accuracy (Zambon et al., 2006) of life-form classification (Wahidin et al., 2015). It allows the proper classification of coral reef life-forms in each of the three satellite images. The CTA and SVM algorithms produce different OA by averagely 12%, which, in terms of OA, is significant. The SVM algorithm provides a low accuracy because not all coral reef life-forms are classified. On the contrary, the CTA algorithm has a better capability to classify coral reef life-forms, especially in schemes that create a large number of classes. Although the accuracy derived in this study is merely 40‒60%, the results are in line with previous similar studies (Phinn et al., 2012; Roelfsema et al., 2013; Wahidin et al., 2015; Wicaksono, 2016). Although Sentinel-2A MSI (10m) can create a coral reef life-form map with higher accuracy than other images, the level of information obtained from it is somewhat general. Meanwhile, PlanetScope (3m) has lower accuracy, but it can map coral reef life-forms in more detail and involve precise information compared with Sentinel-2A MSI (10m). Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Furthermore, the difference in OA can be caused by the quality of the used satellite image. For instance, despite the high spatial resolution, the pixel sharpness of the PlanetScope (3m) image is still lower than Sentinel-2A MSI (10m) that has a lower resolution. Furthermore, the results obtained from the three satellite images have different UA and PA. 392 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Conflict of interest There is no conflict of interest with any financial, personal, or other relationships with other people or organizations related to the material discussed in the article. 4. Conclusion Our results have shown that both PlanetScope (3m) and Sentinel-2A MSI (10m) can be used to map coral reef with higher complexity. This finding indicates that multiresolution images can be used to produce complex coral reef life-form maps with different levels of information details. In our case, the classification scheme on multiresolution images shows that the coral reef life-forms in PlanetScope and Sentinel-2A MSI images can be mapped using the 10m scheme, whereas the ones in PlanetScope Resampling (6m) image are mapped according to the 3m scheme. Parang Island has eight types of coral reef life-forms that can be classified consistently in each satellite image, namely branching, branching staghorn, tabular branching, massive branching, massive staghorn, massive tabular, staghorn, and tabular. Based on the statistical analysis and accuracy assessment, the maximum overall accuracies of the coral reef life-form maps produced from PlanetScope (3m), PlanetScope Resampling (6m), and Sentinel-2A MSI (10m) images are 48.39% (10m scheme), 40.83% (3m scheme), and 60.78% (10m scheme), respectively. The accuracy obtained in our work can be used as a reference for future works of coral reef life-forms mapping. Possibly, variations in accuracy is expected since coral reefs life-forms may vary significantly between areas. Finally, the future availability of detailed coral reef map at regular basis will be beneficial to assist coastal manager in determining and monitoring the effective action to managed their area sustainably. 393 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Acknowledgements This research is funded by “Direktorat Riset dan Pengabdian Masyarakat – Direktorat Jenderal Penguatan Riset dan Pengembangan – Kementerian Riset, Teknologi, dan Pendidikan Tinggi Republik Indonesia” via Penelitian Dasar Unggulan Perguruan Tinggi – PDUPT Scheme Grant Number 153/UN1/DITLIT/DIT-LIT/LT/2018. 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Measuring Biases of Word Embeddings: What Similarity Measures and Descriptive Statistics to Use?
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Proceedings of the First Workshop on Trustworthy Natural Language Processing, pages 8–14 June 10, 2021. ©2021 Association for Computational Linguistics Abstract gate biases of word embeddings (Liang et al., 2020; Ravfogel et al., 2020). Word embeddings are widely used in Natural Language Processing (NLP) for a vast range of applications. However, it has been consis- tently proven that these embeddings reflect the same human biases that exist in the data used to train them. Most of the introduced bias in- dicators to reveal word embeddings’ bias are average-based indicators based on the cosine similarity measure. In this study, we examine the impacts of different similarity measures as well as other descriptive techniques than averaging in measuring the biases of contex- tual and non-contextual word embeddings. We show that the extent of revealed biases in word embeddings depends on the descriptive statis- tics and similarity measures used to measure the bias. We found that over the ten categories of word embedding association tests, Maha- lanobis distance reveals the smallest bias, and Euclidean distance reveals the largest bias in word embeddings. In addition, the contextual models reveal less severe biases than the non- contextual word embedding models with GPT showing the fewest number of WEAT biases. Different approaches have been used to present and quantify corpus-level biases of word embed- dings. Bolukbasi et al. (2016) proposed to mea- sure the gender bias of word representations in Word2Vec and GloVe by calculating the projections into principal components of differences of embed- dings of a list of male and female pairs. Basta et al. (2019) adapted the idea of "gender direction" of (Bolukbasi et al., 2016) to be applicable to contex- tual word embeddings such as ELMo. In (Basta et al., 2019) first, the gender subspace of ELMo vector representations is calculated and then, the presence of gender bias in ELMo is identified. Go- nen and Goldberg (2019) introduced a new gender bias indicator based on the percentage of socially- biased terms among the k-nearest neighbors of a target term and demonstrated its correlation with the gender direction indicator. Caliskan et al. (2017) developed Word Embed- ding Association Test (WEAT) to measure bias by comparing two sets of target words with two sets of attribute words and documented that Word2Vec and GloVe contain human-like biases such as gender and racial biases. May et al. (2019) generalized the WEAT test to phrases and sentences by inserting individual words from WEAT tests into simple sen- tence templates and used them for contextual word embeddings. 2 Method Implicit Association Test (IAT) was first intro- duced by Greenwald et al. (1998a) in psychology to demonstrate the enormous differences in response time when participants are asked to pair two con- cepts they deem similar, in contrast to two con- cepts they find less similar. For example, when subjects are encouraged to work as quickly as pos- sible, they are much likely to label flowers as pleas- ant and insects as unpleasant. In IAT, being able to pair a concept to an attribute quickly indicates that the concept and attribute are linked together in the participants’ minds. The IAT has widely been used to measure and quantify the strength of a range of implicit biases and other phenomena, including attitudes and stereotype threat (Karpinski and Hilton, 2001; Kiefer and Sekaquaptewa, 2007; Stanley et al., 2011). d = |s(x, A, B) −s(y, A, B)| std-devw∈X∪Y s(w, A, B) It is worth mentioning that d is a measure used to calculate how separated two distributions are and is basically the standardized difference of the means of the two distributions (Cohen, 2013). Controlling for the significance, a larger effect size reflects a more severe bias. WEAT and almost all the other studies inspired by it (Garg et al., 2018; Brunet et al., 2018; Gonen and Goldberg, 2019; May et al., 2019) use the fol- lowing approach to measure the association of a single target word with the two sets of attributes (equation 1). First, they use cosine similarity to measure the target word’s similarity to each word in the attribute sets. Then they calculate the average of the similarities over each attribute set. Inspired by IAT, Caliskan et al. (2017) intro- duced WEAT to measure the associations between two sets of target concepts and two sets of attributes in word embeddings learned from large text cor- pora. A hypothesis test is conducted to demon- strate and quantify the bias. The null hypothesis states that there is no difference between the two sets of target words in terms of their relative dis- tance/similarity to the two sets of attribute words. A permutation test is performed to measure the null hypothesis’s likelihood. where: where: s(w, A, B) = fa∈A(s(−→ w , −→a )) −fb∈B(s(−→ w , −→b )) (1) In other words, s(w, A, B) quantifies the associ- ation of a single word w with the two sets of at- tributes, and s(X, Y, A, B) measures the differen- tial association of the two sets of targets with the two sets of attributes. Denoting all the partitions of X ∪Y with (Xi, Yi)i, the one-sided p-value of the permutation test is: Pri(s(Xi, Yi, A, B) > s(X, Y, A, B)) The magnitude of the association of the two target sets with the two attribute sets can be measured with the effect size as: 1 Introduction Word embedding models including Word2Vec (Mikolov et al., 2013), GloVe (Pennington et al., 2014), BERT (Devlin et al., 2018), ELMo (Peters et al., 2018), and GPT (Radford et al., 2018) have become popular components of many NLP frame- works and are vastly used for many downstream tasks. However, these word representations pre- serve not only statistical properties of human lan- guage but also the human-like biases that exist in the data used to train them (Bolukbasi et al., 2016; Caliskan et al., 2017; Kurita et al., 2019; Basta et al., 2019; Gonen and Goldberg, 2019). It has also been shown that such biases propagate to the downstream NLP tasks and have negative impacts on their performance (May et al., 2019; Leino et al., 2018). There are studies investigating how to miti- Kurita et al. (2019) proposed a new method to quantify bias in BERT embeddings based on its masked language model objective using simple template sentences. For each attribute word, us- ing a simple template sentence, the normalized probability that BERT assigns to that sentence for each of the target words is calculated, and the dif- ference is considered the measure of the bias. Ku- rita et al. (2019) demonstrated that this probability- based method for quantifying bias in BERT was more effective than the cosine-based method. 8 Motivated by these recent studies, we compre- hensively investigate different methods for bias ex- posure in word embeddings. Particularly, we inves- tigate the impacts of different similarity measures and descriptive statistics to demonstrate the degree of associations between the target sets and attribute sets in the WEAT. First, other than cosine similarity, we study Euclidean, Manhattan, and Mahalanobis distances to measure the degree of association be- tween a single target word and a single attribute word. Second, other than averaging, we investigate minimum, maximum, median, and a discrete (grid- based) optimization approach to find the minimum possible association to report between a single tar- get word and the two attribute sets in each of the WEAT tests. We consistently compare these bias measures for different types of word embeddings including non-contextual (Word2Vec, GloVe) and contextual ones (BERT, ELMo, GPT, GPT2). observed difference. Let X and Y be two sets of target word embeddings and A and B be two sets of attribute embeddings. 1 Introduction The test statistics is defined as: s(X, Y, A, B) = | P x∈X s(x, A, B) −P y∈Y s(y, A, B)| 2 Method This test computes the probability that target words’ random permuta- tions would produce a greater difference than the In this paper we investigate the impacts of other functions such as min(·), mean(·), median(·), or max(·) for function f(·) in equation (1) (originally only mean(·) has been used). Also, in this paper in addition to cosine similarity, we consider Euclidean and Manhattan distances as well as the following measures for the s(−→ w, −→a ) in equation (1). Mahalanobis distance: introduced by P. C. Ma- halanobis (Mahalanobis, 1936) this distance mea- sures the distance of a point from a distribution: s(−→ w, −→a ) = ((−→ w −−→a )T Σ−1 A (−→ w −−→a )) 1 2 . It is 9 in Table 2. We used publicly available pre-trained models. For contextual word embeddings, we used single word sentences as input instead of using simple template sentences used in other studies (May et al., 2019; Kurita et al., 2019). The sim- ple template sentences such as "this is TARGET" or "TARGET is ATTRIBUTE" used in other stud- ies do not really provide any context to reveal the contextual capability of embeddings such as BERT or ELMo. This way, the comparisons between the contextual embeddings and non-contextual embed- dings are fairer as both of them only get the target or attribute terms as input. For each model, we performed the WEAT tests using four similarity metrics mentioned in section 2: cosine, Euclidean, Manhattan, Mahalanobis. For each similarity met- ric, we also used min(·), mean(·), median(·), or max(·) as the f(·) in equation (1). Also, as ex- plained in section 2, we discretely optimized the association measure and found the minimum asso- ciation in equation (1). In these experiments (Table 3 and Table 4), the larger and more significant ef- fect sizes imply more severe biases. worth noting that the Mahalanobis distance takes into account the distribution of the set of attributes while measuring the association of the target word w with an attribute vector. Discrete optimization of the association mea- sure: In equation (1), s(w, A, B) quantifies the association of a single target word w with the two sets of attributes. Model Median is close to the mean with (µ = 0.74, σ = 0.48) among all its effect sizes. The effect sizes for minimum (µ = 0.68, σ = 0.48) and maximum (µ = 0.65, σ = 0.48) are close to each other, but smaller than mean and median. The discretely optimized association measure (Eq. 2) provides the smallest effect sizes (µ = 0.39, σ = 0.3) and reveals the least number of implicit biases. These differences as the result of apply- ing different descriptive statistics in the association measure (Eq. (1)) show that the revealed biases depend on the applied statistics to measure the bias. 6 Male vs female names with Career vs family 7 Math vs arts with male vs female terms 8 Science vs arts with male vs female terms 9 Mental vs physical disease with temporary vs permanent 10 Young vs old people’s name with pleasant vs unpleasant Table 1: The associations studied in the WEAT As described in section 2, we need each attribute set’s covariance matrix to compute Mahalanobis distance. To get stable covariance matrix estima- tion due to the high dimension of the embeddings we first created larger attribute sets by adding syn- onym terms. Next, we estimated the sparse covari- ance matrices as the number of samples in each attribute set is smaller than the number of features. To enforce sparsity, we estimated the l1 penalty using k-fold cross validation with k=3. The discretely optimized association measure (Eq. 2) provides the smallest effect sizes (µ = 0.39, σ = 0.3) and reveals the least number of implicit biases. These differences as the result of apply- ing different descriptive statistics in the association measure (Eq. (1)) show that the revealed biases depend on the applied statistics to measure the bias. 2 Method To quantify the minimum pos- sible association of a target word w with the two sets of attributes, we first calculate the distance of w from all attribute words in A and B, then calcu- late all possible differences and find the minimum difference. s(w, A, B) = min a∈A,b∈B |s(−→ w , −→a ) −s(−→ w , −→b )| (2) (2) Model WEAT Association 1 Flowers vs insects with pleasant vs unpleasant 2 Instruments vs weapons with pleasant vs unpleasant 3 Eur.-American vs Afr.-American names with Pleasant vs unpleasant (Greenwald et al., 1998b) 4 Eur.-American vs Afr.-American names (Bertrand and Mul- lainathan, 2004) with Pleasant vs unpleasant (Greenwald et al., 1998b) 5 Eur.-American vs Afr.-American names (Bertrand and Mul- lainathan, 2004) with Pleasant vs unpleasant (Nosek et al., 2002) 6 Male vs female names with Career vs family 7 Math vs arts with male vs female terms 8 Science vs arts with male vs female terms 9 Mental vs physical disease with temporary vs permanent 10 Young vs old people’s name with pleasant vs unpleasant Table 1: The associations studied in the WEAT 2 Instruments vs weapons with pleasant vs unpleasant 3 Eur.-American vs Afr.-American names with Pleasant vs unpleasant (Greenwald et al., 1998b) 4 Eur.-American vs Afr.-American names (Bertrand and Mul- lainathan, 2004) with Pleasant vs unpleasant (Greenwald et al., 1998b) Table 2: Word embedding models, used representa- tions, and their dimensions. 5 Eur.-American vs Afr.-American names (Bertrand and Mul- lainathan, 2004) with Pleasant vs unpleasant (Nosek et al., 2002) Impacts of different descriptive statistics: Our first goal was to report the changes in the mea- sured biases when we change the descriptive statis- tics. The range of effect sizes was from 0.00 to 1.89 (µ = 0.65, σ = 0.5). Our findings show that mean has a better capability to reveal biases as it provides the most cases of significant effect sizes (µ = 0.8, σ = 0.52) across models and dis- tance measures. Median is close to the mean with (µ = 0.74, σ = 0.48) among all its effect sizes. The effect sizes for minimum (µ = 0.68, σ = 0.48) and maximum (µ = 0.65, σ = 0.48) are close to each other, but smaller than mean and median. Impacts of different descriptive statistics: Our first goal was to report the changes in the mea- sured biases when we change the descriptive statis- tics. The range of effect sizes was from 0.00 to 1.89 (µ = 0.65, σ = 0.5). Our findings show that mean has a better capability to reveal biases as it provides the most cases of significant effect sizes (µ = 0.8, σ = 0.52) across models and dis- tance measures. 3 Biases studied We studied all ten bias categories introduced in IAT (Greenwald et al., 1998a) and replicated in WEAT to measure the biases in word embeddings. The ten WEAT categories are briefly introduced in Table 1. For more detail and example of target and attribute words, please check Appendix A. Although WEAT 3 to 5 have the same names, they have different target and attribute words. Model Embedding Dim GloVe (840B tokens, web corpus) - 300 Word2Vec (GoogleNews-negative) - 300 ELMo (original) First hidden layer 1024 BERT (base, cased) Sum of last 4 hidden layers in [CLS] 768 GPT Last hidden layer 768 GPT2 Last hidden layer 768 Table 2: Word embedding models, used representa- tions, and their dimensions. 4 Results of experiments p We examined the 10 different types of biases in WEAT (Table 1) for word embedding models listed 10 biases in 10 WEAT categories (Table 3). Using mean of Euclidean, our results confirm all the re- sults by Caliskan et al. (2017), which used mean of cosine in all WEAT tests. The difference is that with the mean of Euclidean measure, the biases are revealed as being more severe. (smaller p-values). Using mean of Euclidean, GPT and ELMo show the fewest number of implicit biases. GPT model shows bias in WEAT 2, 3, and 5. ELMo’s signifi- cant biases are in WEAT 1, 3, and 6. Using mean Euclidean, almost all models (except for ELMo) confirm the existence of a bias in WEAT 3 to 5. Moreover, all contextualized models found no bias in associating female with arts and male with sci- ence (WEAT 7), mental diseases with temporary attributes and physical diseases with permanent at- tributes (WEAT 9), and young people’s name with pleasant attribute and old people’s name with un- pleasant attributes (WEAT 10). For example, in the cosine distance of Word2Vec, if we change the descriptive statistic from mean to minimum, the biases for WEAT 3 and WEAT 4 will become insignificant (no bias will be reported). In another example, in GPT model, while the result of mean cosine is not significant for WEAT 3 and WEAT 4, they become significant for median co- sine. Moreover, almost for all models, the effect size of the discretely optimized minimum distance is not significant. Our intention for considering this statistic was to report the minimum possible association of a target word with the attribute sets. If this measure is used for reporting biases, one can misleadingly claim that there is no bias. Impacts of different similarity measures: The effect sizes for cosine, Manhattan, and Euclidean are closer to each other and greater than the Ma- halanobis distance (cosine: (µ = 0.72, σ = 0.49), Euclidean: (µ = 0.67, σ = 0.5), Manhattan: (µ = 0.63, σ = 0.48), Mahalanobis: (µ = 0.58, σ = 0.45)). Mahalanobis distance also detects the fewest number of significant bias types across all models. As an example, while mean and median effect sizes for WEAT 3 and WEAT 5 in GloVe and Word2Vec are mostly significant for cosine, Euclidean, and Manhattan; the same results are not significant for the Mahalanobis distance. 4 Results of experiments That means with the Mahalanobis distance as the mea- sure of the bias, no bias will be reported for WEAT 3 and WEAT 5 tests. This emphasizes the impor- tance of chosen similarity measures in detecting biases of word embeddings. More importantly, as the Mahalanobis distance considers the distribution of attributes in measuring the distance, it may be a better choice than the other similarity measures for measuring and revealing biases with GPT showing fewer number of biases. Model mean cosine mean Euc mean Maha Maha Eq.2 GloVe 9 9 3 0 (1.39, 0.21) (1.41, 0.2) (0.79, 0.53) (0.34, 0.27) Word2Vec 7 7 5 0 (1.13, 0.54) (1.13, 0.55) (0.84, 0.52) (0.32, 0.33) ELMo 3 3 3 0 (0.64, 0.51) (0,65, 0.52) (0.61, 0.42) (0.36, 0.23) BERT 5 5 2 2 (0.74, 0.5) (0.74, 0.48) (0.47, 0.5) (0.55, 0.52) GPT 2 3 4 0 (0.61, 0.48) (0.65, 0.42) (0.59, 0.35) (0.29, 0.27) GPT2 3 4 3 3 (0.73, 0.46) (0.71, 0.46) (0.69, 0.49) (0.66, 0.49) Table 3: Number of revealed biases out of the 10 WEAT bias types for the studied word embeddings along with the (µ, σ) of their effect sizes. The larger the effect size the more severe the bias. 5 Conclusions We studied the impacts of different descriptive statistics and similarity measures on association tests for measuring biases in contextualized and non-contextualized word embeddings. Our find- ings demonstrate that the detected biases depend on the choice of association measure. Based on our experiments, mean reveals more severe biases and the discretely optimized version reveals fewer number of severe biases. In addition, cosine dis- tance reveals more severe biases and the Maha- lanobis distance reveals less severe ones. Report- ing biases with mean of Euclidean/Mahalanobis distances identifies more/less severe biases in the models. Furthermore, contextual models show less biases than the non-contextual ones across all 10 WEAT tests with GPT showing the fewest number of biases. Biases in different word embedding models: Using any combination of descriptive statistics and similarity measures, all the contextualized mod- els have less significant biases than GloVe and Word2Vec. In Table 3 the number of tests with significant implicit biases out of the 10 WEAT tests along with the mean and standard deviation of the effect sizes for all embedding models have been reported. The complete list of effect sizes along with their p-value are provided in Table 4. Following our findings in the previous sections, we choose mean of Euclidean to reveal biases. 5 Conclusions By doing so, GloVe and Word2Vec show the most num- ber of significant biases with 9 and 7 significant 11 Cosine Euclidean Manhattan Mahalanobis Model WEAT Mean Median Min Max Eq.2 Mean Median Min Max Eq.2 Mean Median Min Max Eq.2 Mean Median Min Max Eq.2 GloVe 1 1.50∗∗∗∗ 1.34∗∗∗∗ 1.35∗∗∗ 1.41∗∗∗∗ 0.27 1.52∗∗∗∗ 1.47∗∗∗∗ 1.31∗∗∗∗ 1.23∗∗∗∗ 0.03 1.50∗∗∗∗ 1.46∗∗∗∗ 1.32∗∗∗∗ 0.90∗∗ 0.15 1.53∗∗∗∗ 1.54∗∗∗∗ 1.19∗∗∗∗ 1.61∗∗∗∗ 0.00 2 1.53∗∗∗∗ 1.37∗∗∗∗ 0.83∗ 1.57∗∗∗∗ 0.08 1.53∗∗∗∗ 1.42∗∗∗∗ 1.42∗∗∗∗ 0.03 0.13 1.51∗∗∗∗ 1.43∗∗∗∗ 1.44∗∗∗∗ 0.27 0.24 1.61∗∗∗∗ 1.63∗∗∗∗ 1.49∗∗∗∗ 0.98∗∗∗ 0.28 3 1.41∗∗∗∗ 1.13∗∗∗∗ 1.53∗∗∗∗ 1.41∗∗∗∗ 0.60∗ 1.37∗∗∗∗ 0.98∗∗∗∗ 1.51∗∗∗∗ 0.09 0.31 0.82∗∗ 0.37 1.24∗∗∗∗ 0.69∗ 0.21 0.57 0.66∗ 0.37 0.89∗∗ 0.13 4 1.50∗∗∗∗ 1.02∗ 1.55∗∗∗∗ 1.47∗∗∗∗ 0.17 1.51∗∗∗∗ 0.40 1.58∗∗∗∗ 0.32 0.06 0.93∗ 0.36 1.14∗∗∗ 0.80∗ 0.05 0.30 0.57 0.04 0.67 0.31 5 1.28∗∗∗ 1.39∗∗∗ 0.45 1.29∗∗∗ 0.57 1.30∗∗∗∗ 1.62∗∗∗∗ 1.13∗∗ 0.36 0.61 0.54 1.03∗ 0.17 0.11 0.37 0.17 0.36 0.01 0.69 0.35 6 1.81∗∗∗ 1.83∗∗∗ 1.70∗∗∗ 1.67∗∗∗ 1.01 1.80∗∗∗∗ 1.75∗∗∗∗ 1.75∗∗∗∗ 1.56∗∗∗ 0.17 1.78∗∗∗∗ 1.78∗∗∗∗ 1.71∗∗∗∗ 1.46∗∗ 0.86 1.17∗ 0.83 1.27∗ 0.60 0.43 7 1.06 0.85 0.61 1.05 0.18 1.10 0.65 0.26 0.70 0.16 0.70 0.03 0.55 0.63 0.50 0.20 0.80 0.02 0.23 0.10 8 1.24∗ 0.93 1.29∗ 1.16∗ 0.36 1.23∗ 1.07 1.12 0.92 0.21 1.03 0.81 0.99 0.83 0.13 0.92 0.71 0.86 0.26 0.26 9 1.38∗ 0.83 0.37 1.47∗ 1.03 1.47∗ 1.04 1.20 1.32∗ 0.90 1.50∗ 0.26 1.18 1.42∗ 0.61 0.99 0.93 1.20 0.55 0.85 10 1.21∗ 1.05 1.01 0.75 0.99 1.26∗ 1.42∗ 0.84 0.64 0.41 0.70 0.90 0.34 0.46 0.25 0.47 0.83 0.45 0.60 0.71 word2vec 1 1.54∗∗∗∗ 1.34∗∗∗∗ 0.55 1.49∗∗∗∗ 0.16 1.50∗∗∗∗ 1.30∗∗∗∗ 1.31∗∗∗∗ 0.95∗∗ 0.31 1.49∗∗∗∗ 1.34∗∗∗∗ 1.38∗∗∗∗ 0.75∗ 0.26 0.84∗ 1.06∗∗∗ 0.79∗ 0.34 0.13 2 1.63∗∗∗∗ 1.49∗∗∗∗ 1.19∗∗∗∗ 1.60∗∗∗∗ 0.22 1.58∗∗∗∗ 1.36∗∗∗∗ 1.37∗∗∗∗ 0.68∗ 0.10 1.44∗∗∗∗ 1.24∗∗∗∗ 1.19∗∗∗∗ 0.70∗ 0.36 1.39∗∗∗∗ 0.99∗∗∗ 0.39 0.15 0.05 3 0.58∗ 0.46 0.10 0.81∗∗ 0.38 0.78∗∗ 0.46 0.82∗∗ 0.62∗ 0.19 0.82∗∗ 0.56 0.68∗ 0.63∗ 0.17 0.24 0.41 0.98∗∗∗∗ 0.68∗ 0.19 4 1.31∗∗∗∗ 1.21∗∗∗ 0.44 1.27∗∗∗∗ 0.09 1.49∗∗∗∗ 0.80∗ 1.66∗∗∗∗ 0.60 0.35 1.44∗∗∗∗ 1.13∗∗∗ 1.37∗∗∗ 0.55 0.86∗ 0.55 0.16 1.30∗∗∗∗ 0.49 0.28 5 0.72 0.68 0.58 0.41 0.19 0.43 0.38 0.41 0.08 0.25 0.27 0.23 0.11 0.05 0.09 0.02 0.61 0.11 0.12 0.24 6 1.89∗∗∗ 1.87∗∗∗ 1.76∗∗∗ 1.65∗∗∗ 0.91 1.88∗∗∗∗ 1.88∗∗∗∗ 1.63∗∗ 1.70∗∗∗∗ 0.85 1.89∗∗∗∗ 1.87∗∗∗∗ 1.39∗ 1.76∗∗∗∗ 0.39 1.21∗ 0.24 1.49∗∗ 0.29 0.01 7 0.97 0.98 0.52 0.71 0.67 0.92 0.45 1.11∗ 1.27∗ 0.70 1.06 0.87 1.04 1.27∗ 1.29∗ 0.97 0.90 0.55 1.35∗ 0.08 8 1.24∗ 1.23∗ 1.18∗ 0.99 0.59 1.25∗ 1.09 1.21∗ 1.49∗∗ 0.60 1.47∗ 1.36∗ 1.33∗ 1.67∗∗∗ 0.00 0.40 0.30 0.48 0.52 0.88 9 1.30∗ 0.69 0.14 1.31 0.42 1.32∗ 1.18 1.07 0.92 0.55 1.08 0.92 0.92 0.46 0.09 1.55∗∗∗∗ 1.23 0.59 0.41 0.94 10 0.09 0.01 0.19 0.66 0.76 0.15 0.01 0.39 0.14 0.43 0.24 0.12 0.36 0.34 0.05 1.20∗ 1.24∗ 1.60∗∗∗ 0.03 0.44 ELMo 1 1.25∗∗∗∗ 1.15∗∗∗ 0.77∗ 0.68∗ 0.03 1.25∗∗∗∗ 1.03∗∗∗ 0.51 0.35 0.48 1.24∗∗∗∗ 1.04∗∗∗ 0.50 0.27 0.19 0.28 0.17 0.28 0.26 0.57 2 1.46∗∗∗∗ 1.37∗∗∗∗ 0.87∗∗ 1.37∗∗∗∗ 0.08 1.46∗∗∗∗ 1.28∗∗∗∗ 1.14∗∗∗∗ 0.71∗ 0.51 1.50∗∗∗∗ 1.22∗∗∗∗ 1.25∗∗∗∗ 0.75∗ 0.27 0.67∗ 0.11 0.79∗ 0.11 0.15 3 0.19 0.19 0.06 0.10 0.30 0.12 0.30 0.20 0.06 0.14 0.16 0.02 0.15 0.12 0.19 0.24 0.29 0.37 0.07 0.27 4 0.29 0.22 0.66 0.44 0.02 0.39 0.07 0.03 0.35 0.19 0.39 0.00 0.02 0.35 0.34 0.33 0.29 0.25 0.08 0.48 5 0.11 0.01 0.27 0.57 0.43 0.14 0.12 0.46 0.14 0.09 0.03 0.04 0.55 0.20 0.85∗ 0.40 0.04 0.71 0.52 0.34 6 1.24∗ 0.95 0.61 0.10 1.00 1.27∗ 0.50 0.02 0.44 0.53 0.30 0.59 0.51 0.20 0.49 1.34∗ 1.10 0.06 0.50 0.22 7 0.32 0.30 0.56 0.25 0.81 0.29 0.48 0.02 0.62 0.81 0.24 0.25 0.41 0.36 0.03 1.34∗ 1.49∗∗ 0.72 0.95 0.88 8 0.28 0.42 0.00 0.38 0.29 0.37 0.14 0.14 0.86 0.66 0.64 0.38 0.61 0.99 0.35 0.18 1.06 0.15 0.06 0.15 9 0.91 0.24 0.67 1.28∗ 0.68 0.93 0.59 1.04 0.69 0.10 1.06 0.65 0.98 0.77 0.38 0.71 0.55 0.94 0.23 0.38 10 0.37 0.81 0.53 0.56 0.23 0.33 0.93 0.36 0.13 0.62 0.28 0.74 0.49 0.06 0.62 0.61 0.73 0.26 0.48 0.20 BERT 1 0.00 0.21 0.12 0.05 0.72∗ 0.01 0.21 0.09 0.11 0.18 0.02 0.32 0.05 0.18 0.46 0.10 0.11 0.24 0.27 0.06 2 0.62 0.39 0.90∗∗ 0.55 0.32 0.63 0.45 0.56 1.02∗∗ 0.24 0.58 0.45 0.23 0.79∗ 0.27 1.31∗∗∗∗ 1.33∗∗∗∗ 1.35∗∗∗∗ 1.25∗∗∗∗ 1.21∗∗∗∗ 3 1.04∗∗∗ 1.02∗∗∗ 0.75∗ 0.83∗∗ 0.58∗ 1.05∗∗∗∗ 1.07∗∗∗∗ 0.77∗∗ 0.72∗ 0.21 1.04∗∗∗∗ 1.09∗∗∗∗ 0.82∗∗ 0.76∗∗ 0.01 0.24 0.30 0.23 0.29 0.17 4 1.19∗∗∗ 1.19∗∗∗ 1.06∗∗∗ 1.08∗∗ 0.26 1.23∗∗∗ 0.97∗ 1.00∗∗ 1.16∗∗∗∗ 0.65 1.20∗∗∗ 1.07∗∗ 0.88∗ 1.10∗∗∗ 0.13 0.27 0.31 0.44 0.17 0.05 5 0.94∗ 0.93∗ 0.30 0.77∗ 0.06 0.88∗ 0.95∗ 0.58 0.11 0.41 0.85∗ 0.98∗ 0.71 0.02 0.45 0.29 0.23 0.04 0.34 0.19 6 1.36∗ 1.20∗ 1.32∗ 0.13 0.22 1.30∗ 1.15∗ 0.20 1.45∗∗ 0.96 1.12 0.83 0.16 1.34∗ 0.82 0.03 0.15 0.24 0.61 0.38 7 1.14∗ 0.85 0.75 1.01 0.07 1.18∗ 0.75 1.03 0.95∗ 0.40 1.20∗ 0.90 1.09 1.02∗ 0.85 0.29 0.09 0.49 0.18 0.77 8 0.24 0.37 0.11 0.55 0.17 0.24 0.02 0.50 0.73 0.37 0.12 0.14 0.13 0.34 0.04 0.47 0.42 0.61 0.27 0.60 9 0.02 0.16 0.03 1.04 0.69 0.12 0.32 0.97 0.00 0.25 0.17 0.34 0.72 0.16 0.66 1.48∗ 1.38∗ 1.52∗ 1.54∗ 1.61∗ 10 0.83 0.76 0.89 0.50 1.28∗ 0.80 0.89 0.40 0.90 0.22 0.91 1.16∗ 0.57 0.72 0.53 0.24 0.28 0.09 0.54 0.47 GPT 1 0.47 0.29 0.57 0.08 0.24 0.58 0.39 0.10 0.10 0.50 0.57 0.25 0.11 0.01 0.10 0.40 0.00 0.54 0.45 0.13 2 1.11∗∗∗ 0.99∗∗ 0.94∗∗ 0.53 0.38 1.15∗∗∗∗ 0.74∗ 0.13 0.23 0.01 1.16∗∗∗∗ 0.82∗ 0.01 0.16 0.28 0.84∗∗ 0.69∗ 0.06 1.01∗∗ 0.05 3 0.09 0.97∗∗∗ 0.64∗ 1.24∗∗∗∗ 0.20 0.70∗ 0.99∗∗∗ 1.21∗∗∗∗ 0.27 0.02 0.06 1.05∗∗∗∗ 1.17∗∗∗∗ 0.56 0.01 0.69∗ 0.97∗∗∗ 1.24∗∗∗∗ 0.90∗∗∗ 0.13 4 0.33 1.54∗∗∗∗ 0.88∗ 1.48∗∗∗∗ 0.30 0.51 1.51∗∗∗∗ 1.37∗∗∗∗ 0.28 0.10 0.31 1.52∗∗∗∗ 1.33∗∗∗∗ 0.50 0.29 0.91∗∗ 1.36∗∗∗∗ 1.26∗∗∗ 1.07∗∗∗ 0.42 5 1.65∗∗∗∗ 1.40∗∗∗∗ 1.57∗∗∗∗ 1.58∗∗∗∗ 0.69 1.57∗∗∗∗ 1.14∗∗∗∗ 1.49∗∗∗∗ 1.65∗∗∗∗ 0.26 1.54∗∗∗∗ 1.23∗∗∗∗ 1.49∗∗∗∗ 1.50∗∗∗∗ 0.57 1.26∗∗∗∗ 1.23∗∗∗∗ 0.98∗∗∗ 1.40∗∗∗∗ 0.06 6 0.67 0.02 0.75 0.89 0.20 0.50 0.25 0.23 0.66 0.08 0.49 0.04 0.34 0.45 0.09 0.66 0.01 1.14∗ 0.27 0.19 7 0.24 0.11 0.02 0.09 0.70 0.20 0.30 0.00 0.15 0.45 0.28 0.16 0.32 0.03 0.18 0.29 0.63 0.22 0.57 0.06 8 0.10 0.16 0.35 0.13 0.40 0.08 0.10 0.32 0.03 0.93∗ 0.12 0.14 0.37 0.08 0.47 0.19 0.13 0.22 0.58 0.43 9 0.70 0.92 1.01 0.01 0.18 0.58 0.63 0.17 0.07 0.42 0.59 0.62 0.40 0.01 0.44 0.50 0.59 0.66 0.03 0.62 10 0.72 0.39 0.73 0.68 0.67 0.61 0.25 0.55 1.03 0.27 0.52 0.34 0.48 0.76 0.77 0.19 0.17 0.07 0.27 0.81 GPT2 1 0.11 0.06 0.20 0.20 0.19 0.06 0.21 0.14 0.04 0.01 0.08 0.05 0.05 0.07 0.04 0.27 0.27 0.26 0.23 0.28 2 0.64 0.28 0.50 0.24 0.04 0.51 0.63 0.21 0.61∗ 0.02 0.47 0.69∗ 0.18 0.53 0.56 0.44 0.45 0.45 0.41 0.34 3 1.27∗∗∗∗ 0.70∗ 1.07∗∗∗∗ 1.15∗∗∗∗ 0.30 1.25∗∗∗∗ 1.21∗∗∗∗ 0.29 1.30∗∗∗∗ 0.48 1.34∗∗∗∗ 1.24∗∗∗∗ 0.39 1.12∗∗∗∗ 0.11 1.25∗∗∗∗ 1.25∗∗∗∗ 1.27∗∗∗∗ 1.22∗∗∗∗ 1.24∗∗∗∗ 4 1.19∗∗ 0.64 1.28∗∗∗ 0.83∗ 0.56 1.17∗∗∗ 1.24∗∗∗∗ 0.39 1.13∗∗ 0.57 1.17∗∗∗ 1.10∗∗ 0.28 1.05∗∗ 0.44 1.29∗∗∗∗ 1.29∗∗∗∗ 1.28∗∗∗∗ 1.28∗∗∗ 1.31∗∗∗∗ 5 1.17∗∗ 1.15∗∗ 1.31∗∗∗ 1.02∗∗ 0.06 1.17∗∗∗ 1.21∗∗∗∗ 0.92∗ 1.13∗∗ 0.77∗ 1.14∗∗ 1.18∗∗∗ 0.13 1.15∗∗ 0.42 1.29∗∗∗ 1.29∗∗∗∗ 1.28∗∗∗∗ 1.30∗∗∗∗ 1.29∗∗∗∗ 6 0.79 1.06 0.94 0.11 0.20 0.86 0.90 0.28 0.94 0.54 0.66 0.74 0.19 0.11 0.63 0.94 0.55 0.52 0.89 0.71 7 0.03 0.49 0.21 0.69 0.17 0.12 0.10 0.24 0.00 0.07 0.23 0.42 0.34 0.01 0.16 0.16 0.17 0.16 0.16 0.14 8 0.42 1.08 0.79 0.42 0.36 0.44 0.37 0.08 0.59 0.32 0.30 0.78 0.22 0.21 0.23 0.36 0.35 0.36 0.37 0.38 9 0.57 0.68 0.16 0.17 0.73 0.36 0.41 0.39 0.64 0.24 0.04 0.10 1.19 0.32 0.05 0.06 0.05 0.14 0.21 0.04 10 1.14 1.12 0.77 0.63 0.03 1.13∗ 1.11 0.09 1.12 0.14 1.12 1.12 0.24 1.13 0.71 0.80 0.83 0.82 0.65 0.86 Table 4: WEAT effect size, *: significance at 0.01, **: significance at 0.001, ***: significance at 0.0001, ****: significance at 0.00001. 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A The studied associations: 10 WEAT categories A The studied associations: 10 WEAT categories WEAT Association NT NA 1 Flowers vs insects with pleasant vs unpleasant Example: {aster, clover} vs {ant, caterpillar} with {caress, freedom} vs { abuse, crash} 25 × 2 25 × 2 2 Instruments vs weapons with pleasant vs unpleasant Example: {bagpipe, cello} vs {arrow, club} with {caress, freedom} vs { abuse, crash} 25 × 2 25 × 2 3 Eur.-American vs Afr.-American names with Pleasant vs unpleasant Example: {Adam, Harry} vs {Alonzo, Jamel} with {caress, freedom} vs {abuse, crash} 32 × 2 25 × 2 4 Eur.-American vs Afr.-American names with Pleasant vs unpleasant Example: {Brad, Brendan} vs {Darnell, Hakim} with {caress, freedom} vs {abuse, crash} 16 × 2 25 × 2 5 Eur.-American vs Afr.-American names with Pleasant vs unpleasant Example: {Brad, Brendan} vs {Darnell, Hakim} with {joy, love} vs {agony, terrible} 16 × 2 8 × 2 6 Male vs female names with Career vs family Example: {John, Paul} vs {Amy, Joan} with {executive, management} vs {home, parents} 8 × 2 8 × 2 7 Math vs arts with male vs female terms Example: {math, algebra} vs {poetry, art} with {male, man} vs {female, woman} 8 × 2 8 × 2 8 Science vs arts with male vs female terms Example: {science, technology} vs {art, Shakespeare} with {brother, father} vs {sister, mother} 8 × 2 8 × 2 9 Mental vs physical disease with temporary vs permanent Example: {sad, hopeless} vs {sick, illness} with {impermanent, unstable} vs {stable, always} 6 × 2 7 × 2 10 Young vs old people’s name with pleasant vs unpleasant Example: {Tiffany, Michelle} vs {Ethel, Bernice} with {love, peace} vs {agony, terrible} 8 × 2 8 × 2 References 13 A The studied associations: 10 WEAT categories WEAT Association NT NA 1 Flowers vs insects with pleasant vs unpleasant Example: {aster, clover} vs {ant, caterpillar} with {caress, freedom} vs { abuse, crash} 25 × 2 25 × 2 2 Instruments vs weapons with pleasant vs unpleasant Example: {bagpipe, cello} vs {arrow, club} with {caress, freedom} vs { abuse, crash} 25 × 2 25 × 2 3 Eur.-American vs Afr.-American names with Pleasant vs unpleasant Example: {Adam, Harry} vs {Alonzo, Jamel} with {caress, freedom} vs {abuse, crash} 32 × 2 25 × 2 4 Eur.-American vs Afr.-American names with Pleasant vs unpleasant Example: {Brad, Brendan} vs {Darnell, Hakim} with {caress, freedom} vs {abuse, crash} 16 × 2 25 × 2 5 Eur.-American vs Afr.-American names with Pleasant vs unpleasant Example: {Brad, Brendan} vs {Darnell, Hakim} with {joy, love} vs {agony, terrible} 16 × 2 8 × 2 6 Male vs female names with Career vs family Example: {John, Paul} vs {Amy, Joan} with {executive, management} vs {home, parents} 8 × 2 8 × 2 7 Math vs arts with male vs female terms Example: {math, algebra} vs {poetry, art} with {male, man} vs {female, woman} 8 × 2 8 × 2 8 Science vs arts with male vs female terms Example: {science, technology} vs {art, Shakespeare} with {brother, father} vs {sister, mother} 8 × 2 8 × 2 9 Mental vs physical disease with temporary vs permanent Example: {sad, hopeless} vs {sick, illness} with {impermanent, unstable} vs {stable, always} 6 × 2 7 × 2 10 Young vs old people’s name with pleasant vs unpleasant Example: {Tiffany, Michelle} vs {Ethel, Bernice} with {love, peace} vs {agony, terrible} 8 × 2 8 × 2 A The studied associations: 10 WEAT categories Association Table 1: The 10 WEAT categories. 14 14
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English
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Ear Decomposition and Balanced Neighborly Simplicial Manifolds
˜The œElectronic journal of combinatorics/˜The œjournal of combinatorics
2,020
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9,535
Abstract We find the first non-octahedral balanced 2-neighborly 3-sphere and the bal- anced 2-neighborly triangulation of the lens space L(3, 1). Each construction has 16 vertices. We show that there exists a balanced 3-neighborly non-spherical 5- manifold with 18 vertices. We also show that the rank-selected subcomplexes of a balanced simplicial sphere do not necessarily have an ear decomposition. Mathematics Subject Classifications: 05E45 Ear decomposition and balanced neighborly simplicial manifolds Hailun Zheng Department of Mathematics University of Michigan Ann Arbor, MI, 48109, USA hailunz@umich.edu Hailun Zheng Department of Mathematics University of Michigan Ann Arbor, MI, 48109, USA hailunz@umich.edu Submitted: Nov 26, 2018; Accepted: Dec 22, 2019; Published: Jan 10, 2020 c⃝The author. Released under the CC BY-ND license (International 4.0). Submitted: Nov 26, 2018; Accepted: Dec 22, 2019; Published: Jan 10, 2020 c⃝The author. Released under the CC BY-ND license (International 4.0). 1 Introduction A simplicial complex is called k-neighborly if every subset of vertices of size at most k is the set of vertices of one of its faces. Neighborly complexes, especially neighborly polytopes and spheres, are interesting objects to study. In the seminal work of McMullen [12] and Stanley [19], it was shown that in the class of polytopes and simplicial spheres of a fixed dimension and with a fixed number of vertices, the cyclic polytope simultaneously maximizes all the face numbers. The d-dimensional cyclic polytope is ⌊d 2⌋-neighborly. Since then, many other classes of neighborly polytopes have been discovered. We refer to [4], [16] and [18] for examples and constructions of neighborly polytopes. Meanwhile, the notion of neighborliness was extended to other classes of objects: for instance, neighborly cubical polytopes were defined and studied in [8], [9], and [17], and neighborly centrally symmetric polytopes and spheres were studied in [1], [3], [7] and [14]. In this paper we discuss a similar notion for balanced simplicial complexes. Balanced complexes were defined by Stanley in [20], where they were called completely balanced. A (d−1)-dimensional simplicial complex is called balanced if its graph is d-colorable. For instance, the barycentric subdivision of regular CW complexes and order complexes are 1 the electronic journal of combinatorics 27(1) (2020), #P1.10 balanced. We say that a balanced simplicial complex is balanced k-neighborly if every set of k or fewer vertices with distinct colors forms a face. For example, if ∆1 and ∆2 are balanced k-neighborly spheres, then the join ∆= ∆1 ∗∆2 is also a balanced neighborly k-sphere, and we call ∆join-decomposable. However, apart from the cross-polytopes, it is not known whether other join-indecomposable balanced k-neighborly polytopes or spheres exist. To the best of our knowledge, no examples of such objects appear in the current literature, even for k = 2. As for balanced 2-neighborly manifolds, one such construction that triangulates the sphere bundle is given in [11]; it is also a minimal balanced triangulation of the underlying topological space. This more or less explains why so far there is even no plausible sharp upper bound conjecture for balanced spheres or manifolds. The goal of this paper is to partially rem- edy this situation by searching for balanced neighborly spheres and manifolds of lower dimensions. 1 Introduction It turns out that even in the lower dimensional cases balanced neighborly spheres or manifolds with a given number of vertices do not always exist. • The octahedral 3-sphere is the only balanced 2-neighborly 3-sphere with less than 16 vertices. • The octahedral 3-sphere is the only balanced 2-neighborly 3-sphere with less than 16 vertices. • There is a unique balanced 2-neighborly 4-sphere with 15 vertices, known as 4155 2 in [10]. • There is a unique balanced 2-neighborly 4-sphere with 15 vertices, known as 4155 2 in [10]. • There exists a balanced 3-neighborly non-spherical 5-manifold with 18 vertices • There are two constructions of balanced 2-neighborly 3-manifolds with 16 vertices; one triangulates the sphere, and the other triangulates the lens space L(3, 1). • There are two constructions of balanced 2-neighborly 3-manifolds with 16 vertices; one triangulates the sphere, and the other triangulates the lens space L(3, 1). In a different direction, it is also interesting to ask whether every rank-selected sub- complex of a balanced simplicial polytope or sphere has a convex ear decomposition. This statement, if true, would imply that rank-selected subcomplexes of balanced simpli- cial polytopes possess certain weak Lefschetz properties, see Theorem 3.9 in [22]. As a consequence, it would also provide an alternative proof of the balanced Generalized Lower Bound Theorem, see Theorem 3.3 and Remark 3.4 in [13]. We present an example giving a negative answer to this question for 3-dimensional spheres. The structure of this manuscript is as follows. In Section 2, after reviewing basic definitions, we establish basic properties of balanced neighborly spheres; in particular, we prove that for some values of f0, such spheres cannot exist. In Section 3 we discuss how to find balanced k-neighborly (2k −1)-manifolds from less neighborly balanced (2k −2)- spheres, for k = 2, 3. In Section 4, we construct a balanced 2-neighborly 3-sphere with 16 vertices. In Section 5, we present the balanced 2-neighborly triangulation of L(3, 1) with 16 vertices. In Section 6 we provide a way to construct balanced spheres whose rank-selected subcomplex does not have an ear decomposition. 2 Basic properties of balanced neighborly spheres A simplicial complex ∆with vertex set V is a collection of subsets σ ⊆V , called faces, that is closed under inclusion, and such that for every v ∈V , {v} ∈∆. For σ ∈∆, let the electronic journal of combinatorics 27(1) (2020), #P1.10 2 dim σ := |σ|−1 and define the dimension of ∆, dim ∆, as the maximum dimension of the faces of ∆. A facet is a maximal face under inclusion. We say that a simplicial complex ∆is pure if all of its facets have the same dimension. If ∆is a simplicial complex and σ is a face of ∆, the star of σ in ∆is st∆σ := {τ ∈ ∆: σ ∪τ ∈∆}. We also define the link of σ in ∆as lk∆σ := {τ −σ ∈∆: σ ⊆τ ∈∆}, and the deletion of a subset of vertices W from ∆as ∆\W := {σ ∈∆: σ ∩W = ∅}. If ∆1 and ∆2 are simplicial complexes on disjoint vertex sets, then the join of ∆1 and ∆2, denoted ∆1 ∗∆2, is the simplicial complex with vertex set V (∆1) ∪V (∆2) whose faces are {σ1 ∪σ2 : σ1 ∈∆1, σ2 ∈∆2}. If ∆is a pure (d −1)-dimensional complex such that every (d −2)-dimensional face of ∆is contained in at most two facets, then the boundary complex of ∆consists of all (d −2)-dimensional faces that are contained in exactly one facet, as well as their subsets. A simplicial complex ∆is a simplicial sphere (resp. simplicial ball) if the geometric realization of ∆is homeomorphic to a sphere (resp. ball). The boundary complex of a simplicial d-ball is a simplicial (d −1)-sphere. A simplicial sphere is called polytopal if it is the boundary complex of a convex polytope. For instance, the boundary complex of an octahedron is a polytopal sphere; we will refer to it as an octahedral sphere. For a fixed field or group k, we say that ∆is a (d −1)-dimensional k-homology sphere if ˜Hi(lk∆σ; k) ∼= ˜Hi(Sd−1−|σ|; k) for every face σ ∈∆(including the empty face) and i ⩾−1. A homology d-ball (over k) is a d-dimensional simplicial complex ∆such that (i) ∆has the same homology as the d-dimensional ball, (ii) for every face F, the link of F has the same homology as the (d−|F|)-dimensional ball or sphere, and (iii) the boundary complex ∂∆is a homology (d −1)-sphere. 2 Basic properties of balanced neighborly spheres The classes of simplicial (d −1)-spheres and homology (d −1)-spheres coincide when d ⩽3. From now on all homology are computed with coefficients in Z and we will omit it from our notation. Next we define a special structure that exists in some pure simplicial complexes. Definition 1. An ear decomposition of a pure (d −1)-dimensional simplicial complex ∆ is an ordered sequence ∆1, ∆2, · · · , ∆m of pure (d −1)-dimensional subcomplexes of ∆ such that: 1. ∆1 is a simplicial (d −1)-sphere, and for each j = 2, 3, · · · , m, ∆j is a simplicial (d −1)-ball. 1. ∆1 is a simplicial (d −1)-sphere, and for each j = 2, 3, · · · , m, ∆j is a simplicial (d −1)-ball. 2. For 2 ⩽j ⩽m, ∆j ∩(∪j−1 i=1∆i) = ∂∆j. 3. ∪m i=1∆i = ∆. We call ∆1 the initial complex, and each ∆j, j ⩾2, an ear of this decompostion. No- tice that this definition is more general than Chari’s original definition of a convex ear decomposition, see [2, Section 3.2], where the ∆i’s are required to be subcomplexes of the boundary complexes of polytopes. In particular, if a complex has no ear decompo- sition, then it has no convex ear decomposition. However, by the Steinitz theorem, all simplicial 2-spheres are polytopal, and hence also all simplicial 2-balls can be realized as subcomplexes of the boundary complexes of 3-dimensional polytopes. So for 2-dimensional 3 the electronic journal of combinatorics 27(1) (2020), #P1.10 simplicial complexes, the notion of an ear decomposition coincides with that of a convex ear decomposition. A (d −1)-dimensional simplicial complex ∆is called balanced if the graph of ∆is d-colorable, or equivalently, there is a coloring map κ : V →[d] such that κ(x) ̸= κ(y) for any edge {x, y} ∈∆. Here [d] = {1, 2, · · · , d} is the set of colors. We denote by Vi the set of vertices of color i. A balanced simplicial complex is called balanced k- neighborly if every set of k or fewer vertices with distinct colors forms a face. We say e is a missing colored edge if e /∈∆and the vertices of e have distinct colors. For S ⊆[d], the subcomplex ∆S := {F ∈∆: κ(F) ⊆S} is called the rank-selected subcomplex of ∆. 2 Basic properties of balanced neighborly spheres We also define the flag f-vector (fS(∆) : S ⊆[d]) and the flag h-vector (hS(∆) : S ⊆[d]) of ∆, respectively, by letting fS(∆) := #{F ∈∆: κ(F) = S}, where f∅(∆) = 1, and hS(∆) := P T⊆S(−1)#S−#TfT(∆). The usual f-numbers and h-numbers can be recovered from the relations fi−1(∆) = P #S=i fS(∆) and hi(∆) = P #S=i hS(∆). # # In the remainder of this section, we establish some restrictions on the possible size of color sets of balanced neighborly spheres. Lemma 2. Let ∆be a balanced k-neighborly homology (2k −1)-sphere. Then ∆has the same number of vertices of each color. In particular, f0(∆) = 2kℓfor some ℓ⩾2. Proof. Let W ⊆[2k] be an arbitrary subset of the set of the colors with |W| = k. Since ∆is balanced k-neighborly, ∆W is also balanced k-neighborly, and hence ∆W is the join of k color sets of colors in W, each considered as a 0-dimensional complex. By the definition of the join and the flag h-numbers, we have fU∪{i}(∆) = fU(∆)f{i}(∆) and hence hU∪{i}(∆) = hU(∆)h{i}(∆) for all i ∈W, U ⊂W and i /∈U. Therefore, Y i∈W (|Vi| −1) = Y i∈W h{i}(∆) = hW(∆) (∗) = h[2k]\W(∆) = Y i∈[2k]\W h{i}(∆) = Y i∈[2k]\W (|Vi| −1), where (∗) follows from the Dehn-Sommerville relations. Since W is an arbitrary k-subset of [2k], it follows that each color set in ∆must have the same size. Remark 3. Lemma 2 not only holds for homology (2k −1)-spheres but also for orientable homology (2k −1)-manifolds. Indeed by replacing the flag h-numbers with the flag h′′- numbers (see [6] for definition), Theorem 4.1 in [6] gives h′′ W(∆) = h′′ [2k]\W(∆), which further implies that hW(∆) = h[2k]\W(∆) since both W and [2k]\W are of size k. The rest of the proof is the same. Unfortunately, the above lemma is not sufficient to tell whether a balanced k-neighborly homology (2k −1)-sphere or manifold with 2kℓvertices can exist for given k, ℓ⩾2. 3 Balanced neighborly (d −1)-manifolds with 3d vertices In this section, we consider balanced ⌊d 2⌋-neighborly (d −1)-manifolds (for d = 3, 4, 5) with each color set of size 3. We begin with the following lemma. In this section, we consider balanced ⌊d 2⌋-neighborly (d −1)-manifolds (for d = 3, 4, 5) with each color set of size 3. We begin with the following lemma. 4 4 the electronic journal of combinatorics 27(1) (2020), #P1.10 w3 z3 u2 w2 z2 u3 z1 w1 u1 u3 w3 z3 z2 u2 w2 z1 u1 w1 Figure 1: Left: triangulation of the vertex link lk∆vi for vi ∈V4, where {u1, u2, u3}, {w1, w2, w3} and {z1, z2, z3} are the three other color sets. Right: the complex Σ. w3 z3 u2 w2 z2 u3 z1 w1 u1 u3 w3 z3 z2 u2 w2 z1 u1 w1 u3 u3 Figure 1: Left: triangulation of the vertex link lk∆vi for vi ∈V4, where {u1, u2, u3}, {w1, w2, w3} and {z1, z2, z3} are the three other color sets. Right: the complex Σ. Lemma 4. Let d ⩾4. If ∆is a balanced homology (d −1)-sphere and Vd = {v1, v2, v3} is the set of vertices of color d, then lk∆vi ∩lk∆vj is a homology (d −2)-ball for any 1 ⩽i < j ⩽3, and ∩3 k=1 lk∆vk is a homology (d −3)-sphere. Lemma 4. Let d ⩾4. If ∆is a balanced homology (d −1)-sphere and Vd = {v1, v2, v3} is the set of vertices of color d, then lk∆vi ∩lk∆vj is a homology (d −2)-ball for any 1 ⩽i < j ⩽3, and ∩3 k=1 lk∆vk is a homology (d −3)-sphere. Proof. Let {i, j, k} = [3] be distinct, Σ = lk∆vi ∩lk∆vj and Γ = ∩3 k=1 lk∆vk. We first prove that Σ and Γ have the same homology as a simplicial (d −2)-ball and simplicial (d −3)-sphere respectively. Since each (d −2)-face of ∆is contained in exactly 2 facets, it follows that lk∆vi ∪lk∆vj = ∆[d−1]. By the Mayer-Vietoris sequence, for any n ⩾0, · · →Hn+1(∆[d−1]) →Hn(Σ) →Hn(lk∆vi) ⊕Hn(lk∆vj) →Hn(∆[d−1]) →· · · . ( (1) Note that ∆[d−1] is a deformation retract of ∆minus three points, hence βd−2(∆[d−1]) = 2 and βk(∆[d−1]) = 0 for 0 ⩽k ⩽d −3. We conclude from (1) that βk(Σ) = 0 for all k ⩾0. 3 Balanced neighborly (d −1)-manifolds with 3d vertices Hence all vertex links in ∆are combinatorially isomorphic. p Since fi(∆[3]) = fi(∆)−P3 j=1 fi−1(lk∆vj) for all i ⩽2, we have f(∆[3]) = (1, 9, 27, 21). Let i, j, k ∈[3] be distinct and Σ := lk∆vi∩lk∆vj. Any facet F of Σ are 2-dimensional, for otherwise if F is an edge, then lk∆F is either a 4-cycle or 6-cycle, where in both cases vi and vj share at least one common neighbor w, i.e., F ∪{w} ∈Σ, a contradiction. Similarly, the facet cannot be 0-dimensional. Also the facets of Σ do not belong to lk∆vk. Hence Σ is a pure 2-dimensional subcomplex of lk∆vi with 9 vertices and f2(∆[3]) −f2(lk∆vk) = 7 triangles. On the other hand, for any u ∈Σ, the vertex link lk∆u is isomorphic to Figure 1 and vi, vj ∈lk∆u. The intersection of links of arbitrary two vertices of the same color has the following property (∗): lkΣ u = lklk∆u vi ∩lklk∆u vj is either an edge, or a path of length 3. Since Σ is 2-dimensional, each connected component of Σ has at least 3 vertices. If there are three components, then Σ is the disjoint union of three triangles, contradicting that f2(Σ) = 7. Otherwise, if Σ is connected, then by observation (∗) we have that Σ is a triangulated 2-manifold (with boundary). Since 21 = 3f2(Σ) = P u∈Σ f1(lkΣ u) and by observation (∗) f1(lkΣ u) ∈{1, 3}, the links of three vertices in Σ are single edges, while the rest are paths of length 3. However, enumeration based on observation (∗) yields that there is no such complex Σ. The last case is that Σ has two connected components. From observation (∗) we see that each component cannot have 4 or 5 vertices. If one component is the triangle, then the other component (as a 6-triangle subcomplex of the 9-vertex balanced 2-sphere) must be the triangulated annulus as shown in Figure 1. By symmetry lk∆vi ∩lk∆vk and lk∆vj ∩lk∆vk are also isomorphic to Σ. In this way we determine ∆[3] = lk∆vi ∪ lk∆vj: it is the union of three octahedral 2-spheres, each having a pair of antipodal facets (F1, F2), (F2, F3) and (F3, F1), respectively. This also determines ∆, which is the balanced triangulation of the nonorientable S2-bundle over S1 known as 31283 2 in [10]. Remark 7. 3 Balanced neighborly (d −1)-manifolds with 3d vertices Since lk∆vk ∪Σ = ∆[d−1] and lk∆vk ∩Σ = Γ, by the Mayer-Vietoris sequence we obtain Note that ∆[d−1] is a deformation retract of ∆minus three points, hence βd−2(∆[d−1]) = 2 and βk(∆[d−1]) = 0 for 0 ⩽k ⩽d −3. We conclude from (1) that βk(Σ) = 0 for all k ⩾0. Since lk∆vk ∪Σ = ∆[d−1] and lk∆vk ∩Σ = Γ, by the Mayer-Vietoris sequence we obtain · · · →Hn+1(∆[d−1]) →Hn(Γ) →Hn(lk∆vk) ⊕Hn(Σ) →Hn(∆[d−1]) →· · · . Hence βd−3(Γ) = 1 and βk(Γ) = 0 for 0 ⩽k ⩽d −4. βd 3( ) βk( ) ⩽ ⩽ Next, for any τ ∈Γ, we have lkΣ τ = lklk∆τ vi ∩lklk∆τ vj and lkΓ τ = ∩3 i=1 lklk∆τ vi. Since lk∆τ is a balanced homology (d−1−|τ|)-sphere, using the same argument as above, we may show that lkΣ τ and lkΓ τ have the same homology as a (d −2 −|τ|)-ball and (d−3−|τ|)-sphere respectively. Therefore Γ is a homology (d−3)-sphere. Finally, for any interior face σ of Σ, lkΣ σ = lklk∆vi σ = lklk∆vj σ, and hence lkΣ σ is a homology sphere. By definition we conclude that Σ is a homology (d −2)-ball. Remark 5. The complex Γ in Lemma 4 is not balanced, since Γ is (d−1)-colorable instead of being (d −2)-colorable. Remark 5. The complex Γ in Lemma 4 is not balanced, since Γ is (d−1)-colorable instead of being (d −2)-colorable. Proposition 6. The only balanced 2-neighborly 3-manifold with 12 vertices triangulates the nonorientable S2-bundle over S1. Proof. Let ∆be a balanced 2-neighborly 3-manifold with 12 vertices. Its f-vector is f(∆) = (1, 12, 54, 84, 42). By Lemma 2 and Remark 3, each color set of ∆has three the electronic journal of combinatorics 27(1) (2020), #P1.10 5 vertices. We let V4 = {v1, v2, v3} be the set of vertices of color 4. Since ∆is balanced 2-neighborly, each lk∆vi is a 2-sphere with 9 vertices, its f-vector is (1,9,21,14). Further- more, the balancedness of ∆implies that every vertex u ∈lk∆vi has deglk∆vi u = 4 or 6. If x is the number of vertices of degree 6 in lk∆vi, then 4(9 −x) + 6x = X u∈lk∆vi deg(lklk∆vi u) = 2f1(lk∆vi) = 42, and hence x = 3. A balanced 2-sphere with 9 vertices, 3 of which have degree 6, is unique up to isomorphism, as shown in Figure 1. 3 Balanced neighborly (d −1)-manifolds with 3d vertices The balanced 2-neighborly manifold 31283 2 is also known as BM4 defined in [11]. In particular in [11, Proposition 6.9] it is shown that 31283 2 is the only balanced 12-vertex 3-manifold with β1 ̸= 0. See [11] and [23] for extension in higher dimensional cases. Next we characterize all balanced 3-spheres with each color sets of size 3. 6 the electronic journal of combinatorics 27(1) (2020), #P1.10 Lemma 8. Up to an isomorphism, there are four triangulations of balanced 3-spheres with each color set of size 3. Lemma 8. Up to an isomorphism, there are four triangulations of balanced 3-spheres with each color set of size 3. Lemma 8. Up to an isomorphism, there are four triangulations of balanced 3-spheres with each color set of size 3. Proof. Let ∆be such a sphere and let V4 = {v1, v2, v3}. Each vertex link of ∆is a balanced 2-sphere with at most 9 vertices, hence it is either the octahedral sphere, the suspension of a 6-cycle, or the connected sum of two octahedral spheres. We denote these three 2-spheres as Σ1, Σ2 and Σ3 respectively. By Lemma 4, ∆[3] is the union of three triangulated 2-balls Bi = lk∆vj ∩lk∆vk, where {i, j, k} = [3], glued along their common boundary complex c. Assume that f0(lk∆vi) ⩽f0(lk∆vj) when i ⩽j. An easy counting leads to f0(∆[3]) = f0(c) + 3 X i=1 f0(Bi\c) = 9, f0(lk∆vi) = f0(c) + f0(Bj\c) + f0(Bk\c) ∈{6, 8, 9}, (2) f0(c) + 3 X i=1 f0(Bi\c) = 9, f0(lk∆vi) = f0(c) + f0(Bj\c) + f0(Bk\c) ∈{6, 8, 9}, (2) (2) ( ) where f0(Bi\c) counts the number of interior vertices of Bi. By the Dehn-Sommerville relations, the f-vector of any triangulated 3-manifold satisfies that f1 = f3 + f0. Since every facet of ∆contains exactly one vertex from V4, we have that f3(∆) = P3 i=1 f2(lk∆vi) and hence f1(∆) = 3 X i=1 f2(lk∆vi) + f0(∆) = 3 X i=1 (2f0(lk∆vi) −4) + 12 = 2 3 X i=1 f0(lk∆vi) ⩽54, we enumerate the combinatorial type of each f0(lk∆vi) as follows: Case 1: lk∆v1 ∼= Σ1. It follows that lk∆v3 is obtained from lk∆v2 by a cross flip (see [5] for a reference). Since f0(∆[3]) = 9, either lk∆v2 ∼= Σ1, lk∆v3 ∼= Σ3, and the cross flip replaces a 2-face of lk∆v2 with its complement in the octahedral sphere. 3 Balanced neighborly (d −1)-manifolds with 3d vertices Or lk∆v2 ∼= Σ2, lk∆v3 ∼= Σ3, and the cross flip replaces the union of three 2-faces of lk∆v2 with its complement in the octahedral sphere. In the first case the 3-sphere is the connected sum of two octahedral 3-spheres, which we denote as S1. In the second case we obtain a 3- sphere S2 (with lkS2 vi ∼= Σi for i ∈[3]). Their f-vectors are f(S1) = (1, 12, 42, 60, 30) and f(S2) = (1, 12, 46, 68, 34). ( ) ( ) Case 2: lk∆v1 ∼= Σ2. In this case the number of missing colored edges in ∆[3] is 9·6 2 −f1(∆[3]) = 27 −P3 i=1 f0(lk∆vi), which equals either 1,2 or 3. 2 [ ] i 1 Subcase 1: lk∆v2 ∼= lk∆v3 ∼= Σ2. By conditions (2), c is a 6-cycle and ∆[3]\c consists of three disjoint vertices of degree either 4 or 6. Note that in Σ2 every pair vertices of degree 4 and degree 6 forms an edge. Since ∆[3] has only three missing edges between vertices of different colors, the vertices in ∆[3]\c are of the same color and has degree 6. Hence ∆[3] is the join of c and three disjoint vertices and ∆is the join of two 6-cycles. Denote this sphere as S3; its f-vector is (1, 12, 48, 72, 36). 2 [ ] i Subcase 1: lk∆v2 ∼= lk∆v3 ∼= Σ2. By conditions (2), c is a 6-cycle and ∆[3]\c consists of three disjoint vertices of degree either 4 or 6. Note that in Σ2 every pair vertices of degree 4 and degree 6 forms an edge. Since ∆[3] has only three missing edges between vertices of different colors, the vertices in ∆[3]\c are of the same color and has degree 6. Hence ∆[3] is the join of c and three disjoint vertices and ∆is the join of two 6-cycles. Denote this sphere as S3; its f-vector is (1, 12, 48, 72, 36). ( ) Subcase 2: lk∆vi ∼= Σi for i = 2, 3. Then c is a 7-cycle; furthermore, B3 has no interior vertices and B1, B2 have a unique interior vertex b1, b2 respectively. Since ∆[3] has two missing colored edges, and three vertices of degree 6 form an empty triangle in lk∆v3 ∼= Σ3, WLOG assume that deg b1 = 4 and deg b2 = 6. the electronic journal of combinatorics 27(1) (2020), #P1.10 3 Balanced neighborly (d −1)-manifolds with 3d vertices The only vertex b3 not connected to b2 in B2 must be the vertex of degree 6 in lk∆v1 ∼= Σ2. Hence B3 is the join Subcase 2: lk∆vi ∼= Σi for i = 2, 3. Then c is a 7-cycle; furthermore, B3 has no interior vertices and B1, B2 have a unique interior vertex b1, b2 respectively. Since ∆[3] has two missing colored edges, and three vertices of degree 6 form an empty triangle in lk∆v3 ∼= Σ3, WLOG assume that deg b1 = 4 and deg b2 = 6. The only vertex b3 not connected to b2 in B2 must be the vertex of degree 6 in lk∆v1 ∼= Σ2. Hence B3 is the join 7 the electronic journal of combinatorics 27(1) (2020), #P1.10 of b3 with the path of length 5, which is c\b3. But then b3 is also the degree-6 vertex in lk∆v2 ∼= Σ2, and there is no way to triangulate B1 such that it shares no common interior edge with B2. of b3 with the path of length 5, which is c\b3. But then b3 is also the degree-6 vertex in lk∆v2 ∼= Σ2, and there is no way to triangulate B1 such that it shares no common interior edge with B2. Subcase 3: lk∆v2 ∼= lk∆v3 ∼= Σ3. Then c is an 8-cycle and only B1 has an interior vertex a. Also ∆[3] has one missing colored edge, so a is of degree 6. Three vertices of degree 6 in Σ3 are of different colors, hence in lk∆v2 and lk∆v3 the other two vertices of degree 6 must be two pairs of antipodal vertices in lk∆[3] a (the other pair of antipodal vertices in lk∆[3] a is the missing edge in ∆[3]). In this way we recover lk∆v2 = B1 ∪ B3, lk∆v3 = B1 ∪B2, where B1, B2, B3 are the 2-balls shown in the figure below (from left to right); in particular, labels of the vertices represent the color and the blue (red resp.) edges form the missing triangle in lk∆v2 (lk∆v3 resp.). We call this 3-sphere S4. Its f-vector is (1, 12, 52, 80, 40). ( ) Case 3: lk∆v1 ∼= Σ3. This is not possible by Proposition 6. the electronic journal of combinatorics 27(1) (2020), #P1.10 3 Balanced neighborly (d −1)-manifolds with 3d vertices 3 1 2 3 2 1 3 2 3 1 2 3 2 1 3 2 1 3 1 2 3 2 1 3 2 3 1 2 3 2 1 3 2 Theorem 9. There exists a unique balanced 2-neighborly homology 4-sphere 4155 2 with each color set of size 3. Theorem 9. There exists a unique balanced 2-neighborly homology 4-sphere 4155 2 with each color set of size 3. Theorem 9. There exists a unique balanced 2-neighborly homology 4-sphere 4155 2 with each color set of size 3. Proof. Let ∆be such a sphere and let its color set V5 = {v1, v2, v3}. By Alexander Duality, ˜Hi(∆{4,5}) ∼= ˜H3−i(∆[3]). In particular, since ∆{4,5} is balanced 2-neighborly, β2(∆[3]) = β1(∆{4,5}) = 4 and β1(∆[3]) = 0. Hence f2(∆[3]) = (f1 −f0 + χ)(∆[3]) = 9 · 6 2 −9 + 5 = 23. By double counting, P3 i=1 f1(lk∆vi) = P W={i,j,5}⊆[5] f2(∆W) = 4 2  f2(∆[3]) = 138. By Proposition 6 and Lemma 8, f1(lk∆vi) ∈{42, 46, 48, 52}, it follows that either 138 = 42 + 48 · 2, that is, lk∆v1 ∼= S1 and lk∆v2, lk∆v3 ∼= S3; or 138 = 46 · 3 and lk∆vi ∼= S2 for all i. By double counting, P3 i=1 f1(lk∆vi) = P W={i,j,5}⊆[5] f2(∆W) = 4 2  f2(∆[3]) = 138. By Proposition 6 and Lemma 8, f1(lk∆vi) ∈{42, 46, 48, 52}, it follows that either 138 = 42 + 48 · 2, that is, lk∆v1 ∼= S1 and lk∆v2, lk∆v3 ∼= S3; or 138 = 46 · 3 and lk∆vi ∼= S2 for all i. Consider the first case above. S1 is the connected sum of two octahedral 3-spheres. For any 2-subset W ⊂[4], the induced subcomplex (S1)W is the union of two 4-cycles glued along an edge, so f1((S1)W) = 7. Similarly, S3 is the join of two 6-cycles, so we have (S3)W is either a 6-cycle or the bipartite graph K3,3, i.e., f1((S3)W) = 6 or 9. Hence 23 = f2(∆W∪{5}) = 3 X i=1 f1((lk∆vi)W) ∈{19, 22, 25}, the electronic journal of combinatorics 27(1) (2020), #P1.10 8 a contradiction. Now we consider the second case, where all vertex links in ∆are isomorphic to S2. Let Γ = lk∆v1 ∩lk∆v2 ∩lk∆v3. 3 Balanced neighborly (d −1)-manifolds with 3d vertices The proof of Lemma 8 implies that for any vertex p /∈V5, lkΓ p = lklk∆p v1 ∩lklk∆p v2 ∩lklk∆p v3 is a 5-cycle (as the boundary of the union of three 2-faces, where we apply the cross-flip). Hence Γ must be the boundary of the icosahedron. Since all lk∆vi are isomorphic, by Lemma 4 Γ divides the 3-sphere lk∆v1 into two 3-balls, each having the same number of facets. If the facets of lk∆v1 are labeled as in the link of vertex 1 in 4155 2 (this is a vertex-transitive triangulation of 4-sphere whose vertex links are isomorphic to S2, see [10]), then one such Γ is the intersection of vertices 1, 6, 8 in 4155 2; we rename it to Γ1. In this case, lk∆v1 = B ∪Γ1 B′, where B, B′ are isomorphic 3-balls. We check by sage [21] that all other subcomplexes in lk∆v1 that are isomorphic to Γ1 are of the form σ(Γ1), where σ is an element in the permutation group of lk∆v1 (of order 8). So it suffices to consider just Γ1. To reconstruct lk∆v2 and hence ∆, note that lk∆u2 has the decomposition lk∆u2 = B′ ∪Γ1 B′′ ∼= S2 for some 3-ball B′′, and furthermore B′′ ∼= B′. To decide B′′ it is equivalent to finding a balanced simplicial isomorphism f : B′ →B′′ with B′ ∩B′′ = B ∩B′′ = Γ1 and f(Γ1) = Γ1; in other words, f is a permutation in Aut(Γ1). We check by sage [21] that the links of vertex 6,8 in 4155 2 are the only candidates for lk∆v2. Hence ∆= 4155 2. Indeed ∆is balanced: the color sets are {1, 6, 8}, {2, 4, 9}, {3, 7, 11}, {5, 10, 15} and {12, 13, 14}. Theorem 10. There exists a balanced 3-neighborly non-spherical 5-manifold with each color set of size 3. Theorem 10. There exists a balanced 3-neighborly non-spherical 5-manifold with each color set of size 3. Proof. By Theorem 9, if such 5-manifold exists, then all vertex links are isomorphic to 4155 2, which we denote as Γ. Based on the list of facets of Γ in [15], we take a color- preserving permutation σ = (1, 6, 8)(2, 4, 9)(11, 3, 7)(10, 15, 5)(13, 14, 12). 3. The f-vector of ∆is (1, 18, 135, 540, 1035, 918, 306). 3. The f-vector of ∆is (1, 18, 135, 540, 1035, 918, 306). Furthermore by the Dehn-Sommerville relations, any balanced 3-neighborly 5-manifold having 3 vertex in each color set also has the f-vector (1, 18, 135, 540, 1035, 918, 306). Let Σ be such a complex, {v1, v2, v3} a color set, Mi = lkΣ vj ∩lkΣ vk for {i, j, k} = [3] and N = M1 ∩M2 ∩M3. If F is an interior face of Mi, then F ∪{vi} /∈Σ. Since Σ is balanced 3-neighborly, lkΣ v1 = M2 ∪M3 ∼= M1 ∪M3 = lkΣ v2 and they have 102 facets, it follows that each Mi has no interior vertices or edges and with 51 facets. Also since all vertex links in Σ are isomorphic to 4155 2, the same argument as in the proof of Theorem 9 implies that N is a 3-manifold whose vertex links are isomorphic to the boundary of the icosahedron; indeed 31515 1 is one such example. We haven’t checked if there exist other balanced 3-neighborly 5-manifolds. (It is not known if there exist 15-vertex non- vertex-transitive 3-manifolds whose vertex links are all isomorphic to the boundary of the icosahedron.) 3 Balanced neighborly (d −1)-manifolds with 3d vertices We choose σ in such a way that σ /∈Aut(Γ) and furthermore, Γ ∩σ(Γ), Γ ∩σ2(Γ) and σ(Γ) ∩σ2(Γ) are isomorphic homology manifolds with no interior faces of dimension < 2 and with a common boundary C. By computer we check that Γ∪σ(Γ)∪σ2(Γ) is balanced 3-neighborly and C is the vertex-transitive 3-manifold 31515 1 that triangulates S3/Q as in [15]. Finally let ∆= (Γ ∗{16}) ∪(σ(Γ) ∗{17}) ∪(σ2(Γ) ∗{18}), where {16, 17, 18} are the vertices of color 6. By sage [21] one verifies that all vertex links of ∆is isomorphic to Γ, which is known as a combinatorial 4-sphere (we say a simplicial complex is a combinatorial sphere if it is PL homeomorphic to the boundary of the simplex). Hence ∆is a combinatorial manifold that is balanced 3-neighborly. Remark 11. The following properties of the balanced 3-neighborly 5-manifold found in the proof of Theorem 10 are verified by sage: Remark 11. The following properties of the balanced 3-neighborly 5-manifold found in the proof of Theorem 10 are verified by sage: 1. It is vertex-transitive and has the following generators of the automorphism group (of order 1080): 1. It is vertex-transitive and has the following generators of the automorphism group (of order 1080): (2, 15)(4, 5)(9, 10)(12, 17)(13, 18)(14, 16), (1, 2)(3, 15)(4, 6)(5, 7)(8, 9)(10, 11), (1, 3)(6, 7)(8, 11)(12, 17)(13, 18)(14, 16), (1, 13)(3, 18)(6, 14)(7, 16)(8, 12)(11, 17), (1, 6, 8)(2, 3, 17)(4, 7, 18)(5, 15, 10)(9, 11, 16). 9 9 the electronic journal of combinatorics 27(1) (2020), #P1.10 2. The homology groups of ∆are given by (Z, 0, Z2, 0, 0, Z). 4 Balanced 2-neighborly 3-sphere with 16 vertices In this section we provide a balanced 2-neighborly triangulation of the 3-sphere. The construction is motivated by Lemma 4. w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 Figure 2: Discs A, B and C (from left to right) w3 u3 v3 w1 u2 v1 w2 v4 u4 w4 v2 w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 Figure 2: Discs A, B and C (from left to right) w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 v3 u2 v1 u3 u3 u1 w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 v3 Figure 2: Discs A, B and C (from left to right) 10 10 the electronic journal of combinatorics 27(1) (2020), #P1.10 w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 w3 u3 v3 w1 u2 v2 u1 w2 v4 u4 w4 v1 Figure 3: Left: disc D′. Right: disc D obtained after rearranging the boundary of D′. w3 u3 v3 w1 u2 v1 u1 w2 v4 u4 w4 v2 w3 u3 v3 w1 u2 v2 u1 w2 v4 u4 w4 v1 u1 gure 3: Left: disc D′. Right: disc D obtained after rearranging the boundary of D′ w3 u3 v3 w1 u2 v2 u1 w2 v4 u4 w4 v1 w3 u3 v3 w1 u2 v2 u1 w2 v4 u4 w4 v1 Figure 4: Left: disc A′. Right: disc B′. Notice that ∂A′ = ∂B′ = ∂D. w3 u3 v3 w1 u2 v2 u1 w2 v4 u4 w4 v1 w3 u3 v3 w1 u2 v2 u1 w2 v4 u4 w4 v1 w4 v4 Figure 4: Left: disc A′. Right: disc B′. Notice that ∂A′ = ∂B′ = ∂D. Construction 12. Assume V1 = {u1, u2, u3, u4}, V2 = {v1, v2, v3, v4}, V3 = {w1, w2, w3, w4} and V4 = {z1, z2, z3, z4} are the four color sets of a balanced 3-sphere Γ. 4 Balanced 2-neighborly 3-sphere with 16 vertices We let lkΓ z1 = A ∪∂A∼∂C C and lkΓ z3 = B ∪∂B∼∂C C, where A, B and C are triangulated 2-balls sharing the same boundary as shown in Figure 2. All possible edges that do not appear in A, B and C are shown in Figure 3 as solid red edges in disc D′. Notice that the dashed edges in D′ are edges in discs A and B, so we may rearrange the boundary of D by switching the positions of vertices v1 and v2, and then replacing the edges containing v1 or v2 in ∂D′ by the dashed edges. In this way, we obtain a triangulation of a 12-gon D as shown in Figure 3. Furthermore, ∂D ⊆A ∪B, and ∂D divides the sphere = A ∪∂A∼∂B B into two discs A′ and B′ as shown in Figure 4. Construction 12. Assume V1 = {u1, u2, u3, u4}, V2 = {v1, v2, v3, v4}, V3 = {w1, w2, w3, w4} and V4 = {z1, z2, z3, z4} are the four color sets of a balanced 3-sphere Γ. We let lkΓ z1 = A ∪∂A∼∂C C and lkΓ z3 = B ∪∂B∼∂C C, where A, B and C are triangulated 2-balls sharing the same boundary as shown in Figure 2. All possible edges that do not appear in A, B and C are shown in Figure 3 as solid red edges in disc D′. Notice that the dashed edges in D′ are edges in discs A and B, so we may rearrange the boundary of D by switching the positions of vertices v1 and v2, and then replacing the edges containing v1 or v2 in ∂D′ by the dashed edges. In this way, we obtain a triangulation of a 12-gon D as shown in Figure 3. Furthermore, ∂D ⊆A ∪B, and ∂D divides the sphere = A ∪∂A∼∂B B into two discs A′ and B′ as shown in Figure 4. We let lkΓ z2 = A′ ∪∂A′∼∂D D and lkΓ z4 = B′ ∪∂B′∼∂D D. Since both stΓ z1 ∩stΓ z3 = C and stΓ z2 ∩(stΓ z1 ∪stΓ z3) = A′ are simplicial 2-balls, it follows that Σ = ∪3 i=1 stΓ zi is a simplicial 3-ball. Furthermore, the boundary of Σ is exactly lkΓ z4. Hence Γ = Σ ∪stΓ z4 is indeed a balanced 2-neighborly 3-sphere. Remark 13. 2. The automorphism group of Γ has two generators (The second generator is given by switching vertices of color 1 and 3, and color 2 and 4, but with the same subscript.) Hence Aut(Γ) has 8 elements. (The second generator is given by switching vertices of color 1 and 3, and color 2 and 4, but with the same subscript.) Hence Aut(Γ) has 8 elements. 3. The complex Γ given in Construction 12 is shellable. For lkΓ z1 = A∪∂A∼∂C C, there exist two shellings c1, . . . , c10, a1, . . . , a10 and a′ 1, . . . , a′ 10, c′ 1, . . . , c′ 10 such that for any 1 ⩽i ⩽10, ci, c′ i are facets from C and ai, a′ i are facets from A. Similarly, there exist two shellings c1, . . . , c10, b1, . . . , b10 and b′ 1, . . . , b′ 10, c′ 1, . . . , c′ 10 for lkΓ z3 = B∪∂B∼∂CC, where bi, b′ i are facets from B. Then 3. The complex Γ given in Construction 12 is shellable. For lkΓ z1 = A∪∂A∼∂C C, there exist two shellings c1, . . . , c10, a1, . . . , a10 and a′ 1, . . . , a′ 10, c′ 1, . . . , c′ 10 such that for any 1 ⩽i ⩽10, ci, c′ i are facets from C and ai, a′ i are facets from A. Similarly, there exist two shellings c1, . . . , c10, b1, . . . , b10 and b′ 1, . . . , b′ 10, c′ 1, . . . , c′ 10 for lkΓ z3 = B∪∂B∼∂CC, where bi, b′ i are facets from B. Then a′ 1 ∗z1, . . . , a′ 10 ∗z1, c′ 1 ∗z1, . . . , c′ 10 ∗z1, c1 ∗z3, . . . , c10 ∗z3, b1 ∗z3, . . . , b10 ∗z3 gives a shelling of stΓ z1∪stΓ z3. We may extend this shelling into a complete shelling of Γ by constructing two similar shellings of lkΓ z2 and lkΓ z4. However, we tried some computer tests and failed to prove either polytopality or non-polytopality. Remark 14. 2. The automorphism group of Γ has two generators It is easy to see that if ∆1 is a balanced 2-neighborly (d1 −1)-sphere and ∆2 is a balanced 2-neighborly (d2 −1)-sphere, then ∆1 ∗∆2 is a balanced 2-neighborly (d1+d2−1)-sphere. Hence by taking joins, we find balanced 2-neighborly (4k−1)-spheres with 16k vertices for any k ⩾1. Question 15. Let d ⩾4 and m ⩾5 be arbitrary integers. Is there a balanced 2-neighborly simplicial (d−1)-sphere all of whose color sets have the same size m? Is there a polytopal sphere with these properties? the electronic journal of combinatorics 27(1) (2020), #P1.10 4 Balanced 2-neighborly 3-sphere with 16 vertices Here we provide some properties of Γ in Construction 12. 1. (A ∪B, C, D) is an ear decomposition of Γ[3]. 1. (A ∪B, C, D) is an ear decomposition of Γ[3]. 1. (A ∪B, C, D) is an ear decomposition of Γ[3]. 11 the electronic journal of combinatorics 27(1) (2020), #P1.10 5 Balanced 2-neighborly L(3, 1) with 16 vertices In this section we present our first construction of a balanced 2-neighborly lens space L(3, 1) with 16 vertices. We denote it by ∆. Each color set of ∆has four vertices. Construction 16. Denote the color sets of ∆by V1 = {u1, u2, u3, u4}, V2 = {v1, v2, v3, v4}, V3 = {w1, w2, w3, w4} and V4 = {z1, z2, z3, z4}. In Figure 5 we illustrate the construction of the vertex links lk∆zi for i = 1, . . . , 4. All these links are realized as cylinders. Two links lk∆z1 and lk∆z2 share the same top and bottom, which are triangulated hexagons spanned by vertices {ui, vi, wi : i = 1, 3} and {ui, vi, wi : i = 2, 4}, respectively. To construct lk∆z3 from lk∆z1, we switch the positions of vertices u3, v3, w3 with vertices u4, v4, w4 respectively and form a new cylinder. The new top and bottom hexagons contain the 2-faces {u1, v1, w1} and {u2, v2, w2}. Similarly, we construct the link lk∆z4 from lk∆z2 by switching the positions of vertices u3, v3, w3 with vertices u4, v4, w4 and letting {u1, v1, w1} and {u2, v2, w2} be the 2-faces that appear in the triangulation of the top and bottom hexagons. It follows that lk∆z3 and lk∆z4 also share the same top and bottom. 12 the electronic journal of combinatorics 27(1) (2020), #P1.10 u4 v2 w4 u2 v4 w2 w1 u3 v1 w3 u1 v3 (a) lk∆z1 u2 v4 w2 u4 v2 w4 w1 u3 v1 w3 u1 v3 (b) lk∆z2 u2 v3 w2 u3 v2 w3 w1 u4 v1 w4 u1 v4 (c) lk∆z3 u3 v2 w3 u2 v3 w2 w1 u4 v1 w4 u1 v4 (d) lk∆z4 Figure 5: Four vertex links of ∆ u4 v2 w4 u2 v4 w2 w1 u3 v1 w3 u1 v3 (a) lk∆z1 u2 v4 w2 u4 v2 w4 w1 u3 v1 w3 u1 v3 (b) lk∆z2 (b) lk∆z2 u2 v3 w2 u3 v2 w3 w1 u4 v1 w4 u1 v4 (c) lk∆z3 u3 v2 w3 u2 v3 w2 w1 u4 v1 w4 u1 v4 (d) lk∆z4 (d) lk∆z4 Figure 5: Four vertex links of ∆ Now since ∆is balanced 2-neighborly, by our construction, it only remains to show that ∆triangulates the lens space L(3, 1). position 18. The complex ∆is a balanced vertex minimal triangulation of L(3, 1) Proof. By Proposition 6.1 in [11], each color set of ∆is of size at least 3. If there are exactly three vertices v1, v2, v3 of color 1 in ∆, apply the Mayer-Vietoris sequence on the triple (st∆v1 ∪st∆v2, st∆v3, ∆) and we obtain that 0 = H1(lk∆v3) →H1(st∆v1 ∪st∆v2) ⊕H1(st∆v3) →H1(∆) →H0(lk∆v3) = 0. Hence H1(st∆v1 ∪st∆v2) ∼= H1(∆) = Z/3Z. However, this is impossible since H1(st∆v1 ∪ st∆v2) ∼= H0(st∆v1 ∩st∆v2), which cannot be Z/3Z. Hence H1(st∆v1 ∪st∆v2) ∼= H1(∆) = Z/3Z. However, this is impossible since H1(st∆v1 ∪ st∆v2) ∼= H0(st∆v1 ∩st∆v2), which cannot be Z/3Z. The same argument as above also shows that the balanced triangulation of any lens space L(p, q) with p > 1 must have at least 16 vertices. 5 Balanced 2-neighborly L(3, 1) with 16 vertices The geometric realizations of st∆z1 and st∆z2 are filled cylinders that share top and bottom. So their union A := st∆z1 ∪st∆z2 is a filled torus (that is, a genus-1 handlebody); so is the union B := st∆z3 ∪st∆z4. Note that these two handlebodies have identical boundary complexes, thus they provide a Heegaard splitting of a lens space. To identify which lens space ∆triangulates, we need to determine the homeomorphism φ : ∂A →∂B. Consider two generators γ, δ of π1(A ∩B) = π1(∂A), where γ is the 6- cycle (u3, v1, w3, u1, v3, w1) and δ is the 4-cycle (u1, w2, u4, w3). In particular, δ is also a generator of π1(A). From the construction we see that φ(γ) is a loop running around the equator of ∂B thrice and the meridian of ∂B once. Also φ(δ) runs around the equator of ∂B twice and the meridian of ∂B once. Hence it is indeed the lens space L(3, 1). Remark 17. Our construction ∆has the following properties: 1. All vertex links are combinatorially equivalent. 2. From Figure 5 we see lk∆zi ∩lk∆zj has two connected components when {i, j} = {1, 2} or {3, 4} (they are the top and bottom hexagons as shown in Figure 2); and it 2. From Figure 5 we see lk∆zi ∩lk∆zj has two connected components when {i, j} = {1, 2} or {3, 4} (they are the top and bottom hexagons as shown in Figure 2); and it 13 the electronic journal of combinatorics 27(1) (2020), #P1.10 has three connected components when i ∈{1, 2} and j ∈{3, 4} (each component is the union of two facets along the side of the cylinders). In general, the intersection of two vertex links, where the vertices are of the same color, always has at least two connected components. 3. There are three group actions on the vertices of ∆: 3. There are three group actions on the vertices of ∆: (a) Fix the subscript and rotate the corresponding vertices of color 1, 2 and 3 respectively. The generator is given by (u1v1w1)(u2v2w2)(u3v3w3). (a) Fix the subscript and rotate the corresponding vertices of color 1, 2 and 3 respectively. The generator is given by (u1v1w1)(u2v2w2)(u3v3w3). (b) Rotate vertices of the same color. The generator is The automorphism group of ∆is of size 96. The automorphism group of ∆is of size 96. Proposition 18. The complex ∆is a balanced vertex minimal triangulation of L(3, 1). (u1u3u2u4)(v1v3v2v4)(w1w3w2w4)(z1z3z2z4). (c) Exchange lk∆z1 and lk∆z2, lk∆z3 and lk∆z4, by exchanging vi and wi (or ui and wi, ui and vi) for all i ∈[4]. The generators are (c) Exchange lk∆z1 and lk∆z2, lk∆z3 and lk∆z4, by exchanging vi and wi (or ui and wi, ui and vi) for all i ∈[4]. The generators are (z1z2)(z3z4)(v1w1)(v2w2)(v3w3)(v4w4), (z1z2)(z3z4)(u1w1)(u2w2)(u3w3)(u4w4) and (z1z2)(z3z4)(u1v1)(u2v2)(u3v3)(u4v4). (z1z2)(z3z4)(v1w1)(v2w2)(v3w3)(v4w4), (z1z2)(z3z4)(u1w1)(u2w2)(u3w3)(u4w4) and (z1z2)(z3z4)(u1v1)(u2v2)(u3v3)(u4v4). The automorphism group of ∆is of size 96. 6 Balanced spheres and ear decomposition In this section our goal is to construct a balanced 3-sphere whose rank-selected sub- complexes do not have ear decompositions. The motivation is from the balanced 2- neighborly construction of L(3, 1) in Section 5. Indeed, we want to construct a balanced 3-dimensional complex ∆so that 1) each vertex link is a 2-sphere; 2) for a fixed color set V4 = {v1, · · · , vk}, the intersection of any two vertex links lk∆vi ∩lk∆vj always has at least two connected components (as the property listed in Remark 17); and 3) ∪4 i=1 st∆vi is 3-ball, which together with the condition 1) guarantees that ∆is a 3-sphere. 14 the electronic journal of combinatorics 27(1) (2020), #P1.10 In the following we take k = 5 and give such a construction. Figure 6 illustrates the links lk∆v1, · · · , lk∆v4. Every label represents the color of the vertex. Also each connected component of lk∆v1 ∩lk∆v2 is colored in green, lk∆vi ∩lk∆v3 is colored in blue for i = 1, 2, and lk∆vj ∩lk∆v4 is colored in pink for j = 1, 2, 3. Immediately we check that all these intersections of vertex links have 2 or 3 connected components. 3 2 3 2 3 2 1 1 1 1 2 1 3 1 2 3 1 3 2 3 2 3 2 1 1 1 1 2 1 2 2 3 2 3 3 1 (a) lk∆v1 and lk∆v2 3 3 1 3 1 3 1 3 2 1 2 1 2 2 1 2 1 2 1 3 1 3 1 (b) lk∆v3 2 3 3 2 1 2 1 2 1 3 1 3 3 1 1 (c) lk∆v4 Figure 6: Four vertex links as triangulated 2-spheres. For simplicity’s sake, we omit some diagonal edges in the quadrilaterals in (b), and some labels of vertices in (c). 6 Balanced spheres and ear decomposition 2 3 3 2 1 2 1 2 1 3 1 3 3 1 1 (c) lk∆v4 3 3 1 3 1 3 1 3 2 1 2 1 2 2 1 2 1 2 1 3 1 3 1 (b) lk∆v3 3 2 3 2 3 2 1 1 1 1 2 1 3 1 2 3 1 3 2 3 2 3 2 1 1 1 1 2 1 2 2 3 2 3 3 1 (a) lk∆v1 and lk∆v2 (a) lk∆v1 and lk∆v2 (b) lk∆v3 (c) lk∆v4 Figure 6: Four vertex links as triangulated 2-spheres. For simplicity’s sake, we omit some diagonal edges in the quadrilaterals in (b), and some labels of vertices in (c). (a) lk∆v1 ∪lk∆v2 (b) ∪3 i=1 lk∆vi (c) ∪4 i=1 lk∆v4 Figure 7: how the links are glued together. (b) ∪3 i=1 lk∆vi (c) ∪4 i=1 lk∆v4 (c) ∪4 i=1 lk∆v4 (b) ∪3 i=1 lk∆vi (a) lk∆v1 ∪lk∆v2 Figure 7: how the links are glued together. Figure 7 shows how ∆\V4 is formed from these links. First we glue lk∆v1 and lk∆v2 along two green triangles. The resulting complex lk∆v1 ∪lk∆v2 is shown in Figure 7a. Then we place lk∆v3 on top of lk∆v1 ∪lk∆v2. As we see from Figure 7b, the boundary complex of ∪3 i=1 st∆vi is a triangulated torus. Finally, we place lk∆v4 on top of ∪3 i=1 lk∆vi so that st∆v4 “covers the 1-dimensional hole” in ∪3 i=1 st∆vi, see Figure 7c. We denote the subspace of R3 enclosed by lk∆vi as Si for 1 ⩽i ⩽4, and let S5 := ∪i⩽4Si. From our construction it follows that the boundary complex of S5 is a 2-sphere; we let it be lk∆v5. 15 the electronic journal of combinatorics 27(1) (2020), #P1.10 Indeed ∆is a 3-sphere since ∆is the union of two 3-balls S5 and st∆v5 glued along their common boundary lk∆v5. Since each lk∆vi ∩lk∆vj has at least two connected components for 1 ⩽i ̸= j ⩽4, the Mayer-Vietoris sequence implies that Si ∪Sj is not contractible for all 1 ⩽i ̸= j ⩽4. A similar inspection of lk∆vi ∪lk∆vj ∪lk∆vk also implies that the boundary complexes of Si ∪Sj ∪Sk’s cannot be triangulated 2-spheres for distinct 1 ⩽i, j, k ⩽4. Proposition 19. Not all rank-selected subcomplexes of balanced simplicial spheres have ear decompositions. Proof. Consider the complex ∆constructed above. 6 Balanced spheres and ear decomposition We denote the union of interior faces of a complex τ by int τ. Suppose ∆\V4 has an ear decomposition (Γ1, Γ2, · · · , Γk). Since |V4| = 5 and β2(∆\V4) = 4, k must be 4. Notice first that ∪i⩽4 lk∆vi divides R3 into five subspaces, namely, S1, · · · , S4 and the complement of S5, each having lk∆vi as the boundary complex for 1 ⩽i ⩽5 respectively. The complex Γ1 is the union of 2-balls B1, B2 with ∂B1 = ∂B2 = Γ1 ∩Γ2. By the Jordan theorem, B1 ∪Γ2 is a triangulated 2- sphere that separates R3 into two connected components. Hence the bounded component must be either Si ∪Sj or Si ∪Sj ∪Sk for some 1 ⩽i, j, k ⩽4. (We may assume that it is not Si, since otherwise we may consider the 2-sphere ∪i⩽3Γi −∪1⩽i̸=j⩽3 int(Γi ∩Γj) instead of Γ1 ∪Γ2 −int(Γ1 ∩Γ2), where the subset enclosed by this sphere in R3 cannot be Si anymore.) This contradicts the fact that the boundaries of Si ∪Sj or Si ∪Sj ∪Sk are not 2-spheres. Remark 20. One can think of all the figures illustrated above as projections of a subcom- plex of ∆−st∆v5 onto R3. However, we do not know whether the complex provided in this section can be realized as the boundary of a 4-polytope. Acknowledgements The author was partially supported by a graduate fellowship from NSF grant DMS- 1361423. I thank Moritz Firsching for pointing out the automorphism groups of the constructions in Section 3 and 4 and running some computational tests to decide whether the constructions are polytopal. Many thanks to Lorenzo Venturello and the anonymous referees for pointing out mistakes in an earlier version and contributing to a few remarks, improvement of the proofs in this paper. [2] M. K. Chari. Two decompositions in topological combinatorics with applications to matroid complexes. Trans. Amer. Math. Soc., 349:3925–3943, 1997. [3] D. L. Donoho and J. Tanner. Counting the faces of randomly-projected hypercubes and orthants, with applications. Discrete Comput. Geom., 43:522–541, 2010. [1] G. Burton. The non-neighbourliness of centrally symmetric convex polytopes having many vertices. J. Combin. Theory Ser. A, 58:321–322, 1991. [1] G. Burton. The non-neighbourliness of centrally symmetric convex polytopes having many vertices. J. Combin. Theory Ser. A, 58:321–322, 1991. [2] M. K. Chari. 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Hereditary Angioedema: The Economics of Treatment of an Orphan Disease
Frontiers in medicine
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Citation: Lumry WR (2018) Hereditary Angioedema: The Economics of Treatment of an Orphan Disease. Front. Med. 5:22. doi: 10.3389/fmed.2018.00022 Keywords: health economics, orphan disease, hereditary angioedema, burden, treatment Hereditary angioedema (HAE) is a rare autosomal-dominant genetic disease (1, 2). It occurs in approximately 1:30,000–80,000 individuals and affects less than 8,000 individuals in the United States (USA), 15,000 in Europe, and 200,000 worldwide (3, 4) Disease-specific therapy for HAE, although available in other countries since 1979 (5), only recently became available in the USA. With the Food and Drug Administration (FDA) approval of a human nano-filtered human plasma- derived C1 inhibitor administered intravenously for routine prevention of HAE attacks on October 10, 2008, a new era in therapy for HAE began (6). Since 2008, four therapies to treat HAE attacks have received FDA approval, including nano-filtered pasteurized human plasma-derived C1 inhibitor and ecallantide in 2009, icatibant in 2011, and recombinant C1 inhibitor in 2014 (7–11). These therapies are currently registered in many countries around the world. A concentrated human plasma-derived C1 inhibitor administered subcutaneously for routine prevention of HAE attacks was approved on June 22, 2017 by the FDA and other preventive therapies are currently in development (12).hi Reviewed by: Reviewed by: Coen Maas, University Medical Center Utrecht, Netherlands Allen P. Kaplan, Medical University of South Carolina, United States y y This burst of drug development and approvals has greatly benefited individuals with HAE, their families, caregivers, physicians, and healthcare providers (13, 14). The burden of HAE has been reduced, quality of life improved and utilization of urgent care, emergency facilities, and hospitals decreased significantly (14, 15). Concerns raised by healthcare payers in the USA and healthcare sys- tems in other countries about the financial impact of these newly approved therapies on healthcare payment systems have led to barriers to and limitation of access to these life changing and potentially life saving therapies (16–20).hh *Correspondence: William Raymond Lumry lumrymd@allergyspecialists.us Specialty section: This article was submitted to Hematology, a section of the journal Frontiers in Medicine Specialty section: This article was submitted to Hematology, a section of the journal Frontiers in Medicine Received: 15 September 2017 Accepted: 23 January 2018 Published: 16 February 2018 This review will discuss the cost burden of HAE on patients, healthcare systems, and society. The impact of availability of and access to novel and specific therapies on morbidity, mortality, and the overall burden of disease will be explored along with potential changes in treatment paradigms to improve effectiveness and reduce cost of treatment. The prevalence of orphan diseases, legislative incentives to encourage development of orphan disease therapies, and the impact of orphan disease treatment on healthcare payment systems will be discussed. Received: 15 September 2017 Accepted: 23 January 2018 Published: 16 February 2018 Review Review published: 16 February 2018 doi: 10.3389/fmed.2018.00022 Hereditary Angioedema: The Economics of Treatment of an Orphan Disease William Raymond Lumry* This review will discuss the cost burden of hereditary angioedema on patients, health- care systems, and society. The impact of availability of and access to novel and specific therapies on morbidity, mortality, and the overall burden of disease will be explored along with potential changes in treatment paradigms to improve effectiveness and reduce cost of treatment. The prevalence of orphan diseases, legislative incentives to encourage development of orphan disease therapies and the impact of orphan disease treatment on healthcare payment systems will be discussed. Keywords: health economics, orphan disease, hereditary angioedema, burden, treatment Edited by: Edited by: Alvin H. Schmaier, Case Western Reserve University, United States BURDEN OF HAE In addition to the direct cost of medical care, indirect costs must be considered. Missed time from work or school, decreased productivity at work and loss of opportunity are significant costs to the patient and society. Patients report a decrease of productivity on the job due to HAE (26). A national audit in the United Kingdom of patients with HAE and acquired angioedema revealed patients lost on average 9 days of work per year rang- ing from 0 to 43 days (29). A European Union (EU) study of the socioeconomic burden of HAE revealed both patients and caregivers were affected with each losing an average of 20 days from work or school per year (24). In addition to the expense of specific HAE therapies, other costs of caring for an individual with HAE are high. These costs include the direct cost of providing medical care to these patients and the indirect costs of the disease on patients, their families, and on society. Patients with HAE consume more health care and are more costly to the health care system. In a United Kingdom study, the cost of care for an HAE patient was 160% more for primary care and 447% more for secondary care than a patient without HAE even if the costs of specialist care and medications were excluded (23). During swelling attacks, patients with HAE and their caregivers miss a significant amount of time from work and school, and when “well” are often less productive at work. Many report being unable to achieve their educational and career goals, or even maintain employment as a result of their disease (24, 25). Loss of educational and career opportunity is commonly reported. In the HAEA survey conducted in 2007, 57% of HAE patients reported having career advancement hindered, 69% felt that they could not consider certain types of jobs because of their disease, 100% felt that educational advancement had been hindered, 55% had to limit their educational choices, and 48% had not achieved the level of education that they desired (25). Even after disease-specific therapies became available, decreased opportunity remains a problem. IMPACT OF NEW THERAPIES FOR HAE Patients with HAE have benefited from approval of novel, disease-specific drugs that treat and prevent swelling attacks. The benefits include improvement in health and quality of life (QoL), increased February 2018  |  Volume 5  |  Article 22 Frontiers in Medicine  |  www.frontiersin.org 1 HAE Treatment Economics Lumry Lumry ability to work and pursue educational and career goals, reduced disability and reduction of costly urgent care visits, and hospitali- zations and longer survival (13, 14, 15).h ability to work and pursue educational and career goals, reduced disability and reduction of costly urgent care visits, and hospitali- zations and longer survival (13, 14, 15).h cost of having and treating HAE in the USA was also assessed in this survey (26). The average annual cost per patient was $44,597. Cost varied depending on the severity of the disease ranging from $11,587 for mildly affected patients to $28,764 and $104,857, respectively, for those moderately or severely affected. The total mean annual expenditure per HAE patient of $44,597 included direct medical costs of $29,177 comprised of $17,381 for hospital admissions, $2,827 for emergency department (ED) visits, $3777 for outpatient care, and $5,194 for medications and indirect costs of $15,420, including $5,157 for reduced productivity, $6,417 for reduced income, $3,402 for missed work, and $4,444 for travel and childcare (26). Accounting for inflation, the average annual cost per patient in 2017 would be more than $65,000. The economic cost of these therapies is high. In 2012, the annual cost of nano-filtered C1 inhibitor (Cinryze®, Shire, Lexington, MA, USA) indicated for routine prevention of HAE attacks, when used at approved dosage and interval, was $487,000 USD per patient, the most expensive drug for any orphan disease treated in the USA (21). In 2015, sales of Cinryze® generated the second highest revenue per patient treated to a pharmaceutical company, $210,000 USD, of all orphan drugs (22). Current USA average wholesale prices (AWP) for treatments of swelling attacks range from $5,000 to more than $12,000 per attack treated. The cost of these therapies is generally less outside of the USA with some national health systems able to acquire them at a significant discount to USA prices. Emergency department usage and hospitalization before 2008 was high as only supportive treatment, including intravenous fluids, anti-emetics, analgesics, and airway support was available. BURDEN OF HAE In an EU socioeconomic burden of disease report published in 2014 after current therapies became available, 42% patients reported their educational advancement was hindered, 40% were prevented from applying for certain jobs and 36% felt that their career advancement was diminished, 9% switched positions within their company, and 10% left their position permanently because of this disabling disease (24). HAE patients also suffer from anxiety and depression at much higher rates than the normal population. Results from two independ- ent studies suggest that between 14 and 43% of patients of HAE IMPACT OF NEW THERAPIES FOR HAE In 2007, there were 2,705 ED visits in the USA with HAE as the primary diagnosis with 40.9% resulting in hospitalization. At a mean cost of $1,479 per ED visit $3,727,080 was spent for urgent care (27). Between 2004 and 2007, there were 10,125 hospitaliza- tions where HAE was the primary or secondary diagnosis. The average length of stay was 5.1 days and average cost per hospi- talization was $8383 resulting in $21,220,000 annual expenditure for hospital services (28). Although not commonly seen in the orphan disease drug market the increasing number of approved therapies for HAE may result in competition between pharmaceutical companies decreasing the cost of these therapies. The first example of this occurred recently with the pricing of Haegarda®, a subcutane- ous C1 inhibitor concentrate (C1INH) for routine prevention of HAE attacks approved in June, 2017. Despite approximately four times the amount of C1 inhibitor being required to treat a patient with Haegarda® compared to Cinryze®, the AWP per treatment with Haegarda® is 15% less. As other drugs are developed and approved for this indication hopefully competitive pricing will continue to push costs down.i INNOVATIVE TREATMENT PARADIGMS Although disease-specific treatments for HAE are costly, appro- priate and timely treatment decreases ED visits, hospitalizations, lost time from school and work, and prevents death lowering the overall cost of the disease to the healthcare system and to soci- ety. ED visits and hospitalization for supportive treatment were the norm before availability of home and self-administration of treatment (25). Self-administration for treatment and preven- tion of attacks is safe, effective and encouraged. Patients can accurately recognize and safely self-treat HAE attacks leading to earlier treatment, earlier resolution of symptoms, decreased ED and hospital visits, and improved quality of life and cost savings (33–39). An example of this problem is found in Argentina. Members of the Argentine HAE Patient Association completed question- naires about the availability and their access to HAE treatments in 2009, 2013, and 2016. Despite C1INH being registered and approved for treatment of HAE attacks before 2009, C1INH was available to only 26% of those responding to the 2009 survey. This increased to 55% by 2016. Despite being approved only 10% had access to icatibant in 2016. Reimbursement for these medications in 2016 was also a challenge with only 64% reimbursed at 100% with 19% having no reimbursement available at all. The majority of the patients received treatment from health personnel or at the hospital and over 50% reported not receiving treatment until their attack was severely painful. Reordering and resupply of medica- tion was difficult for 66% of patients with only 20 per reporting a fast replacement and 53% being able to obtain replacement within 10 days (44). Innovative treatment paradigms may lower the cost and the burden of disease further. Italian HAE patients who self- administered C1INH decreased their mean annual number of hospitalizations from 16.8 to 2.1, time to administration of treat- ment from 3.2 to 1.9 h, time to beginning of symptom improve- ment from 84 to 54 min, time to complete symptom resolution from 12.8 to 10.8 h and number of missed days of work or school from 23.3 to 7.1 compared to hospital or ED administered therapy. The total cost of therapy, including both direct and indirect cost, was approximately 30,010 Euros ($36,000 USD) per patient when the therapy was administered in a hospital or clinic setting and 26,621 Euros ($32,000 USD) when treatment was administered at home. COST OF HAE CARE BEFORE SPECIFIC THERAPIES WERE AVALIABLE IN THE USA As disease-specific therapies for HAE were not available in the USA before 2008, there is an opportunity to review costs and impact of the disease before current therapies became available. An internet survey completed by members of the USA HAE Association (HAEA) in 2007 found patients experienced an aver- age of 26.9 swelling attacks per year lasting 61.3 h each. Over 80% were considered moderate to severe in intensity (25). Total annual February 2018  |  Volume 5  |  Article 22 Frontiers in Medicine  |  www.frontiersin.org 2 HAE Treatment Economics Lumry experience clinically significant depression further adding to their cost of treatment and disability (25, 30). with 84% reduction in ED visits in this group. In the self-treated patients, there was no need for tracheotomy, no deaths reported and an improved QoL in all physical and psychological domains. Despite a 300% increase in use of newer “high cost” treatments, the cost to treat an average of 36 attacks per patient per year was a manageable at $16,766 (35). A fatal HAE attack with loss of an individual both from the work force and from society is the ultimate burden of this disease and results in significant hardship both socially and economically to the patient’s family and society. Unfortunately, patients with HAE continue to succumb to this treatable disease. In a review of all USA death certificates from 1999 to 2010, HAE was considered a contributing factor or the underlying cause of death in 600 people with 270 of these deaths directed attributed to HAE (31). In a German study which included 728 patients from 182 families with HAE, 214 deaths were recorded. The mean age of death from an HAE attack was 40.6 years (32). Unfortunately in many countries, implementation of a home or self-administration policy is not possible. In Japan, Greece and most of Eastern Europe acute therapies are only available at the hospital or in specialty clinics, if available at all. In Brazil and Mexico, home therapy is available but is not reimbursed.i Specific treatments for HAE attacks are available in many countries as shown in Table  1. Treatments to prevent attacks are less widely available. What is not obvious from this table is although these treatments may be registered and approved for marketing in a particular country, they may not be accessible to the patients that need them. INNOVATIVE TREATMENT PARADIGMS This represented savings of 11% or 57,619 Euros ($69,000 USD) for the 17 patients reported in the study (40). Despite the advances in the treatment for HAE with availabil- ity of new effective on demand and attack preventing treatments, these medications are not approved in many countries around the world. Even if approved, they are often not accessible to the patients who need them. Despite improvement of treatment out- comes and cost savings, when the treatment is self-administered or given in the home setting, in many countries treatment of attacks is only available in hospital and clinic settings. Spanish investigators estimated patients who self-treated their HAE attacks with icatibant compared to health care set- ting administration would save an average of 121 Euros ($145) per attack in direct and indirect costs, a 9.2% decrease in costs. Reduction in direct costs accounted for 74% of the savings. This would achieve an annual health system savings of 551,371 Euros ($661,600) (41). An USA study reported a $650,000 savings when 249 HAE attacks over 5  months were treated with ecallantide given at home by an infusion nurse compared to the cost of treating these attacks in the ED or hospital (42). In the United Kingdom, home administration of icatibant compared to hospital administration of C1INH saved $861–$1167/attack (43). COST OF HAE CARE BEFORE SPECIFIC THERAPIES WERE AVALIABLE IN THE USA Barriers to access include require- ment for health care system or judicial approval, availability only in specialized treatment centers or hospitals, limits on reimburse- ment, and limits on number of treatments allowed or resupply of medication to providers or patients. Frontiers in Medicine  |  www.frontiersin.org ORPHAN DRUG DEVELOPMENT POLICIES AND REIMBURSEMENT ISSUES Individuals with HAE and other rare and burdensome diseases have benefited greatly from the development of therapies made economically feasible by the orphan drug policies. Orphan drug policies have been established in the USA, the EU and Japan to encourage the development of safe and effective therapies for rare diseases. The USA Orphan Drug Act was enacted in 1983 (45). In Denmark, 80 HAE patients were followed prospectively for 10 years. By 2012, 49% were self-administering C1NH or icatibant February 2018  |  Volume 5  |  Article 22 Frontiers in Medicine  |  www.frontiersin.org 3 SUMMARY Orphan drug policies are in place to encourage development of safe and effective therapies for orphan diseases. With a limited number of patients to treat, the cost per patient of orphan drugs to recover research and development costs is high. Orphan diseases affect approximately 8–10% of the population. Overall expendi- tures for drugs to treat orphan diseases remain proportional to the incidence of these diseases in the population. Expenditures for orphan drugs are currently less than 10% of pharmaceutical expenditures and 1% of the total healthcare costs in the USA. Despite these facts, payers in the USA and healthcare authorities around the world perceive treatment of orphan diseases puts an inordinate burden on their payment systems and have put barri- ers in place to limit use of new disease specific therapies.i These policies have been successful in encouraging companies to develop therapies for a wide variety of rare conditions. In January, 1983 there were 38 drugs approved in the USA for orphan disorders (22). On July 1, 2017, 635 approved drugs were listed in the Orphan Disease Therapeutic Registry (47). and 98 designated orphan drugs were approved for marketing in the EU (48).h There are 25–30 million individuals, 8–10% of the population of the USA, affected by one of the 7,000 diseases designated as orphan, who may benefit from provisions of the Orphan Drug Act (16, 49). Although treatments for these rare diseases have provided great benefits to affected individuals and their families, the high cost of these therapies has led to the perception by pay- ers and some in society that treatment of orphan diseases places an inordinate burden on healthcare payment systems (16, 17). This concern about high cost of these treatments has resulted in barriers to limit access to them, including formulary approval, high coinsurance and copayment rates, prior authorization and multiple reauthorizations, step therapy, and limits on supply and resupply of medication (16, 17, 20). Orphan drugs benefit many people with previously under- served orphan diseases. These drugs offer significant value to patients and society in terms of improvements in health, reduced disability, increased productivity, including the ability to continue working, reduced healthcare utilization, and improved quality of life and survival. Patients with HAE are not an exception and have benefited greatly. Availability of new and novel therapies to treat and prevent swelling attacks has dramatically decreased the burden of this disease. ute V V V V V V V V V V V V C C C Indication Age/groups Route Acute treatment Prophylaxis Home Therapy Children, <12 years old Adolescence, 12–18 years old STP LTP ✓ ✓ – ✓ ✓ ✓ IV ✓ – – ✓ ✓ ✓ IV bia, ✓ ✓ – ✓ ✓ ✓ IV ✓ ✓ – ✓ – ✓ IV ✓ ✓ – ✓ ✓ ✓ IV ✓ ✓ – ✓ – ✓ IV ✓ ✓ – ✓ ✓ ✓ IV ✓ – – – – – IV ✓ – – ✓ – ✓ IV ✓ ✓ ✓ ✓ – ✓ IV – – ✓ ✓ – ✓ IV ✓ ✓ ✓ ✓ – – IV ✓ ✓ ✓ SC ✓ – – ✓ – – SC ✓ – – ✓ – – SC bia) ✓ – – ✓ – – SC ✓ – – ✓ – – SC ✓ – – ✓ – – SC – – – – – – SC ✓ – – ✓* – ✓ SC – ✓ ✓ ✓ – ✓ Oral – – ✓ ✓ – – Oral bia) ✓ – – ✓ – – Oral – ✓ ✓ ✓ ✓ ✓ Oral – – – ✓ – – Oral bia) ✓ – – ✓ – – Oral – – – ✓ – – Oral – – – – – – Oral bia) – – – – – – Oral HAE Treatment Economics Lumry Under this act, a drug is given orphan designation if the disease it treats affects less than 200,000 (approximately 1:1,650) individuals or if there is no reasonable expectation of profitability for the drug. Incentives provided include tax credits for research costs, grants to aid in clinical research, and a 7-year marketing exclusivity for approved orphan drugs.i Under this act, a drug is given orphan designation if the disease it treats affects less than 200,000 (approximately 1:1,650) individuals or if there is no reasonable expectation of profitability for the drug. Incentives provided include tax credits for research costs, grants to aid in clinical research, and a 7-year marketing exclusivity for approved orphan drugs.i them registered and approved. In 2007, $13.3 billion was spent in the USA accounting for 4.3% of $311 billon total expenditures for pharmaceuticals that year. By 2013, this had increased to $25.8 billion, 7.7% of $337 billion pharmaceutical spend. IMS Health Market Prognosis has forecasted USA total drug expenditures of $465.0 billion in 2018 with orphan drugs accounting for $44.10 billion, 9.5% of this amount, representing an increase of 0.7% over four years (52, 53). In 1999, the EU enacted its orphan drug policy that defines an orphan disease as one with 5 or less patients per 100,000 (1:20,000) individuals. Research incentives are available within the EU and its member States and fees are waived for approval of the marketing application and a 10-year marketing exclusivity is provided (46).h Frontiers in Medicine  |  www.frontiersin.org SUMMARY As the number of approved therapies for HAE increases the cost of therapy may decrease in response to competition. The first example of this occurred with the pricing of Haegarda®, a subcutaneous C1INH for routine prevention of HAE attacks approved in June, 2017. Prudent therapeutic choices, utilization of new treatment paradigms, and improvement in availability and accessibility to these new therapies will continue to improve the lives of patients with HAE and other orphan diseases. Coverage by payers and healthcare systems of these life altering and potentially life saving therapies should continue with barriers to access being appropriately addressed.hi Drug research and development are time-consuming and expensive activities. The average cost of developing and win- ning market approval for a new prescription drug was recently estimated to be $2.6 billion with $1.4 billion of this amount spent on research and development (50, 51). Orphan drugs are no exception. With many fewer patients to treat, the cost per patient treated must be higher in order to recover research and development expenditures. For the top 100 drugs in 2016, the annual median drug cost per patient in the USA for treatment of non-orphan conditions was $27,756 compared to $140,443 per patient treated with an orphan disease (49). Although the cost per patient to treat an orphan disease is often high, the perception that cost of treatment of rare diseases as a whole has an inordinate impact on total pharmaceutical expenditures and health care costs is inaccurate. Analyses in the USA and EU have shown this impact is minimal and in line with the 8–10% prevalence of these diseases in the population. In 2014, total expenditures for pharmaceuticals in the USA accounted for only 9.8% of total healthcare expenditures of $3.0 trillion. Expenditures on orphan drugs for orphan indications was approximately $33.5 billion representing less than 10% of pharmaceutical expenditures and only 1% of the total healthcare expenditures (22, 52). The conversation about the high cost for benefit obtained of orphan drugs with payers, society, and the patients needs to change to recognition of the benefits of timely and appropriate care. We need to solve the access problem by working with health systems, patients, and their advocates on the most cost-effective and efficient ways to deliver this care. We need to continue to disseminate knowledge regarding benefits to patients and to society of effective and safe treatments for HAE and all rare diseases. REFERENCES Fatal laryngeal attacks and mortality in heredi- tary angioedema due to C1-INH deficiency. J Allergy Clin Immunol (2012) 130(3):692–7. doi:10.1016/j.jaci.2012.05.055 10. Firazyr Prescribing Information. Shire (2017). Available from: http://labeling. cslbehring.com/PI/US/Berinert/EN/Berinert-Prescribing-Information.pdf 33. Aberer W, Maurer M, Reshef A, Longhurst H, Kivity S, Bygum A, et al. 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Longhurst HJ, Farkas H, Craig T, Aygören-Pürsün E, Bethune C, Bjorkander J, et al. HAE international home therapy consensus document. Allergy Asthma Clin Immunol (2010) 6:22. doi:10.1186/1710-1492-6-22 16. Handfield R, Feldstein J. Insurance companies’ perspectives on the orphan drug pipeline. Am Health Drug Benefits (2013) 6(9):589–98. 38. Levi M, Choi G, Picavet C, Hack CE. Self-administration of C1-inhibitor concentrate in patients with hereditary or acquired angioedema caused by C1-inhibitor deficiency. J Allergy Clin Immunol (2006) 117(4):904–8. doi:10.1016/j.jaci.2006.01.002 i 17. Hyde R, Dobrovolny D. Orphan drug pricing and payer management in the United States: are we approaching the tipping point? Am Health Drug Benefits (2010) 3(1):15–23. 39. Hernández Fernandez de Rojas D, Ibañez E, Longhurst H, Maurer M, Fabien V, Aberer W, et al. REFERENCES quality of life, productivity, and depression. Allergy Asthma Proc (2010) 31(5):407–14. doi:10.2500/aap.2010.31.3394 quality of life, productivity, and depression. Allergy Asthma Proc (2010) 31(5):407–14. doi:10.2500/aap.2010.31.3394 1. Agostoni A, Cicardi M. Hereditary and acquired C1-inhibitor deficiency: biological and clinical characteristics in 235 patients. Medicine (1992) 71:206–15. doi:10.1097/00005792-199207000-00003 1. Agostoni A, Cicardi M. Hereditary and acquired C1-inhibitor deficiency: biological and clinical characteristics in 235 patients. Medicine (1992) 71:206–15. doi:10.1097/00005792-199207000-00003 26. Wilson DA, Bork K, Shea EP, Rentz AM, Blaustein MB, Pullman WE. Economic costs associated with acute attacks and long-term management of hereditary angioedema. Ann Allergy Asthma Immunol (2010) 104(4):314–20. doi:10.1016/j.anai.2010.01.024 2. Frank MM, Gelfand JA, Atkinson JP. Hereditary angioedema: the clin- ical syndrome and its management. Ann Intern Med (1976) 84:580–93. doi:10.7326/0003-4819-84-5-580 27. 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Jolles S, Williams P, Carne E, Mian H, Huissoon A, Wong G, et al. A UK national audit of hereditary and acquired angioedema. Clin Exp Immunol (2014) 175(1):59–67. doi:10.1111/cei.12159 6. Lunn M, Santos C, Craig T. Cinryze™ as the first approved C1 inhibitor in the USA for the treatment of hereditary angioedema: approval, efficacy and safety. J Blood Med (2010) 1:163–70. doi:10.2147/JBM.S9576 30. Fouche AS, Saunders EF, Craig T. Depression and anxiety in patients with hereditary angioedema. Ann Allergy Asthma Immunol (2014) 112(4):371–5. doi:10.1016/j.anai.2013.05.028 7. Cinryze Prescribing Information. Shire (2017). Available from: http://pi.shire- content.com/PI/PDFs/Cinryze_USA_ENG.pdf 31. Kim SJ, Brooks JC, Sheikh J, Kaplan MS, Goldberg BJ. Angioedema deaths in the United States, 1979-2010. Ann Allergy Asthma Immunol (2014) 113(6):630–4. doi:10.1016/j.anai.2014.09.003 8. Berinert Prescribing Information. CSL Behring. Available from: http://labeling. cslbehring.com/PI/US/Berinert/EN/Berinert-Prescribing-Information.pdf 9. Kalbitor Prescribing Information. Dyax Corporation. (2017). Available from: http://www.kalbitor.com/hcp/rems/pdf/KalbitorFullPrescribingInformation.pdf 32. Bork K, Hardt J, Witzke G. AUTHOR CONTRIBUTIONS Orphan drug expenditures have increased over the last decade due to the increasing price of these therapies and the number of WL researched and wrote the manuscript. February 2018  |  Volume 5  |  Article 22 5 HAE Treatment Economics Lumry REFERENCES Treatment of HAE attacks in the icatibant outcome survey: an analysis of icatibant self-administration versus administration by health care professionals. Int Arch Allergy Immunol (2015) 167:21–8. doi:10.1159/000430864 18. Cardarelli W. Managed care implications of hereditary angioedema. Am J Manag Care (2013) 19(7 Suppl):s119–24. i 19. Morrow T. Insurers will find icatibant lifesaving but expensive treatment. managed care once again faces the all-too-familiar debate about cost and benefit. Manag Care (2011) 20(11):63–4. 40. Petraroli A, Squeglia V, Di Paola N, Barbarino A, Bova M, Spanò R, et al. Home therapy with plasma-derived C1 inhibitor: a strategy to improve clinical out- comes and costs in hereditary angioedema. Int Arch Allergy Immunol (2015) 166(4):259–66. doi:10.1159/000381341 i 20. Robinson SW, Brantley K, Liow C, Teagarden JR. An early examination of access to select orphan drugs treating rare diseases in health insurance exchange plans. J Manag Care Spec Pharm (2014) 20(10):997–1004. 21. Tilles SA, Borish L, Cohen JP. Management of hereditary angioedema in 2012: scientific and pharmacoeconomic perspectives. Ann Allergy Asthma Immunol (2013) 110(2):70–4. doi:10.1016/j.anai.2012.11.014 41. Blasco AJ, Lázaro P, Caballero T, Guilarte M. Social costs of icatibant self-administration vs. health professional-administration in the treat- ment of hereditary angioedema in Spain. Health Econ Rev (2013) 3(1):2. doi:10.1186/2191-1991-3-2 22. EvaluatePharma Orphan Drug Report. 3rd ed. (2015). Available from: http:// info.evaluategroup.com/rs/607-YGS-364/images/EPOD15.pdf 42. Specialty pharmacy news. (2012) 9(7):9–11. 23. Helbert M, Holbrook T, Drogon E, Mannan A. Exploring cost and burden of illness of HAE in the UK. Value in Health (2013) 16(7):A381. 43. NHS Commissioning Board Clinical Commissioning Policy: Treatment of Acute Attacks in Hereditary Angioedema. (2016). Available from: https://www. england.nhs.uk/wp-content/uploads/2013/04/b09-p-b.pdf 24. Aygören-Pürsün E, Bygum A, Beusterien K, Hautamaki E, Sisic Z, Wait S, et al. Socioeconomic burden of hereditary angioedema: results from the hereditary angioedema burden of illness study in Europe. Orphanet J Rare Dis (2014) 9:99–108. doi:10.1186/1750-1172-9-99 44. Menendez A, Malbran A; C1INH-HAE. A Web Based Poll on Accessibility to Acute Attack Treatments in Argentina. Poster HAEI Meeting Madrid. (2016). 45. Food and Drug Administration. Orphan Drug Act FDA. MD: Silver Spring (2017). Available from: http://www.fda.gov/RegulatoryInformation/Legislation/ SignificantAmendmentstotheFDCAct/OrphanDrugAct/ 25. Lumry WR, Castaldo AJ, Vernon MK, Blaustein MB, Wilson DA, Horn PT. The humanistic burden of hereditary angioedema: impact on health-related February 2018  |  Volume 5  |  Article 22 Frontiers in Medicine  |  www.frontiersin.org 6 Lumry Lumry HAE Treatment Economics 46. Frontiers in Medicine  |  www.frontiersin.org February 2018  |  Volume 5  |  Article 22 REFERENCES Regulation (EC) No 141/2000 of the European Parliament and of the Council of 16 December 1999 on orphan medicinal products Official Journal L 018, 22/01/2000 P. 0001 – 0005. Available from: http://eur-lex.europa.eu/legal- content/EN/TXT/?uri=CELEX%3A32000R0141 52. Divino V, Dekoven M, Kleinrock M, Wade RL, Kaura S. Orphan drug expendi- tures in the United States: a historical and prospective analysis 2007-18. Health Aff (2016) 35(9):1588–94. doi:10.1377/hlthaff.2016.0030 ff 53. Health IMS. Market Prognosis 2014–2018, USA. London: IMS Health (2014). 30 p. 47. Food and Drug Administration. Orphan Drug Approval List. MD: Silver Spring FDA (2017). Available from: http://www.accessdata.fda.gov/scripts/ opdlisting/oopd/listResult.cfm Conflict of Interest Statement: WL is a consultant for biocryst, pharming, CSL Behring, and shire. He speaks on behalf of CSL Behring, pharming and shire and has received research grants from biocryst, pharming, dyax, lev, jerini, viropharma, CSL Behring, and shire. He is a member of the medical advisory board of the us hereditary angioedema association. 48. Orphanet. Orphanet: List of Orphan Drugs in Europe. (2017). Available from: http://www.orpha.net/orphacom/cahiers/docs/GB/list_of_orphan_drugs_ in_europe.pdf 49. EvaluatePharma Orphan Drug Report. 4th ed. (2017). Available from: http:// info.evaluategroup.com/rs/607-YGS-364/images/EPOD17.pdf Copyright © 2018 Lumry. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 50. DiMasi JA, Grabowski HG, Hansen RW. Innovation in the Pharmaceutical Industry: New Estimates of R&D Costs. Boston: Tufts Center for the Study of Drug Development (2014). Available from: http://csdd.tufts.edu/news/ complete_story/cost_study_press_event_webcast 51. Avorn J. The $2.6 billion pill – methodologic and policy considerations. N Engl J Med (2015) 372:1877–9. doi:10.1056/NEJMp1500848 51. Avorn J. The $2.6 billion pill – methodologic and policy considerations. N Engl J Med (2015) 372:1877–9. doi:10.1056/NEJMp1500848 February 2018  |  Volume 5  |  Article 22 Frontiers in Medicine  |  www.frontiersin.org
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Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids
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Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids Aya Yoshimura1,2, Masaki Kawamata3, Yusuke Yoshioka3, Takeshi Katsuda3, Hisae Kikuchi4, Yoshitaka Nagai4,5, Naoki Adachi2,6, Tadahiro Numakawa2,7, Hiroshi Kunugi2, Takahiro Ochiya3 & Yoshitaka Tamai1 received: 24 March 2016 accepted: 13 July 2016 Published: 19 August 2016 Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum- derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These results suggested that this Tg rat model should provide significant information for understanding the intercellular transfer and/or mother-child transfer of EVs in vivo. Extracellular vesicles (EVs) are small membrane vesicles (40–200 nm in diameter) that are secreted from many different cell types in the body. EVs are originated from the plasma membrane and endosomes1. The former are vesicles called as microvesicles generated by outward budding from plasma membrane. The latter are formed by invagination and budding of early endosomes and the endosomes containing a number of vesicles inside the lumen are called multivesicular bodies (MVBs). The MVBs release vesicles called exosomes into the extracel- lular environment by fusion with the plasma membrane. EVs can contain lipids, proteins and RNAs, including microRNAs (miRs), and the compositions depend on the cellular origin. EVs collected from the cerebrospinal fluid (CSF) of brain-injured patients include inflammation-related proteins2, and EVs from the CSF of glioblas- toma patients exhibits increased miR-21 levels, which is a highly overexpressed miR in glioblastoma cells3. These results have suggested that EVs may offer important information on pathological conditions as a novel biomarker. The EVs released into the extracellular space can be incorporated into the internal components to target cells; alternatively, the receptors and antigens (e.g., proteins, ligands) on EVs can activate cell surface receptors of target cells4. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids The ability of EVs to affect gene expression extends not only to neighbour cells but also to distant cells, because EVs circulate through the bloodstream and other body fluids5,6. g yl To facilitate further investigation of EV functions in cell-cell communication in vivo, we generated a new transgenic (Tg) rat model using EVs labelled with green fluorescent protein (GFP). Whereas most relevant studies have focused on EV functions in vitro, its roles in vivo such as EV transfer among tissues in the body of an indi- vidual or from mother to child via breast milk or placenta are still uncertain. Several studies have suggested that EVs target specific cells depending on the type of cell from which the EVs are released. Frühbeis and colleagues have reported that oligodendroglial-derived EVs are mainly uptaken by microglia and neurons but not by astro- cytes and oligodendrocytes7. It is essential to identify the releasing cells (i.e., donor cells) and/or the target cells 1Division of Laboratory Animals Resources, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan. 2Department of Mental Disorder Research, National Institute of Neuroscience, NCNP, Tokyo, Japan. 3Division of Molecular and Cellular medicine, National Cancer Center Research Institute (NCC), Japan. 4Department of Degenerative Neurological Diseases, National Institute of Neuroscience, NCNP, Tokyo, Japan. 5Department of Neurotherapeutics, Osaka University Graduate School of Medicine, Osaka, Japan. 6Department of Biomedical Chemistry, School of Science and Technology, Kwansei Gakuin University, Sanda, Japan. 7Department of Cell Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan. Correspondence and requests for materials should be addressed to T.O. (email: tochiya@ncc.go.jp) Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 1 www.nature.com/scientificreports/ (i.e., recipient cells) of EVs circulating in the body not only to reveal the transfer mechanism of EVs among cells but also to use EVs as carriers for drug delivery. g y We constructed a human CD63-copGFP gene regulated by the CAG promoter (CAG/human CD63-GFP). The CD63 protein is a member of the transmembrane 4 superfamily (tetraspanin), and is known as an EV marker. CD63 is highly enriched in late endosomes (MVBs) via an intracellular pathway from the trans-Golgi network or via endocytosis from the cell surface8. Within MVBs, CD63 is incorporated into intraluminal vesicles, through a trafficking pathway that requires ceramide in some types of cells9,10; then, the vesicles rich in CD63 are released as exosomes by fusion with the plasma membrane. Results P d i Production of CD63-GFP rats and phenotypic analysis. CAG/human CD63-GFP transgenic rats expressing copGFP-fusion protein to human CD63 under the control of the CAG promoter (Fig. 1a) were gener- ated by gene transfection into rat embryonic stem cells (rESCs) (Fig. 1b) and injection of transfected rESCs into blastocysts (Fig. 1c). We selected the rESC colonies expressing GFP fluorescence, and further selected the rESC lines showing highly fluorescent signals that were comparatively stable after several passages. To generate coat-col- our chimaeras, the transfected rESCs, which originated from a Wistar strain (white-coated), were injected into blastocysts from LEA x LEA mating (brown-coated) or LEA x Wistar mating (brown-coated). We observed that the rESCs were incorporated into the inner cell mass of the blastocysts (arrow in Fig. 1c). The white coat colour from the rESCs appeared mainly in the face (arrowhead in Fig. 1d). The chimaeric rats produced white-coated newborns after mating with Wistar males, showing the successful germline transmission of the rESCs (arrows in Fig. 1d). Furthermore, the transgenic offspring were genotyped using PCR analysis (Fig. 1e). The germline chimaera in this study was confirmed in one of four rESC lines, and it was named the Wistar-esTgN(CAG/CD63-GFP)3NCCRI strain. Tg male offspring were not viable after birth. Tg male foetuses, compared with Tg female foetuses, showed relatively high-level expression of human CD63-GFP in fluorescence microscopy observations (GFP+​ +​; Supplementary Fig. 1a) and western blotting analysis (anti-human CD63 and anti-copGFP; Supplementary Fig. 1b,c). There were no apparent developmental defects during gestation, and nontransgenic and transgenic male litters showed equal Mendelian ratios by genotyping: six males were GFP-negative (No. 2–7), and six males were GFP-positive (No. 1–6) in one litter (Supplementary Fig. 1b); three males were GFP-negative (No. 3–5), and three males were GFP-positive (No. 3, 5, 6) in another litter (Supplementary Fig. 1c). The Tg female rats were fertile, but they were likely to die sooner than usual, after reaching an age of four to six months. We also gener- ated other Tg rats with a human CD63-GFP gene regulated by the Sox2 promoter activated in neural stem cells because the CAG promoter is ubiquitously expressed. The tissue-specific human CD63-GFP Tg rats gave birth to both sexes; these rats were fertile and did not tend to die early (unpublished). y GFP-positive neonatal rats expressed exogenous human CD63-GFP throughout their bodies (Fig. 2a,b). Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids In previous studies, a GFP-tagged CD63 gene (CD63-GFP) has been transfected into cultured cell lines to detect EV transfer and incorporation into recipient cells11,12. In addition, CD63-GFP in vivo transfection into the fruit fly (Drosophila melanogaster) has been reported; this study has shown that CD63-GFP-labelled EVs from the male reproductive glands mediate the interaction between sperm and the female reproductive tract after mating13. The present study is the first report of EVs labelled with CD63-GFP in mammals. We showed that exogenous human CD63-GFP expression was detected in the tissues of Tg rats by immunostaining and biochemical detection. Furthermore, the human CD63-GFP labels were detected in EVs isolated from the blood serum, breast milk, and amniotic fluid (AF) of the Tg rats. Our animal model may potentially provide a new approach to study the intraindividual transfer and/or mother-child transfer of EVs in vivo. Results P d i The endogenous rat CD63 was ubiquitously expressed in main organs except for the thymus and liver, which showed expression signals that were lower and indistinct relative to those of other tissues (Fig. 2b). The heart, kidney and stomach expressed particularly high levels of human CD63-GFP (Fig. 2a xiii” and Fig. 2b). At embry- onic day 18–19 (E18–19), GFP fluorescence was also observed in placentas (arrows in Supplementary Fig. 1a). Interestingly, the placentas of GFP-negative foetuses expressed GFP signals only on the maternal side (arrowhead in Supplementary Fig. 1a). CD63-GFP signals are located in endosomes, and extracellular secretion was attenuated by GW4869. To analyse the human CD63-GFP localization inside cells, primary fibroblast cells were pre- pared from caudal vertebrae of Wistar wild type (Wt) and Tg rats. Human CD63-GFP expression was located close to nuclei (upper panels in Fig. 3a), and GFP signals overlapped with endogenous rat CD63-positive sig- nals (lower panels in Fig. 3a). The cells with low GFP expression showed no human CD63-positive signals, only rat CD63-positive signals (arrows in Fig. 3a). Time-lapse imaging of cells from Tg rats showed the movement of small GFP-positive organelles between plasma membranes and around nuclei (Supplementary Fig. 2). We isolated EVs from the conditioned medium (EV-free FBS) of the cultured fibroblast cells by ultracentrifuga- tion. Both EVs from Wt and Tg cells showed a peak in particle size between 100 and 200 nm (Wt; 152 nm and Tg; 137 nm) by measurement using a NanoSight system (Fig. 3b). It has been suggested that there are multiple molecular machineries involved in the generation of MVB and the sorting in endosomes have different manners (e.g. ceramide-dependent or -independent), and which mechanism predominates depends on the cell type1,14. The tetraspanin CD63 plays in the MVB formation and the endosomal sorting14,15. Next, to examine whether the extracellular secretion of human CD63-GFP proteins was regulated via the same biogenesis pathway as endoge- nous rat CD63-containing exosomes, the fibroblast cells were treated with a neutral sphingomyelinase (nSMase) inhibitor, GW4869, which is known to block ceramide biosynthesis and reduce EV secretion16. The rat CD63 signals inside the Wt and Tg cells increased after incubation with 10 μ​M GW4869 for 24 hours, and the GFP fluorescence signals were also increased in Tg cells after GW4869 treatment (Fig. 3c). Results P d i The expression levels of rat CD63 and human CD63-GFP proteins in the cell lysates remained unchanged after GW4869 treatment (right Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 2 www.nature.com/scientificreports/ Figure 1. Generation of CAG/human CD63-GFP transgenic (Tg) rats. (a) Structure of the transgene construction. The transgene was constructed using human CD63-copGFP under control of the CAG prom (b) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cul rESCs expressed GFP. (c) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rE adherence to the inner cell mass (ICM). BF: bright field. Scale bars =​ 100 μ​m. (d) Adult female chimaeric r from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches w present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with Wistar wild type (Wt) male (arrows). (e) Genotyping by PCR analysis of the extracted DNA from ear snips of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: siz marker. Figure 1. Generation of CAG/human CD63-GFP transgenic (Tg) rats. (a) Structure of the transgene construction. The transgene was constructed using human CD63-copGFP under control of the CAG promoter. (b) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cultured rESCs expressed GFP. (c) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rESC adherence to the inner cell mass (ICM). BF: bright field. Scale bars =​ 100 μ​m. (d) Adult female chimaeric rat from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches were present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with a Wistar wild type (Wt) male (arrows). (e) Genotyping by PCR analysis of the extracted DNA from ear snips of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: size marker. in Fig. 3d). In contrast, the extracellular rat CD63 and human CD63-GFP levels in the EVs collected by ultra- centrifugation decreased with GW4869 in a dose-dependent manner (left in Fig. 3d). The levels of another EV marker, flotillin-1, were also reduced in Wt (10 μ​M GW4869) and Tg cells (1 and 10 μ​M GW4869) compared with cells that did not receive GW4869. These results suggested that GW4869 blocked EV release into the con- ditioned medium but did not affect protein expression. Results P d i These results also suggested that the exogenous human CD63-GFP in fibroblast cells was secreted through the ceramide-dependent biogenesis pathway, as was endoge- nous rat CD63. in Fig. 3d). In contrast, the extracellular rat CD63 and human CD63-GFP levels in the EVs collected by ultra- centrifugation decreased with GW4869 in a dose-dependent manner (left in Fig. 3d). The levels of another EV marker, flotillin-1, were also reduced in Wt (10 μ​M GW4869) and Tg cells (1 and 10 μ​M GW4869) compared with cells that did not receive GW4869. These results suggested that GW4869 blocked EV release into the con- ditioned medium but did not affect protein expression. These results also suggested that the exogenous human CD63-GFP in fibroblast cells was secreted through the ceramide-dependent biogenesis pathway, as was endoge- nous rat CD63. The EVs labelled with CD63-GFP from rat body fluids. EVs were isolated from blood serum, breast milk and AF of Wt and Tg rats using ultracentrifugation methods. Nanoparticle tracking analysis showed a peak between 100 and 200 nm in each sample (Fig. 4a–c). The particles imaged by electron microscopy (EM) were observed to be mainly between 50–150 nm in size (Fig. 4d). Western blot analysis revealed that the EVs of the three Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 3 www.nature.com/scientificreports/ Figure 2. Human CD63-GFP expression analysis in Tg rats (Wister-esTgN(CAG/CD63-GFP)3NCCRI). (a) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative (i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed especially high fluorescent signals (xiii”). (b) Western blotting for endogenous rat CD63 and exogenous human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+​) offspring. Ctx: cortex, cbl: cerebellum, and hip: hippocampus. Figure 2. Human CD63-GFP expression analysis in Tg rats (Wister-esTgN(CAG/CD63-GFP)3NCCRI). (a) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative (i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed especially high fluorescent signals (xiii”). (b) Western blotting for endogenous rat CD63 and exogenous human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+​) offspring. Ctx: cortex, cbl: cerebellum, and hip: hippocampus. body fluids from Wt and Tg rats were positive for EV markers, flotillin-1 and endogenous rat CD63 (Fig. 5a,b). Results P d i Furthermore, signals of antibodies to human CD63 and copGFP in the EVs were detected in the three body fluids of Tg rats but not in those of Wt rats (Fig. 5a,b). To visually confirm the presence of EVs labelled with human CD63-GFP, immunoelectron microscopy analysis was performed using serum-derived EVs. The EVs from Wt rats were not labelled with anti-human CD63 antibody (Fig. 5c, upper and Supplementary Fig. 3a). In contrast, some of the EVs from Tg rats were immunolabelled with anti-human CD63 antibody, and the immunolabelling for human CD63 was detected on EVs 50–100 nm in sizes (Fig. 5c, lower and Supplementary Fig. 3b–d). The negative staining without the first antibody showed no immunoreactivity on the Tg EVs (Supplementary Fig. 3e). These results suggested that human CD63 was enriched in the subpopulation of EVs in Tg rats. Transfer of the labelled EVs with CD63-GFP into recipient cells in vitro. To evaluate the utility of the labelled EVs from the body fluids of Tg rats for monitoring EV transfer between cells, the serum-derived EVs were incubated with primary rat embryonic fibroblast cells (REFs) in vitro for 10–11 hours (Fig. 6a,b). Although it was difficult to chase the incorporated Tg EVs by GFP fluorescent directly, the Tg EVs in the recipient cells were detectable by immunostaining with antibodies to human CD63 and copGFP (Fig. 6a and Supplementary Fig. 4). In general, the EVs collected by ultracentrifugation consists of microvesicles and exosome subpopulations, and the immunoelectron microscopy analysis in this study indicated human CD63-immunopositive and –immunon- egative subpopulations of the EVs in the serum of Tg rats. To examine how the EVs with human CD63-GFP were transferred into REFs, Wt and Tg EVs were prelabelled with PKH67, a green fluorescent lipid dye that stains all EV subpopulations. We did not observe green fluorescent signals in the cells that were incubated with vehicle PBS (−) labelled with PKH67. In contrast, PKH67-labelled EVs showed highly intense fluorescent signals around cells nuclei (Fig. 6a). The human CD63 immunosignals were detected only in Tg EVs and a substantial proportion of EVs incorporated into the recipient cells were human CD63-immunnopositive (Fig. 6a). Furthermore, late endo- some/lysosome marker LAMP1 was colocalized to both PKH67 and human CD63 in the recipient REFs (arrows in Fig. 6b). These results demonstrate a substantial uptake of CD63-enriched EVs by the recipient cells through the endocytic pathway. Results P d i Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Human CD63-GFP localization and extracellular vesicle (EV) analysis in the primary fibroblast cells obtained from the caudal vertebrae of Tg rats. (a) Localization of human CD63-GFP in the cultured Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars =​ 50 μ​m. (b) Size distribution of the EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. (c,d) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals and GFP signals were increased after treatment with 10 μ​M GW4869, a neutral sphingomyelinase (nSMase) inhibitor, for 24 hours (c). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63 and flotillin-1) and human CD63-GFP in the isolated EVs (d; left). However, the expression levels of rat CD63 and human CD63-GFP in the cell lysates were not changed by GW4869 (d; right). β​-actin was used as a loading control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP overexpression (Supplementary Fig. 5). Figure 3. Human CD63-GFP localization and extracellular vesicle (EV) analysis in the primary fibroblast cells obtained from the caudal vertebrae of Tg rats. (a) Localization of human CD63-GFP in the cultured Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars =​ 50 μ​m. (b) Size distribution of the EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. (c,d) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals and GFP signals were increased after treatment with 10 μ​M GW4869, a neutral sphingomyelinase (nSMase) inhibitor, for 24 hours (c). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63 and flotillin-1) and human CD63-GFP in the isolated EVs (d; left). However, the expression levels of rat CD63 and human CD63-GFP in the cell lysates were not changed by GW4869 (d; right). β​-actin was used as a loading control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP overexpression (Supplementary Fig. 5). Discussion EVs are released into the extracellular environment, and they have been found in various body fluids, such as CSF17, breast milk18, saliva18, urine19, AF20, and blood serum. The results of proteome and miR profiling have shown that the EVs from body fluids carry many immune- or development-related components. Furthermore, the analysis of EVs from the blood of mothers during pregnancy21 and from breast milk during lactation22 have provided data showing time-dependent changes in the number of EVs or RNA contents of EVs. However, the studies of EV transfer using EVs isolated from body fluids have been very few in number compared with the experiments using EVs isolated from in vitro culture. Most of these studies injected labelled EVs that were col- lected from conditioned medium of cultured cells into a body to examine the transfer of EV contents23. PKH67 and PKH26 are the most common reagents used to stain EVs. Because they are membrane dyes with a high affinity for lipids, all EVs are labelled with PKH reagents. In addition, body fluids include many more lipid com- ponents than conditioned medium, and these lipids may also be stained by PKH reagents. In this study, we gen- erated and analysed a novel Tg rat expressing human CD63-GFP under the CAG promoter. Human CD63-GFP expression was detected by western blotting analysis in the EVs collected from three body fluids: blood serum, breast milk and AF. CD63 proteins are highly enriched particularly on exosome membranes via trafficking into late endosomes (MVBs)8. We expected that, in combination with other EV markers, human CD63-GFP might be helpful to distinguish EVs from other vesicles and lipid components as well as provide more precise identification of the EVs endocytosed into recipient cells.hf y p This study suggested that the overexpression of CD63 had strong functional effects in vivo, which resulted in embryonic lethality for males and premature death for females. It has been reported that CD63 associates with many protein, and has a key role in intracellular trafficking and cell signalling activity, which indicates that CD63 binding to other membrane proteins regulates various cellular processes, such as cell migration, adhesion and differentiation, via altering complex formation at the plasma membrane8,24–26. In mast cells (MCs), the absence of CD63 results in significantly decreased MC degranulation, which led to a reduction of allergic reactions27. Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 5 www.nature.com/scientificreports/ Figure 4. Discussion Characterization of the EVs isolated from three body fluids of Wt and Tg rats. (a–c) Size distribution of EVs from serum (a), breast milk (b) and amniotic fluid (AF) (c) determined using a NanoSight system. (d) Electron microscopy (EM) images of EVs. Scale bars =​ 200 nm. AF samples were collected from pregnant Tg rats at embryonic day 16–17 (E16–17) after mating with Wt males. GFP−: GFP-negative foetuses. GFP+​: GFP-positive foetuses. Figure 4. Characterization of the EVs isolated from three body fluids of Wt and Tg rats. (a–c) Size distribution of EVs from serum (a), breast milk (b) and amniotic fluid (AF) (c) determined using a NanoSight system. (d) Electron microscopy (EM) images of EVs. Scale bars =​ 200 nm. AF samples were collected from pregnant Tg rats at embryonic day 16–17 (E16–17) after mating with Wt males. GFP−: GFP-negative foetuses. GFP+​: GFP-positive foetuses. Figure 4. Characterization of the EVs isolated from three body fluids of Wt and Tg rats. (a–c) Size distribution of EVs from serum (a), breast milk (b) and amniotic fluid (AF) (c) determined using a NanoSight system. (d) Electron microscopy (EM) images of EVs. Scale bars =​ 200 nm. AF samples were collected from pregnant Tg rats at embryonic day 16–17 (E16–17) after mating with Wt males. GFP−: GFP-negative foetuses. GFP+​: GFP-positive foetuses. Figure 5. Analysis of the EVs labelled with human CD63-GFP in the three body fluids. (a,b) Western blotting analysis of the EVs isolated from serum (a), breast milk and AF (b) of Wt and Tg rats for flotillin-1, rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating with Wt males. GFP−: GFP-negative foetuses. GFP+​: GFP-positive foetuses. (c) Immunoelectron microscopy images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars =​ 200 nm. Figure 5. Analysis of the EVs labelled with human CD63-GFP in the three body fluids. (a,b) Western blotting analysis of the EVs isolated from serum (a), breast milk and AF (b) of Wt and Tg rats for flotillin-1, rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating with Wt males. GFP−: GFP-negative foetuses. GFP+​: GFP-positive foetuses. (c) Immunoelectron microscopy images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars =​ 200 nm. Discussion Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 6 www.nature.com/scientificreports/ Figure 6. Uptake of serum-derived EVs by rat embryonic fibroblast cells (REFs). (a) EVs of Wt rats, Tg rats and vehicle PBS (−​) as a control were prelabelled with PKH67. Then, REFs were incubated with PKH67- labelled EVs for 11 hours. The incorporated EVs in the recipient REFs were detected by PKH67 (green) and anti-human CD63 (red). The Tg EVs are also shown as magnified views (white square region) with different colours (PKH67: blue, human CD63: red, nuclei: gray). Scale bars =​ 20 μ​m. (b) Images of co-localization of EVs with LAMP1 in REFs. EVs of Wt (with PKH67) and Tg rats were incubated with REFs for 10 hours, and the EVs from Tg rats were detected using an antibody to human CD63. Magnified views (arrows) are shown in the insets. Scale bars =​ 25 μ​m. Nuclei were counterstained with Hoechst 33342 (blue). Figure 6. Uptake of serum-derived EVs by rat embryonic fibroblast cells (REFs). (a) EVs of Wt rats, Tg rats and vehicle PBS (−​) as a control were prelabelled with PKH67. Then, REFs were incubated with PKH67- labelled EVs for 11 hours. The incorporated EVs in the recipient REFs were detected by PKH67 (green) and anti-human CD63 (red). The Tg EVs are also shown as magnified views (white square region) with different colours (PKH67: blue, human CD63: red, nuclei: gray). Scale bars =​ 20 μ​m. (b) Images of co-localization of EVs with LAMP1 in REFs. EVs of Wt (with PKH67) and Tg rats were incubated with REFs for 10 hours, and the EVs from Tg rats were detected using an antibody to human CD63. Magnified views (arrows) are shown in the insets. Scale bars =​ 25 μ​m. Nuclei were counterstained with Hoechst 33342 (blue). The Tg rats in this study often exhibited inflammation around the eyes as animals grew (data not shown). Therefore, we considered the possibility that the Tg rats might overexpress CD63 in MCs, which in turn might causing allergic inflammation by increased MC degranulation. We generated another line of Tg rats expressing human CD63-GFP under the Sox2 promoter, which expressed it in the kidney in addition to the brain; a large amount of urine output was observed, but no lethality or infertility was observed in either gender (unpublished). Methods After being blocked with 5% skim milk, the mem- branes were probed with primary antibodies specific to the following proteins: rat CD63 (1:250; AbD Serotec, CA, USA); human CD63 (1:250; Becton Dickinson); copGFP (1:10000; Evrogen, Moscow, Russia); and flotillin-1 (1:500; Becton Dickinson). β​-actin (1:5000; Sigma-Aldrich) was used as a loading control. The membranes were subsequently incubated with secondary antibodies, and the signals were detected using chemiluminescent rea- gents (ImmunoStar and ImmunoStar LD, Wako, Tokyo, Japan). Immunocytochemistry. Cultured cells were fixed with 4% paraformaldehyde (PFA) for 15–20 min at room temperature. After being washed, cells were incubated with primary antibodies in blocking solution containing 0.2% Triton X-100 (Sigma-Aldrich) and 10% FBS in PBS at 4 °C overnight. Primary antibodies specific to the following proteins were used: rat CD63 (1:200; AbD Serotec) and human CD63 (1:200; Becton Dickinson). After the cells were stained with Alexa Fluor 546 Goat anti-Mouse IgG1 antibody (1:2000; Life Technologies), they were imaged with BIOREVO BZ-9000 (Keyence, Osaka, Japan) and Axiovert 200 (Carl Zeiss, Westlar, Germany) fluo- rescence microscopes. DAPI was used to stain cell nuclei. Fibroblast culture and inhibition of EV secretion. Primary fibroblast cells were prepared from caudal vertebrae of Wt and Tg rats (CAG/human CD63-GFP) at postnatal day 21 (P21). The primary cells were main- tained in DMEM (Life Technologies) with 10% FBS and antibiotic-antimycotic solution (Life Technologies) at 37 °C in 5% CO2.i For inhibition of EV secretion, the fibroblast cells were plated on glass-bottom dishes and 10 cm culture dishes; after expansion, GW4869 (Sigma-Aldrich) treatments were performed for 24 hours. The 35 mm glass-bottom dishes were used to observe intracellular rat CD63 as an EV marker and human CD63-GFP expression, and cells on the 10 cm dishes were stored at −​80 °C until protein extraction. Conditioned medium from the 10 cm dishes was used for the isolation of EVs. To isolate EVs, conditioned medium from the 10 cm dishes was replaced with the EV-free FBS (System Biosciences) medium before GW4869 treatment. Culture supernatant was centri- fuged at 1,400 rpm for 10 min and then filtered through a 0.22 μ​m membrane filter (Millipore) to remove cellular debris. EVs were collected by ultracentrifugation at 35,000 rpm for 70 min at 4 °C using a Beckman SW41Ti rotor (Beckman, CA, USA). The pellets were washed with PBS by re-ultracentrifugation and were resuspended in PBS. Methods Animals. All animals (Wistar, LEA and Tg (Wistar-esTgN(CAG/CD63-GFP)3NCCRI) rat strains) used in this study were treated in accordance with the institutional guidelines of the Animal Ethics Committee for the care and use of animals in the National Institute of Neuroscience, National Centre of Neurology and Psychiatry, and National Cancer Centre Research Institute, Japan. Production of CAG/human CD63-GFP Tg rats using rat ES cells. A DNA fragment encoding human CD63-copGFP (human CD63-GFP) from a pCT-CMV-CD63-GFP vector (System Biosciences, CA, USA) was subcloned into a pECFP-1 plasmid (Clontech, Shiga, Japan) containing CAG promoter. The plasmid CAG/ human CD63-GFP was linearized by SalI and was then transfected into Wistar rESCs using a Mouse ES Cell Nucleofector Kit (Lonza, Basel, Swiss) and an Amaxa Nucleofector (A-013 program) as described previously33. pCAG/human CD63-GFP contained a neomycin/kanamycin selection cassette. The rESCs were seeded onto mitomycin-C-treated neomycin-resistant MEFs (Millipore, MA, USA) in YPAC medium33 with 2% Matrigel (Becton Dickinson, NJ, USA). Then, G418 (Geneticin) (Sigma-Aldrich, MO, USA) was added to the culture medium at day 1 for the selection of transfected cells. Colonies showing GFP fluorescence were selected and expanded. The rESCs in this study were derived from the cell lines established by Kawamata and Ochiya37. l ll d d (d ) f d d h Blastocysts were collected at 4.5 days post coitus (dpc) from LEA x LEA or Wistar x LEA mating and injected with approximately 12 transfected rESCs. The injected blastocysts were surgically transferred into the uterine horns of 3.5 dpc pseudopregnant Wistar rats. Chimaeric rats were identified by coat colour. Germline transmis- sion was confirmed by the coat colour of F1 rats resulting from mating with Wistar rats. Transgenic offspring were genotyped by GFP fluorescence and PCR analysis. Sex determination in foetuses was performed by PCR using the male-specific Sry gene as a marker. Immunoblotting. For total protein extraction, the tissues and cultured cells were solubilized in lysis buffer (1% SDS, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 10 mM sodium pyrophospate, 10 mM NaF, 1 mM PMSF and 2 mM Na3VO4). The samples were sonicated on ice, and then the lysates were cleared by centrifugation. Protein concentration was measured using a Pierce BCA Protein Assay Kit (Life Technology, CA, USA). Cell lysates and EVs were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were trans- ferred onto Immobilon-P Transfer membranes (Millipore). Discussion A previous report has found that, although CD63-deficient mice display no lethality or infertility due to com- pensation by other tetraspanins, they exhibit morphologic changes in the kidney, and increased urine output28, similarly to that of our Tg rats. These results suggest that CD63-GFP will be further useful for visualizing EVs using tissue- or stage-specific expression constructs, with consideration of the potential effects on phenotype.l g g pi p pf p yp While the present experiments did not show that serum-derived EVs from the Tg rats retained GFP fluores- cence after transfer into recipient cells via in vitro assay, the EV signals in recipient cells were clearly detectable by using an antibody to human CD63. The immunoelectron microscopy analysis revealed that the immunolabelling with human CD63-specific antibody was not detected on all isolated EVs. In addition, the transfer of EVs into recipient cells in vitro showed the human CD63 immunoreactivity in many EVs. These results suggested that human CD63-GFP labelling would provide a helpful tool for tracing of incorporated exosomes although more detailed experiments are required. This animal model is intended to contribute to research on the transition path- way of EVs using body fluids. Several studies on breast milk have suggested that the composition of milk-derived EV may be a potential immune-regulator for infants29,30 and an activator of postnatal growth31. Immune-related miRs have been identified in the milk-derived EVs29,32. Other than miRs, the milk-derived EVs carry bioactive TGF-β​33, which is known to differentiate naïve T cells into regulatory T cells34 and to promote development of the intestinal barrier35. Nevertheless, the transfer pathway and recipient cells of milk-derived EVs in infants remain poorly understood. A previous report has found that mouse intestinal stem cells show uptake of food (grape)-derived EV-like vesicles that were stained by PKH dye in vivo, and the targeted cells exhibit increased proliferation after taking up the grape-derived vesicles36. Our Tg rats produced the EVs labelled with human CD63-GFP, which were detected only in the tissues of Tg rats and not in those of Wt rats. We expect that study of in vivo uptake of the milk-derived EVs from Tg rats will reveal interesting targets in the intestinal tissues of infants. Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 7 www.nature.com/scientificreports/ The novel rat model that we generated allowed studies of the movement of endosomes before EV release and the detection of EVs released. Discussion Our new animal model shows promise for facilitating further studies to reveal the communication between cells/tissues via EVs. Methods The isolated EVs were quantified by size and count using a NanoSight system (NanoSight, Amesbury, UK) and by measuring their protein concentration using a Pierce BCA Protein Assay Kit. EV isolation from serum, breast milk and AF body fluids. Blood samples were collected from Wt and Tg adult female rats. The blood serum samples were centrifuged at 1,400 rpm for 10 min and then at twice at 10,000 g for 15 min to remove blood cells; this was followed by ultracentrifugation at 35,000 rpm for 70 min at 4 °C. Breast milk samples were collected from the stomachs of GFP-negative newborns (P4-5) taking milk from Wt or Tg mother rats. Collected solid milk samples were homogenized and resuspended in PBS; then, they were centrifuged at 2,000 g for 10 min to remove fat and cell debris. Non-transparent supernatants were filtered through a 0.22 μ​m filter to remove residual fat, producing a clear supernatant (milk whey). Finally, the EVs were Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 8 www.nature.com/scientificreports/ collected by ultracentrifugation. AF samples were collected from pregnant Tg rats at E16-17 that had mated with Wt males. Tg foetuses were identified by the fluorescence of GFP under a fluorescence stereomicroscope (Leica, Wetzlar, Germany). AF samples were centrifuged at 2,000 g for 10 min and then at 10,000 g for 10 min, and EVs were collected by ultracentrifugation. collected by ultracentrifugation. AF samples were collected from pregnant Tg rats at E16-17 that had mated with Wt males. Tg foetuses were identified by the fluorescence of GFP under a fluorescence stereomicroscope (Leica, Wetzlar, Germany). AF samples were centrifuged at 2,000 g for 10 min and then at 10,000 g for 10 min, and EVs were collected by ultracentrifugation. EM analyses. The body fluid-derived EVs were plated on collodion-carbon-coated grids and fixed with 2% PFA. For immunoelectron microscopy, the serum-derived EVs were permeabilized with 0.1% saponin, stained with anti-human CD63 (1:50, Becton Dickinson), and then incubated in secondary anti-mouse antibody con- jugated with 10-nm gold particles (Amersham, Buckinghamshire, UK). Finally, the EVs were post-fixed in 0.2% glutaraldehyde and negatively stained with uranyl acetate. The images were captured using a transmission elec- tron microscope (Tecnai Spirit; FEI, OR, USA). Transfer of serum EVs into REFs. REFs were prepared from Wt rats at E14.5. Methods Embryos were dissoci- ated by trypsin treatment (Sigma-Aldrich), and the single cells were cultured in DMEM including 10% FBS and antibiotic-antimycotic solution at 37 °C in 5% CO2. Serum-derived EVs from Wt or Tg rats were isolated using ultracentrifugation. The EVs were visualized using a PKH67 fluorescence labelling kit (Sigma-Aldrich). Serum EVs were incubated with REFs on 24-well glass bottom plates for 10–11 hours at 37 °C in 5% CO2. After being washed, the cultured cells were fixed with 4% PFA. The EVs from Tg rats were detected using anti-human CD63 antibody after having been transferred into REFs. To analyse localization of EVs in REFs, anti-LAMP1 antibody (1:250, Sigma-Aldrich) was used. After the cells were stained with Alexa Fluor 546 Goat anti-Mouse IgG1 (1:2000; Life Technologies), Alexa Fluor 546 Goat anti-Rabbit IgG (1:2000; Life Technologies) and Alexa Fluor 488 Goat anti-Rabbit IgG (1:200; Life Technologies) antybodies, internalization of EVs were captured with a FV1000 confo- cal microscope (OLYMPUS, Tokyo, Japan) and a BIOREVO BZ-9000 fluorescence microscopy (Keyence, Osaka, Japan). Hoechst 33342 was used to stain cell nuclei. J p ) All experimental protocols were approved by the National Institute of Neuroscience, National Center of Neurology and Psychiatry, and National Cancer Centre Research Institute, Japan. p ) All experimental protocols were approved by the National Institute of Neuroscience, National Center o Neurology and Psychiatry, and National Cancer Centre Research Institute, Japan. References Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude mouse models. Adv. Drug Deliv. Rev. 65, 383–390 (2013). 3. Corrigan, L. et al. BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J. Cell Biol. 206 671–688 (2014). 4. Edgar, J. R., Eden, E. R. & Futter, C. E. Hrs- and CD63-dependent competing mechanisms make different sized endosoma intraluminal vesicles. Traffic 5, 197–211 (2014).h ffi 5. Van Niel, G. et al. The tetraspanin CD63 regulates ESCRT-independent and –dependent endosomal sorting during melanogenesis Dev. Cell 21, 708–721 (2011). Trajkovic, K. et al. Ceramide triggers budding of exosome vesicles iii 7. Street, J. M. et al. Identification and proteomic profiling of exosomes in human cerebrospinal fluid. J. Transl. Med. 10, 5 (2012). iil 18. Lässer, C. et al. Human Saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J. Transl. Med. 9, 9 (2011). 19 Keller S et al CD24 is a marker of exosomes secreted into urine and amniotic fluid Kidney Int 72 1095 1102 (2007) iil 18. Lässer, C. et al. Human Saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J. Transl. Med. 9, 9 (2011).l l 20. Bretz, N. P. et al. Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-like rec signaling. J. Cell Biol. 288, 36691–36702 (2013).if g g 1. Salomon, C. et al. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration PLoS ONE 9, e98667 (2014). 22. Hata, T., Murakami, K., Nakatani, H., Yamamoto, Y., Matsuda, T. & Aoki, N. Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs. Biochem. Biophys. Res. Commun. 396, 528–533 (2010). 23. Grapp, M. et al. Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma. Nat. Commun. 4, 2123 (2014).f 4. Iizuka, S. et al. Ameloblastin regulates osteogenic differentiation by inhibiting Src kinase via cross talk between integrin β​1 and CD63. Mol. Cell. Biol. 31, 783–792 (2011). 25. Tugues, S. et al. Tetraspanin CD63 promotes vascular endothelial gowth factor receptor 2-β​1 integrin complex formation, thereby regulating activation and downstream signaling in endothelial cells in vitro and in vivo. J. Cell Biol. 288, 19060–19071 (2013). References 1. Raposo, G. & Stoorvogel, W. Extracellular vesicles: Exosomes, microvesicles, and friends. J. Cell Biol. 18, 373–383 (2013).lt 1. Raposo, G. & Stoorvogel, W. Extracellular vesicles: Exosomes, microvesicles, and friends. J. Cell Biol. 18, 373–383 (2013). 2. de Rivero Vaccari, J. P. et al. Exosome-mediated inflammasome signaling after central nervous system injury. J. Neurochem. 10, 13036 (2015).l ( ) 3. Akers, J. C. et al. miR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): A platform for glioblastoma biomarke development. PLoS ONE 8, e78115 (2013).i p 4. Andaloussi, E. L., Mäger, I., Breakefield, X. O. & Wood, M. J. Extracellular vesicles: biology and emerging therapeutic opportunities Nat. Rev. Drug Discov. 12, 347–357 (2013).t g 5. Takahashi, Y. et al. Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenou injection. J. Biotechnol. 165, 77–84 (2013). j 6. Tominaga, N. et al. Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier. Nat. Commun. 6, 6716 (2015). 7. Frühbeis, C. et al. Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication. PLoS Biol 11, e1001604 (2013).fi 8. Pols, M. S. & Klumperman, J. Trafficking and function of the tetraspanin CD63. Exp. Cell Res. 315, 1584–1592 (2009). 9 K k N I hi H Y hi k Y T k hi F M ki Y & O hi T S h i d i ll l 8. Pols, M. S. & Klumperman, J. Trafficking and function of the te fi 9. Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y. & Ochiya, T. Secretory mechanism and intercellular tra microRNA in living cells. J. Biol. Chem. 285, 17442–17452 (2010). g 0. Kajimoto, T., Okada, T., Miya, S., Zhang, L. & Nakamura, S. Ongoing activation of sphingosine 1-phosphate receptors mediate maturation of exosomal multivesicular endosomes. Nat. Commun. 4, 2712 (2013). 11. Koumangoye, R. B., Sakwe, A. M., Goodwin, J. S., Patel, T. & Ochieng, J. Detachment of breast tumor cells induces rapid secreti exosomes which subsequently mediate cellular adhesion and spreading PLoS ONE 6 e24234 (2011) 11. Koumangoye, R. B., Sakwe, A. M., Goodwin, J. S., Patel, T. & Ochieng, J. Detachment of breast tumor cells induce exosomes which subsequently mediate cellular adhesion and spreading. PLoS ONE 6, e24234 (2011).f 12. Suetsugu, A., Honma, K., Saji, S., Moriwaki, H., Ochiya, T. & Hoffman, R. M. Acknowledgements g We thank Dr. Nobuyoshi Kosaka for expert advice on analysis of extracellular vesicles. This work was supported by grants from the Grant-in-Aid for Young Scientists (B) (A.Y.) in the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant in Aid for the Japan Science and Technology Agency (JST) through the Center of Open Innovation Network for Smart Health (COINS) initiated by the Council for Science and Technology Policy, and a Grant in Aid for the Japan Agency for Medical Research and Development (A-MED) through the Basic Science and Platform Technology Program for Innovative Biological Medicine. References Generation of genetically modified rats from embryonic stem cells. Proc. Natl. Acad. Sci. USA 107, 14223–14228 (2010). Author Contributions A.Y., M.K., Y.T. and T.O. designed the study. A.Y. performed the experimental work, analysed the data and wrote the manuscripts. M.K. assisted in the generation of Tg rats using ES cells. Y.Y. provided technical assistance and helpful discussion on the analysis of extracellular vesicles. H. Kikuchi performed the electron microscopy experiments. T.K. assisted in the culture and provided helpful comments. N.A. performed time-lapse imaging and provided helpful comments. Y.N., T.N. and H. Kunugi provided helpful discussion. The manuscript was finalized by T.O. References 26 L S Y t l TIMP 1 d l t h t i f h l t ll th h CD63 d i t i i li Bi h J 459 g g g g 26. Lee, S. Y. et al. TIMP-1 modulates chemotaxis of human neural stem cells through CD63 and integrin signaling. Biochem. J. 459, 565–576 (2014).thfi Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 9 www.nature.com/scientificreports/ 8. Schröder, J. et al. Deficiency of the tetraspanin CD63 associated with kidney pathology but normal lysosomal function. Mol. Cell Biol. 29, 1083–1094 (2009). Biol. 29, 1083 1094 (2009). 29 Kosaka N Izumi H Sekine K & Ochiya T microRNA as a new immune-regulatory agent in breast milk Silence 1 7 (2010) , ( ) 29. Kosaka, N., Izumi, H., Sekine, K. & Ochiya, T. microRNA as a new immune-regulatory agent in breast milk. Silence 1, 7 (2010 30. Melnik, B. C., John, S. M. & Schmitz, G. Milk: an exosomal microRNA transmitter promoting thymic regulatory T cell matur preventing the development of atopy ? J. Transl. Med. 12, 43 (2014). p g p py 31. Melnik, B. C., John, S. M. & Schmitz, G. Milk is not just food but most likely a genetic transfection system activating mTO signaling for postnatal growth. Nutr. J. 12, 103 (2013). g g p g J , ( ) 32. Zhou, Q. et al. Immune-related microRNAs are abundant in breast milk exosomes. Int. J. Biol. Sci. 8, 118–123 (2012). 33. Pieters, B. C. et al. Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-β​. ONE 10, e0121123 (2015). 4. Chen, W. et al. Conversion of peripheral CD4+​ CD25-naïve T cells to CD4+​ CD25+​ regulatory T cells by TGF-β​ induction o transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003). p p p 5. Hering, N. A. et al. Transforming growth factor-β​, a whey protein component, strengthens the intestinal barrier by upregulating claudin-4 in HT-29/B6 cells. J. Nutr. 141, 783–789 (2004).h 35. Hering, N. A. et al. Transforming growth factor-β​, a whey protein component, strengthens th claudin-4 in HT-29/B6 cells. J. Nutr. 141, 783–789 (2004). 35. Hering, N. A. et al. Transforming growth factor-β​, a whey pro 36. Ju, S. et al. Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colotis. Mol. Ther. 21, 1345–1357 (2013).i ( ) 37. Kawamata, M. & Ochiya, T. Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 Additional Information upplementary information accompanies this paper at http://www.nature.com/srepi Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Yoshimura, A. et al. Generation of a novel transgenic rat model for tracing extracellular esicles in body fluids. Sci. Rep. 6, 31172; doi: 10.1038/srep31172 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 10
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Significant reduction in errors associated with nonbonded contacts in protein crystal structures: automated all-atom refinement with<i>PrimeX</i>
Acta crystallographica. Section D, Biological crystallography
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research papers Jeffrey A. Bell, Kenneth L. Ho and Ramy Farid* All-atom models are essential for many applications in molecular modeling and computational chemistry. Non- bonded atomic contacts much closer than the sum of the van der Waals radii of the two atoms (clashes) are commonly observed in such models derived from protein crystal structures. A set of 94 recently deposited protein structures in the resolution range 1.5–2.8 A˚ were analyzed for clashes by the addition of all H atoms to the models followed by optimization and energy minimization of the positions of just these H atoms. The results were compared with the same set of structures after automated all-atom refinement with PrimeX and with nonbonded contacts in protein crystal structures at a resolution equal to or better than 0.9 A˚ . The additional PrimeX refinement produced structures with reasonable summary geometric statistics and similar Rfree values to the original structures. The frequency of clashes at less than 0.8 times the sum of van der Waals radii was reduced over fourfold compared with that found in the original structures, to a level approaching that found in the ultrahigh-resolution structures. Moreover, severe clashes at less than or equal to 0.7 times the sum of atomic radii were reduced 15-fold. All-atom refinement with PrimeX produced improved crystal structure models with respect to nonbonded contacts and yielded changes in structural details that dramatically impacted on the interpretation of some protein–ligand interactions. Schro¨dinger, 120 West 45th Street, 17th Floor, New York, NY 10036, USA Correspondence e-mail: ramy.farid@schrodinger.com Acta Cryst. (2012). D68, 935–952 # 2012 International Union of Crystallography Printed in Singapore – all rights reserved Significant reduction in errors associated with nonbonded contacts in protein crystal structures: automated all-atom refinement with PrimeX Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 ISSN 0907-4449 Received 15 February 2012 Accepted 19 April 2012 # 2012 International Union of Crystallography Printed in Singapore – all rights reserved research papers angle conformation of the residue backbone have recently been demonstrated (Tronrud et al., 2010). Brunger and coworkers (Fenn et al., 2010, 2011; Schnieders et al., 2011) have combined the all-atom force field AMOEBA with a new refinement scheme and have described the advantages of a more complex molecular description that includes the calcu- lation of electrostatic interactions between protein atoms. Additional recent innovations in the use of geometric infor- mation in refinement include the use of deformable elastic network refinement (Schro¨der et al., 2010), hydropathic force- field terms (Koparde et al., 2011) and jelly-body restraints (Murshudov et al., 2011). space refinement techniques developed for use with crystal structures of small molecules at atomic resolution. They recognized the necessity of using additional chemical infor- mation combined with reciprocal-space refinement to accu- rately determine atomic positions in this situation. A complete system of geometric restraints was devised for the first widely used protein reciprocal-space refinement program, PROLSQ (Konnert, 1976; Konnert & Hendrickson, 1980; Hendrickson, 1985). H atoms were not explicitly considered in this system. The introduction of simulated-annealing refinement led to the widespread adoption of the program X-PLOR (Bru¨nger, 1992). This program featured geometric restraints based on the CHARMM force field (Bru¨nger et al., 1986, 1989; Bru¨nger & Karplus, 1988). Originally, use of this force field required an all-atom model. CHARMM-based restraints evolved in a way that removed the requirements for hydrogen coordinates. This change was associated with an alteration in the representation of nonbonded contacts from a Lennard–Jones potential to a much simpler repulsive function and the elimination of the use of electrostatic potentials. These modifications were partially motivated by electrostatic artifacts that were introduced into the structural results owing to the lack of an implicit solvent model. In addition, the long time required for computation of the complete set of nonbonded interactions was a significant impediment to the refinement of large crystal structures (Nilges et al., 1988; Weis et al., 1990). By the time that X-PLOR was superseded by the program CNS (Bru¨nger et al., 1998), any requirement for explicit H-atom coordinates for protein crystallographic refinement had been eliminated. However, the capability to apply an electrostatic model and more complete nonbonded interactions in an all-atom model remained an essential part of CNS for the determination of structures from NMR data (Linge et al., 2003). (Chen, Arendall et al., 2010). At this point, the question of the source of all-atom models needed for computational work can be addressed more clearly. Currently, such all-atom models are produced by adding H atoms to the united-atom models produced by crystallography. For water molecules and for protein H atoms whose position is subject to some degree of freedom, i.e. non-riding H atoms, either a force-field-dependent or a rule-based method is employed to determine the positions of these H atoms in order to avoid close nonbonded contacts and to form hydrogen bonds as appropriate. Nevertheless, when H atoms are added in this way to a very large majority of protein crystal structures deposited in the Protein Data Bank (Berman et al., 2000), multiple close nonbonded contacts between atoms are observed. One goal of this work is to document this obser- vation and to try to understand why such interactions occur, the recent focus on protein structure validation with H atoms present notwithstanding. Engh & Huber (1991) brought important additional infor- mation to the definition of the geometry for protein crystal structures. Their survey of bond lengths and angles observed in small peptide crystal structures at high resolution has been uniformly adopted as a standard against which protein crystal structure models are judged. It has also become the basis for the restraint system in all of the major refinement programs. Recent developments indicate an interest among crystallo- graphers in the application of more complex descriptions of molecular geometry in refinement to aid in producing better models. The refinement programs REFMAC (Murshudov et al., 2011) and PHENIX (Afonine et al., 2005) may be employed with ‘riding H atoms’, even though the ultimate result to be deposited is a united-atom model. research papers Structure-validation tools for protein geometry, partially based on the Engh & Huber standard, are available in several widely used computer programs, most notably PROCHECK (Laskowski et al., 1993), WHAT_CHECK (Hooft, Vriend et al., 1996), NUCheck (Feng et al., 1998) and SFCHECK (Vaguine et al., 1999). These programs address close non- bonded contacts largely from a united-atom perspective. More recently, the structure-validation programs Reduce and MolProbity (Davis et al., 2007; Chen, Arendall et al., 2010) have become important and popular additions to the toolkit of protein crystallographers. They are based on the concept that better judgments can be made as to the correct positioning of certain groups in the model after the addition of H atoms to a united-atom protein crystal structure and after observing their interactions. Within their software system, interpenetration of van der Waals molecular surfaces by 0.4 A˚ or more constitutes a clash. The authors flatly state that Such large overlaps cannot occur in the actual molecule, but mean that at least one of the two atoms is modeled incorrectly 1. Introduction The majority of protein crystal structures are solved in the resolution range 1.7–2.8 A˚ , a resolution range in which the diffraction experiment does not present sufficient information to accurately place individual atoms without additional chemical information. Electron-density peaks specifically for H atoms are not observed in this resolution range owing to a low signal-to-noise ratio. Therefore, H atoms are usually not explicitly included in molecular models of protein crystal structures. A molecular model without explicit coordinates for H atoms is denoted as an united-atom model, in contrast to an all-atom model. United-atom models are frequently insuffi- cient for molecular modeling and computational chemistry applications (such as structure-based virtual screening or lead optimization). How is the gap bridged between current best crystallographic practices and the requirements of these other disciplines for all-atom structures that include hydrogen coordinates? A brief history of the use of H atoms and chemical restraints in protein crystal structure refinement is useful before answering this question. Jensen and coworkers (Watenpaugh et al., 1973) first demonstrated that moderate-resolution protein crystal structures could benefit from the reciprocal- 935 Acta Cryst. (2012). D68, 935–952 doi:10.1107/S0907444912017453 Acta Cryst. (2012). D68, 935–952 research papers model is still consistent with the experimental data. In other words, one does not know how far is too far. This procedure could be especially dangerous if the original clashes were caused by atoms that were significantly misplaced. those deposited in 2010 to ensure that refinement followed modern practices and that sufficient time had passed for structures to be withdrawn if found to contain gross errors. Reflection data were required to have been deposited with the coordinates. Each entry was restricted to contain one or more protein chains but no DNA or RNA. The reported Rfree values were limited to 0.28 or lower. Structures were required to have a high-resolution limit between 1.5 and 2.8 A˚ . The molecular mass of protein within the asymmetric unit was limited to be between 10 and 300 kDa. Proteins with homologous sequences were removed at 30% identity. The refinement program PrimeX was implemented partially in response to these issues. It applies well established methods of protein crystal structure refinement (Bell et al., 2012) combined with the all-atom OPLS force field (Jorgensen et al., 1996; Kaminski et al., 2001; Banks et al., 2005) for geometric restraints. Aside from the presence or absence of H atoms in the model, these OPLS-based restraints differ in two specific respects from what have become the traditional restraint systems: (i) a Lennard–Jones description of both the attractive and repulsive components of van der Waals interactions replaces the simpler repulsive term of most Engh and Huber- based restraints and (ii) electrostatic interactions are treated, including a Surface Generalized Born model to account for implicit solvent effects (Ghosh et al., 1998; Gallicchio et al., 2002; Zhu et al., 2007; Li, Abel et al., 2011). The net effect of these differences is very significant. In a simpler restraint system, the bond-length targets are each a function of a single parameter according to the atom types involved in the bond. A similar situation occurs for bond angles. However, the bond- length and bond-angle targets specified by OPLS are a func- tion of several parameters that can all affect a single bond length or bond angle. In other words, the restraint target for a particular bond length (or angle) is contingent on the local environment of the atoms involved. 2.2. PrimeX crystallographic calculations and refinement The key features of PrimeX crystal structure refinement have been described in some detail elsewhere (Bell et al., 2012). Only details relevant to this work are described below. 2.2.1. Restrained reciprocal-space minimization. Reciprocal- space coordinate minimization is applied in PrimeX with a maximum-likelihood target, using the formulation of Pannu & Read (1996). The PrimeX implementation follows the general concepts developed by Bru¨nger and coworkers (Bru¨nger, 1989; Bru¨nger et al., 1998). A maximum-likelihood target has been shown to improve the convergence of refinement and to reduce the effects of model bias (Murshudov et al., 1997). Refinement of protein crystal structures with an all-atom model and a complete force field does much more than avoid errors whose remediation may seriously degrade the accuracy of the coordinates. The more detailed accounting for non- bonded interactions within the protein used in PrimeX can also produce a direct positive effect during refinement. While even small changes in the structure near a ligand-binding site can be critical for structure-based drug discovery, examples are presented to show how refinement with an all-atom model can result in large coordinate improvements at such sites. OPLS 2005 (Jorgensen et al., 1996; Kaminski et al., 2001; Banks et al., 2005) is a general-purpose force field for modeling proteins, nucleic acids and small molecules. PrimeX applies this consistent molecular description as geometric restraints during the refinement of the atomic positions for all molecular components of large biological crystal structures. Restrained isotropic B-factor refinement is applied in PrimeX using an approach similar to that used in the program CNS (Bru¨nger et al., 1989). research papers Touw & Vriend (2010) have shown that at least one type of protein bond angle is a complex function of the local environment and is not well described by a single Engh & Huber (1991) target angle. The target geometric values in the well characterized restraint system of Karplus and coworkers depend on the local back- bone conformation of the protein (Tronrud et al., 2010). That any particular force field can reproduce all such dependencies remains to be demonstrated, but potentially a force-field- based restraint system can more effectively adapt to local environments than current protein crystallography restraint systems. In addition, the PrimeX-calculated Rfree was required to exceed the R value by at least 0.008. This requirement ensured that the test set deposited in the PDB was likely to be the one that was actually used in the refinement of the deposited coordinates. (The Rfree calculated using the deposited test set was found to actually be lower than the calculated R factor in several cases, strongly indicating that the deposited test set was not used in the final refinement of the deposited coordi- nates.) Others have made similar observations about test-set entries in the PDB (Joosten et al., 2009; Afonine et al., 2010). A meaningful comparison of Rfree values was not possible without using a single consistent test set. The high-resolution reference data set used in this study was selected from the PDB with the following restrictions: (i) the deposition of diffraction data was required, (ii) coordinate sets were selected from entries containing protein but no DNA or RNA and with a high-resolution limit of 0.9 A˚ or better, (iii) proteins with homologous sequences were removed at 30% identity and (iv) only proteins refined with coordinates for H atoms were included in this set. (Chen, Arendall et al., 2010). (Riding H atoms are those H atoms whose positions can be determined unambiguously from the positions of the non-H atoms; for example, the H atom attached to the O of a serine residue is not a riding H atom since its position depends on the torsion angle of the C—O bond, while the H atom on the C atom of an amino acid is a riding H atom, since all torsion angles affecting its position are determined by non-H atom coordi- nates.) The advantages of a restraint scheme in which geometric target values for a residue depend on the torsion- The usual remedy in computational chemistry to these high- energy close contacts is to minimize the coordinates of the all- atom model against a force field, with non-H atoms restrained to their positions in the crystallography-derived model so that they do not deviate too far from their experimentally deter- mined positions. This solution is less than ideal, because the method produces no feedback as to whether the all-atom 936 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 research papers research papers H-atom positions are a function of the force field acting on all atoms, while the positions of non-H atoms are refined under the joint influence of crystallographic and force-field gradients. Thus, the H-atom coordinates are not biased towards the centre of mass of the electron-density distribution, as may occur in some forms of all-atom refinement (Coulson & Thomas, 1971). For H atoms bonded to more electronegative atoms, elec- trostatic forces are a major determinant of nonbonded inter- actions and they must be evaluated during the calculation of geometric gradients in the refinement. Thus, PrimeX employs the complete molecular-mechanics description of atomic interactions embodied in OPLS, including electrostatic terms (Jorgensen et al., 1996; Kaminski et al., 2001; Banks et al., 2005). Also included in the current PrimeX calculations was an optional implicit solvation term (Ghosh et al., 1998; Gallicchio et al., 2002; Zhu et al., 2007). An overview of the OPLS force field, and a description of the details of the second-generation Surface Generalized Born model used to implicitly account for solvation effects, have been provided by Li, Abel et al. (2011). Both the electrostatic and solvation calculations employ residue-based cutoffs of 15 A˚ for long-range interactions between neutral residues, of 30 A˚ between charged residues and of 20 A˚ between mixed charged and neutral residues. Such approximations and model features should be considered in the context of a trade-off between computational time and rigorous calculations, as discussed by Moulinier et al. (2003), who pioneered the use of the Generalized Born approach in refinement, and by Fenn et al. (2011), who have advocated the use of an alternate elec- trostatic model in refinement. Refinement incorporating a complete electrostatic description has been shown to lead to lower Rfree values compared with refinement excluding these interactions (Knight et al., 2008; Fenn et al., 2010, 2011; Schnieders et al., 2011). 2.2.5. The polish refinement workflow. The all-atom structures produced by PrimeX refinement, as described in Table 4, were the result of application of the ‘polish’ workflow to the united-atom models obtained from the PDB without any human intervention during the refinement process. The refined coordinates that were the result of this process are archived at http://www.schrodinger.com/primex. The purpose of the polish workflow is to produce the best all-atom model possible that is consistent with the diffraction data, starting with an already well refined crystal structure. 2.1. Data-set selection The members of the moderate-resolution protein data set used in this study were selected from the Protein Data Bank (Berman et al., 2000). Candidate structures were limited to Because of its dependence on the OPLS force field, PrimeX operates on all-atom models at all stages of refinement. H-atom coordinates do not participate in crystallographic 937 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 research papers the locations of hydrogen-bond donors and acceptors to define clusters of such sites that might be connected through hydrogen bonds. Within each cluster, hydrogen bonding is evaluated using a rule-based method to find an optimal combination of the variable components of these systems. The structural features adjusted during hydrogen-bond optimiza- tion are (i) alcoholic H-atom positions; (ii) sulfhydryl H-atom positions; (iii) phenolic H-atom positions; (iv) charge and tautomeric states of aspartic acid and glutamic acid side chains; (v) charge states, tautomeric states and orientation (flip) of histidine side chains; (vi) orientation (flip) of aspar- agine and glutamine side chains; and (vii) positions of H atoms in water molecules. The goal of this procedure is to minimize the energy of the system by maximizing the number of hydrogen bonds while avoiding close high-energy nonbonded interactions. This is accomplished by enumerating plausible orientations for each rotatable hydrogen and water molecule by identifying nearby hydrogen-bond donors and acceptors. Initial solutions for the overall local hydrogen-bond network are then generated by iteratively choosing the optimal state for each species in turn until convergence, starting from a variety of random starting conditions. These initial solutions are then recombined with each other and further optimized via simulated annealing. The best solution obtained overall is then chosen. The hydrogen-bond optimization tool in PrimeX performs essentially the same tasks as a number of other hydrogen-bond optimization tools such as NETWORK (Hooft, Sander et al., 1996) and Reduce (Word et al., 1999). calculations and are not influenced directly by the diffraction data. The advantage of this approach is that the under- determined nature of the crystallographic refinement calcu- lation is not made worse by the many more parameters for H-atom coordinates and B factors. H-atom positions are a function of the force field acting on all atoms, while the positions of non-H atoms are refined under the joint influence of crystallographic and force-field gradients. Thus, the H-atom coordinates are not biased towards the centre of mass of the electron-density distribution, as may occur in some forms of all-atom refinement (Coulson & Thomas, 1971). calculations and are not influenced directly by the diffraction data. The advantage of this approach is that the under- determined nature of the crystallographic refinement calcu- lation is not made worse by the many more parameters for H-atom coordinates and B factors. research papers Although this equation is the default method for assigning B-factor restraint weights in the polish workflow, stepwise optimization of this value is provided as an option. Also, minimization performed during weight optimization may optionally be applied towards progress in the refinement of the structure. The current work focuses specifically on interactions among atoms within the proteins since this issue is the central concern for computational chemistry applications. Close interactions with water and solvent molecules will be the focus of future work. Clashes generated from symmetry considerations were not counted for observations on the moderate-resolution data set since they are not optimized by the version of the Protein Preparation Wizard used in this study. Interatomic contacts were calculated after removing hydrogen bonds from consideration. Because of the lack of certainty regarding the positions of H atoms, all donor– acceptor atom pairs that could potentially be involved in a hydrogen bond were also excluded from the list of close contacts, even if an H atom was not found directly between them. This conservative approach avoided over-reporting as clashes any interactions that might actually be hydrogen bonds. Where alternate conformations were found, only the conformation with the higher occupancy was considered for the calculation of clashes. An initial optimization of hydrogen-bond orientation is applied and is followed by separate coordinate and B-factor reciprocal-space minimization steps. The model is then refined with a defined set of operations comprised of reciprocal-space coordinate minimization, hydrogen-bond optimization, reciprocal-space coordinate/B-factor minimization, simulated annealing and a final reciprocal-space coordinate/B-factor minimization. The optimization of X-ray and B-factor restraint weights as described above is repeated after this first refinement round. The same defined set of refinement procedures is then repeated twice more but without simulated annealing. The definition of a clash most commonly used in protein crystallography derives from the work of Jane Richardson, David Richardson and coworkers (Word et al., 1999; Davis et al., 2007; Chen, Arendall et al., 2010). It is simply the overlap of two van der Waals surfaces by 0.4 A˚ or more. It is employed with an atomic radius of 1.00 A˚ for polar and aromatic H atoms and a radius of 1.17 A˚ for all other H atoms, resulting in clashes between two H atoms with the same radii at separa- tions of 1.60 and 1.94 A˚ , respectively. research papers corresponds to the weight employed when the minimum Rfree value is observed. The weights selected for this set of proteins ranged between 0.25 and 1.73. Issues surrounding the selection of restraint weights when using an all-atom force field have been discussed by Fenn & Schnieders (2011). B-factor restraint weights (wB) are estimated from the high-resolution limit (r) of the diffraction data according to the equation was employed in this study. It was based on an analysis of 28 403 structures in the Cambridge Structural Database (Allen, 2002). Their results agreed well with the frequently cited van der Waals radii derived by Bondi (1964) when the available solid-state structural data were not nearly so extensive. The largest difference between the two studies was that the radius of the H atom was determined to be 1.1 A˚ rather than 1.2 A˚ as in the older work. wB ¼ wA102ðr2:2Þ: ð1Þ ð1Þ As observed by Rowland & Taylor (1996), atoms may at times have nonbonded interactions somewhat less than the sum of their van der Waals radii. For the purposes of this work, a center-to-center distance of less than or equal to 0.8 times the sum of the van der Waals radii was defined as a ‘clash’. A reasonable conclusion from the selection of data presented by Rowland and Taylor is that interatomic distances of less than or equal to 0.7 times the sum of van der Waals radii are rare. Such interactions were denoted as ‘severe clashes’ in this work. The functional form of this equation was chosen based on the known B-factor restraint-weight requirements in PrimeX at the bounds of the usual resolution range for refinement. For a low-resolution structure of 2.7 A˚ or worse, a weight of at least ten times wA is required. For refinement at high resolution (better than 1.7 A˚ ), a very low B-factor restraint weight (<0.1 times wA) is required. The values of the two constants in the equation were varied while observing the R factors from refinement in a broad resolution range. The current equation was observed to be as effective within PrimeX as stepwise optimization of wB as described for wA above and required much less computational time. Continued development of this method, such as the exploration of any effect of noncrys- tallographic symmetry restraints, will be reported in future work. research papers Clashes between two H atoms in the current work occur at a separation of 0.8 times the sum of their van der Waals radii, i.e. 1.76 A˚ . For most other atoms the definition applied here is less strict than that applied by the Richardson group. The one exception is for O atoms, which have a smaller radius in the Richardson system, resulting in clashes between two O atoms at 2.40 A˚ separation compared with 2.53 A˚ in the current work. The Richardson atomic parameters were designed from various theoretical and practical considerations (Word et al., 1999) to yield a system in which all observed clashes were exceptional. The approach in the current work was to use the values for atomic radii 2.3. Direct generation of all-atom models from selected structures To generate the all-atom models described in Table 3, H atoms were added to united-atom models from the PDB and the positions of the H atoms were optimized using the hydrogen-bond network optimization function described above (x2.2.4) as implemented in the Protein Preparation Wizard (Maestro v.9.2; Schro¨dinger LLC). The positions of all H atoms were then also optimized through energy minimiza- tion against the OPLS force field, with the positions of all heavier atoms held fixed. research papers The workflow applies reciprocal-space optimization of co- ordinates and thermal factors, simulated-annealing refinement and hydrogen-bond optimization in an automated manner as described below. It does not have as a purpose the remedia- tion of more serious errors in crystal structure fitting such as the choice of the wrong side-chain rotamer, mis-identification of protein electron density as part of the solvent model or the rebuilding of misplaced side chains, which would require the application of additional fitting functions. 2.2.2. Simulated-annealing refinement. Simulated-annealing refinement within PrimeX is implemented through the general- purpose molecular-modeling package IMPACT (Banks et al., 2005), employing concepts for simulated-annealing refinement validated in the program CNS (Adams et al., 1997). PrimeX simulated annealing provides two alternative energy models for dynamic simulation refinement. In the complete energy model, all molecular-mechanics terms are evaluated during the simulation. In the approximate method, the electrostatic and implicit solvation terms are not evaluated, a method similar to that employed in CNS (Adams et al., 1997). All calculations in this work involved the complete energy model. Bond orders are first assigned throughout the structure and H atoms are added. Initial analysis of the input structure provides basic crystallographic statistics for the structure using the bulk water correction in PrimeX (the flat model of Jiang & Bru¨nger, 1994) and overall anisotropic scaling. A detailed analysis of close nonbonded contacts for the input structure is also provided. 2.2.3. Electron-density map calculations. Map calculations in PrimeX are based on the SIGMAA weighting scheme of Read (1986), a data treatment that has been shown to decrease the bias in electron-density maps. As a next step, reciprocal-space minimization is applied at increasing weight on the X-ray terms (wA) in order to optimize this weight for subsequent refinement. The value selected 2.2.4. Hydrogen-bond network optimization. An addi- tional hydrogen-bond optimization tool in PrimeX analyzes 938 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 research papers Table 1 Ch † A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater than 0.7 times that sum. ‡ A severe clash occurs when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. Table 2 Classification of clashes and severe clashes requiring correction in Table 1. Classification No. of clashes and severe clashes Hydroxyl (or sufhydryl) group: rotation around carbon– oxygen (sulfur) bond relieves clash 27 Mistake in positioning of non-H atoms in the electron density 15 Flip of asparagine or glutamine residue relieves clash 4 Flip or alternate tautomer of histidine residue relieves clash 5 (Rowland & Taylor, 1996) unadjusted and to observe from ultrahigh-resolution protein structures how frequently clashes might reasonably be expected to occur owing to the local molecular environment. 2.4.2. Calculation of summary geometry statistics. Bond- length, bond-angle and torsion-angle statistics were calculated in NUCheck (Feng et al., 1998). Side-chain group planarity deviation was calculated using the Protein Reports utility in PrimeX. 3. Results In 37% of the clashes a chemically implausible interaction was observed in which a hydroxyl or sulfhydryl H atom pointed directly at another H atom. The positions of these H atoms were not supported in any obvious way by the observed electron density. The most likely explanation is that these hydrogen positions were oversights in the model-building process. In addition, 21% of the close interactions identified occurred at positions where the heavy atoms to which the H atoms were attached did not fit the electron density well and appeared to be incorrectly positioned. Clashes were included in this category only if the electron-density map provided reasonable doubt as to the correctness of the structure and suggested a more attractive alternate position. A further 12% of close contacts could be removed by flipping or changing the tautomer of an asparagine, glutamine or histidine side chain. 2.4. Structure-validation calculations 2.4.1. Clash detection and van der Waals radii. The Rowland & Taylor (1996) compilation of van der Waals radii 939 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 Acta Cryst. (2012). D68, 935–952 research papers Table 1 Characteristics of ultrahigh-resolution protein structures. Table 1 Ch Table 1 Characteristics of ultrahigh-resolution protein structures. ‘Corrected’ PDB code Resolution (A˚ ) No. of residues Bond- length r.m.s.d. (A˚ ) Bond- angle r.m.s.d. () Side-chain planarity r.m.s.d. (A˚ ) !-Angle standard deviation () Clashes† Severe clashes‡ Clashes† Severe clashes‡ Reference 1byz 0.90 52 0.019 2.0 0.006 2.9 0 1 0 0 Prive´ et al. (1999) 1dy5 0.87 248 0.020 2.6 0.013 6.6 8 2 2 0 Esposito et al. (2000) 1i1w 0.89 303 0.041 2.9 0.016 6.4 1 2 0 0 Natesh et al. (2003) 1m40 0.85 263 0.014 2.6 0.009 6.1 1 0 0 0 Minasov et al. (2002) 1muw 0.86 386 0.016 2.5 0.010 8.2 18 3 7 0 Fenn et al. (2004) 1p9g 0.84 41 0.017 2.4 0.009 6.7 0 0 0 0 Xiang et al. (2004) 1ucs 0.62 64 0.014 2.2 0.010 5.3 0 1 0 0 Ko et al. (2003) 1vyr 0.90 364 0.017 2.3 0.012 5.8 0 0 0 0 Khan et al. (2004) 1yk4 0.69 52 0.022 2.7 0.011 5.7 2 3 0 0 Bo¨nisch et al. (2005) 2b97 0.75 142 0.028 2.8 0.016 7.6 2 0 0 0 Hakanpa¨a¨ et al. (2006) 2h5c 0.82 198 0.025 2.4 0.015 7.3 2 0 2 0 Fuhrmann et al. (2006) 2vb1 0.65 129 0.021 3.1 0.012 7.1 1 0 0 0 Wang et al. (2007) 2wur 0.90 236 0.034 3.3 0.016 6.6 10 5 7 0 Shinobu et al. (2010) 2xu3 0.90 220 0.011 1.4 0.007 5.7 1 1 1 1 Hardegger et al. (2011) 3a38 0.70 83 0.028 2.9 0.010 6.8 0 0 0 0 Takeda et al. (2010) 3g63 0.88 381 0.014 2.1 0.009 6.7 2 0 1 0 Liebschner et al. (2009) 3ip0 0.89 158 0.012 1.5 0.007 6.1 1 0 0 0 Blaszczyk et al. (2003) 3mi4 0.80 223 0.028 2.5 0.015 6.8 3 3 1 0 A. Brzuszkiewicz, M. Dauter & Z. Dauter (unpublished work) Total 3543 52 21 21 1 Mean 0.021 2.5 0.011 6.4 Count per 100 residues 1.5 0.6 0.6 0.03 † A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater than 0.7 times that sum. ‡ A severe clash occurs when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. research papers the close contacts actually present in the structures. Thus, the frequency of bona fide ‘clashes’ and ‘severe clashes’ was 0.6 and 0.03 per 100 residues, respectively. Note that the second value is based on just a single observation (one severe clash in 2xu3). respectively. Overall, clashes in the moderate-resolution structure set were observed at a frequency of 4.0 per 100 residues, over six times the rate of bona fide clashes in the ultrahigh-resolution set. Severe clashes were observed at a frequency of 0.5 per 100 residues, compared with 0.03 for bona fide severe clashes for the reference ultrahigh-resolution data set (Table 1). respectively. Overall, clashes in the moderate-resolution structure set were observed at a frequency of 4.0 per 100 residues, over six times the rate of bona fide clashes in the ultrahigh-resolution set. Severe clashes were observed at a frequency of 0.5 per 100 residues, compared with 0.03 for bona fide severe clashes for the reference ultrahigh-resolution data set (Table 1). ) 3.1.2. Summary geometry statistics. The average r.m.s. deviations (r.m.s.d.s) of bond lengths and angles with respect to the Engh & Huber (1991) standard were 0.021 A˚ and 2.5, respectively (Table 1). The significance of the former number might be questioned since at this resolution the refined bond lengths are likely to reflect the effects of restraints. However, atomic positions are observed accurately enough at this resolution such that bond angles can be precisely determined from the crystallographic results. Thus, the latter value may be significant. However, two observations must be considered in interpreting this bond-angle value. Firstly, the program SHELX (Sheldrick & Schneider, 1997) may apply bond-angle restraints (as 1,3-atom distance restraints) during refinement, although published information rarely allows one to deduce the effect of these possible restraints on bond angles. Normally, one might expect the net effect of restraints would be to narrow the distribution of values observed. At the same time, a critical observation is that high-resolution protein crystal structures very frequently have r.m.s. Z scores (Spronk et al., 2004) greater than 1, i.e. the standard deviation of bond angles for these structures are greater than what would be predicted from the work of Engh & Huber (1991). 3.3. All-atom refinement with PrimeX 3.3.1. Clashes and severe clashes. The additional all-atom refinement in PrimeX applied to the moderate-resolution data set produced the structures characterized in Table 4. The frequency of regular clashes overall was 0.9 per 100 residues, which is well below the frequency originally observed for the ultrahigh-resolution set (1.5 per 100 residues; Table 1), but somewhat higher than the corrected value of 0.6 per 100 residues. The frequency of clashes overall was decreased more than fourfold from all-atom models derived from the coordi- nates as originally deposited. The frequency of severe clashes overall was 0.03 per 100 residues, the same value as obtained for the corrected ultrahigh-resolution structures (Table 1) and 17-fold lower than the frequency in the otherwise remediated moderate-resolution structures (Table 3). Seven of the 94 structures had neither type of clashes after all-atom refine- ment. All structures without clashes were solved at 2.2 A˚ resolution or better. 39 of the 94 structures had both no severe clashes and a lower frequency of clashes than the corrected ultrahigh-resolution structures. The average r.m.s.d. from planarity for side-chain groups was 0.011 A˚ and the average standard deviation of the ! angle was 6.4. For similar reasons, these two values may be considered to be reference points for the geometry of models at moderate resolution. Each of the residual clashes in the PrimeX-refined set was inspected with reference to a 2Fo  Fc composite OMIT map. Clear evidence of a better alternate interpretation of the electron density was present for 14% of the close contacts, owing to either large problems with the main-chain fit or to the need for a substantially different side-chain rotamer. Subtracting the number of clashes attributable to these issues from the total clashes provided an estimate of the frequency of bona fide regular clashes as 0.7 clashes per 100 residues, approaching the corrected frequency found in the ultrahigh- resolution structure set (0.6 clashes per 100 residues). Within this structural survey, many situations were observed to be ambiguous and were not counted. Thus, the level of clashes owing to model errors might actually have been somewhat higher. A very time-intensive comprehensive re-refinement of the structures would be required to confirm this suspicion, which is beyond the scope of the present work. research papers This observation (Joosten et al., 2009) is generally interpreted to mean that the bond angles are too widely distributed in very high resolution structures, possibly because these restraints are faulty owing to variations in bond lengths at high resolu- tion. The possibility that the Engh and Huber parameters predict too narrow a distribution owing to biases in the small- molecule structures from which the parameters were derived is generally discounted. 3.2.2. Summary geometry statistics. As shown in Table 3, the bond-length r.m.s.d.s for the set of proteins varied over a wide range, from 0.004 A˚ for 3phe to 0.031 A˚ for 3lje, with an average of 0.014 A˚ . Bond-angle r.m.s.d.s varied from a minimum of 0.6 (3ni0) to a maximum of 2.5 (3nof), with an average of 1.4. The average r.m.s.d. for side-chain group planarity was 0.005 A˚ and the average peptide torsion-angle standard deviation was 5.1. A more detailed examination and comparison of these summary statistics follows in x3.3.2. 3.1. Ultrahigh-resolution structures Before exploring the close contacts (clashes and severe clashes as defined above) and structural geometry at moderate resolution, some perspective can be obtained on summary geometric statistics and the occurrence of clashes from ultra- high-resolution protein structures. 3.1.1. Clashes and severe clashes. Table 1 shows observa- tions from 18 X-ray crystal structures with at least 0.9 A˚ resolution which were refined (by the authors of the respective structures) with H atoms present. (At this resolution hydrogen positions may have been guided by electron density, although electron density need not have been observed for all H atoms.) Over the entire set of structures, ‘clashes’ and ‘severe clashes’ were observed with a frequency of 1.5 and 0.6 occurrences per 100 residues, respectively. Ultimately, only 30% of the observed clashes withstood critical examination and avoided being included in Table 2. In Table 1, the ‘corrected’ columns offer a better estimate of All of the observed clashes were examined individually to determine their origin. The results are presented in Table 2. 940 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 research papers research papers 942 Bell et al  PrimeX Acta Cryst (2012) D68 935–952 Table 3 Statistics for structures of moderate-resolution data in the PDB. PDB code Resolution (A˚ ) No. of residues Refinement program Bond- length r.m.s.d. (A˚ ) Bond- angle r.m.s.d. () Side-chain planarity r.m.s.d. (A˚ ) !-Angle standard deviation () R factor† Rfree† Clashes‡ Severe clashes§ Reference 2x3k 2.50 1145 REFMAC 0.015 1.5 0.004 6.7 0.216 0.266 58 7 Schmelz (2010) 2xda 1.85 150 REFMAC 0.014 1.3 0.005 5.7 0.189 0.216 2 1 Paz et al. (2011) 2xn8 1.64 409 REFMAC 0.023 1.8 0.009 6.2 0.181 0.220 6 0 Ouellet et al. (2011) 2xpp 1.74 161 REFMAC 0.018 1.6 0.005 4.8 0.208 0.252 5 0 Diebold et al. (2010) 2xs6 2.09 166 REFMAC 0.007 1.0 0.002 4.9 0.219 0.258 4 0 } 2xsn 2.68 1341 BUSTER 0.013 1.6 0.008 2.8 0.224 0.260 13 2 J. R. C. Muniz, C. D. O. Cooper, W. W. Yue, E. Krysztofinska, F. Vondelft, S. Knapp, O. Gileadi, C. H. Arrowsmith, A. M. Edwards, J. Weigelt, C. Bountra, K. L. Kavanagh & U. Oppermann (unpublished work) 2xsq 1.72 179 REFMAC 0.009 1.2 0.004 5.4 0.173 0.191 0 1 } 2xsw 1.90 666 PHENIX 0.011 1.3 0.004 5.6 0.184 0.209 8 0 } 2xsx 1.70 869 REFMAC 0.015 1.5 0.007 5.9 0.177 0.207 11 3 M. Vollmar, E. Krysztofinska, A. Chaikuad, T. Krojer, R. Cocking, F. Vondelft, C. Bountra, C. H. Arrowsmith, J. Weigelt, A. Edwards, W. W. Yue & U. Oppermann (unpublished work) 2xu7 1.90 752 REFMAC 0.023 1.8 0.008 7.1 0.211 0.245 14 1 Lejon et al. (2011) 2xul 2.20 645 REFMAC 0.029 2.0 0.005 6.5 0.196 0.235 20 1 Fokina et al. (2010) 2xvs 1.80 166 REFMAC 0.016 1.5 0.007 5.8 0.200 0.249 0 0 Adams et al. (2012) 2xvv 2.40 582 CNS 0.007 1.2 0.005 1.0 0.211 0.252 35 1 Ryan et al. (2011) 2xxj 1.96 1239 PHENIX 0.007 1.0 0.003 9.0 0.190 0.237 27 3 J. Tickle, E. De Mendoza Barbera & F. M. D. Vellieux (unpublished work) 3acw 1.63 284 REFMAC 0.007 1.5 0.006 4.9 0.228 0.252 5 0 Lin et al. (2010) 3aey 1.92 698 CNS 0.005 1.2 0.004 1.2 0.192 0.214 29 5 Murakawa et al. (2011) 3ajx 1.60 828 CNS 0.004 1.2 0.004 1.3 0.198 0.218 8 0 Orita et al. (2010) 3ale 2.50 1460 CNS 0.007 1.3 0.004 1.3 0.227 0.279 117 15 Morita et al. research papers (2010) 3am9 2.17 2584 REFMAC 0.022 1.9 0.008 6.9 0.182 0.248 99 12 Matsumoto et al. (2010) 3l9w 1.75 695 REFMAC 0.018 1.6 0.007 5.6 0.212 0.239 16 1 Roosild et al. (2010) 3lb4 1.56 274 REFMAC 0.011 1.3 0.005 4.2 0.253 0.281 10 0 Thompson et al. (2010) 3lfl 2.10 686 PHENIX 0.007 1.0 0.002 5.2 0.207 0.266 43 8 Zhou et al. (2011) 3lje 1.75 121 CNS 0.031 2.4 0.010 3.4 0.185 0.217 14 3 Pizzo et al. (2010) 3ljq 1.90 570 CNS 0.012 1.3 0.006 7.3 0.155 0.197 17 1 Wang & Guo (2010) 3lju 1.70 373 REFMAC 0.016 1.4 0.007 6.0 0.203 0.238 1 0 Tong et al. (2010) 3lpf 2.26 1180 REFMAC 0.011 2.2 0.004 8.6 0.275 0.300 241 34 Wallace et al. (2010) 3lre 2.20 589 REFMAC 0.020 1.7 0.006 6.4 0.231 0.278 28 3 Peters et al. (2010) 3lrp 2.50 181 CNS 0.006 1.2 0.004 1.2 0.197 0.263 7 1 Cook et al. (2010) 3lt3 2.10 404 REFMAC 0.006 0.9 0.002 4.3 0.242 0.293 5 0 Biswas et al. (2010) 3m0e 2.63 1729 PHENIX 0.010 1.2 0.003 5.3 0.216 0.250 90 11 Chen, Sysoeva et al. (2010) 3m0h 1.58 1685 CNS 0.004 1.2 0.004 1.3 0.162 0.182 28 6 Yoshida et al. (2010) 3m4z 1.94 309 REFMAC 0.012 1.2 0.004 4.9 0.177 0.202 2 0 Heldman et al. (2010) 3m5o 1.60 410 REFMAC 0.009 1.2 0.004 5.9 0.240 0.270 10 3 Romano et al. (2010) 3m67 1.80 257 REFMAC 0.027 2.0 0.010 7.0 0.178 0.230 9 0 Cˇ apkauskaite˙ et al. (2010) 3mbv 2.00 222 REFMAC 0.016 1.4 0.007 5.1 0.270 0.282 2 1 Borshchevskiy et al. (2010) 3mfa 1.63 194 REFMAC 0.013 1.3 0.004 6.9 0.198 0.223 8 0 Morin et al. (2011) 3mif 2.00 310 REFMAC 0.009 1.1 0.002 6.1 0.261 0.279 11 1 Chufa´n et al. (2010) 3mk9 2.08 173 REFMAC 0.006 1.1 0.005 1.0 0.216 0.242 7 0 Compton et al. (2011) 3mke 1.75 265 REFMAC 0.012 1.3 0.005 5.8 0.158 0.188 1 0 Ke et al. (2011) 3mvi 1.60 698 REFMAC 0.012 1.3 0.004 5.4 0.174 0.211 10 2 Niu et al. (2010) 3mxe 1.85 198 REFMAC 0.009 1.2 0.003 6.1 0.202 0.241 1 0 Ali et al. (2010) 3n2v 1.55 158 REFMAC 0.025 1.8 0.011 6.1 0.187 0.204 1 0 Attolino et al. 3.2. Moderate-resolution crystal structures as deposited 94 crystal structures with a broad range of sizes from several different refinement programs were examined (Table 3). Their most important common characteristics were recent deposi- tion in the PDB and falling into the most highly populated resolution range typical for protein crystal structures (see x2 for further details on the selection of this data set). 3.2.1. Clashes and severe clashes. The occurrence of close contacts was enumerated after addition of H atoms and after careful optimization of H-atom positions without changing the coordinates of any non-H atoms. Table 3 shows the frequency of clashes and severe clashes for each protein. Their rates of occurrence per 100 residues were observed in a very broad range from 20.4 (3lpf) to 0.0 (three instances) and from 2.9 (3lpf) to 0.0 (35 instances) for clashes and severe clashes, Clash frequencies derived from Tables 1, 3 and 4 are compared in Table 5, as well as with respect to the various 941 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 942 Bell et al.  PrimeX research papers (2010) 3nfy 1.94 498 REFMAC 0.019 1.8 0.007 6.3 0.187 0.279 23 6 Patterson et al. (2010) 3ni0 1.60 182 PHENIX 0.003 0.6 0.001 3.2 0.230 0.257 2 1 Swiecki et al. (2011) 3nk4 2.00 581 PHENIX 0.010 1.2 0.003 5.7 0.234 0.242 14 1 Han et al. (2010) 3nl6 2.61 1545 PHENIX 0.007 1.2 0.002 5.2 0.238 0.252 174 22 Paul et al. (2010) 3nm8 2.00 570 REFMAC 0.010 1.4 0.005 2.4 0.230 0.278 53 6 Sendovski et al. (2010) 3nmi 2.01 636 REFMAC 0.008 0.9 0.014 6.9 0.207 0.245 30 1 Radford et al. (2011) 3nof 1.60 213 REFMAC 0.029 2.5 0.011 5.9 0.197 0.225 19 3 Hall et al. (2011) 3nok 1.65 466 REFMAC 0.023 1.9 0.009 7.0 0.199 0.243 11 0 Carrillo et al. (2010) 3nv6 2.20 404 REFMAC 0.016 1.5 0.005 5.9 0.200 0.264 14 2 Yang et al. (2010) 3nxg 1.95 1291 REFMAC 0.008 1.1 0.002 5.9 0.180 0.213 21 0 Neu et al. (2010) 3nxp 2.20 363 REFMAC 0.011 1.3 0.003 6.1 0.213 0.239 11 2 Chen, Pelc et al. (2010) 3o0a 1.77 425 REFMAC 0.009 1.2 0.003 9.0 0.211 0.250 6 1 V. Cura, N. Olieric, E.-D. Wang, D. Moras, G Eriani & J. Cavarelli (unpublished work) 3o3p 1.70 635 PHENIX 0.007 1.1 0.002 5.2 0.221 0.255 39 4 Empadinhas et al. (2011) 3o4h 1.82 2302 REFMAC 0.019 1.7 0.007 6.2 0.244 0.267 71 19 Harmat et al. (2011) 3o79 1.60 202 REFMAC 0.022 1.7 0.009 5.3 0.210 0.237 4 2 Khan et al. (2010) Table 3 Statistics for structures of moderate-resolution data in the PDB. 942 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 research papers refinement programs. Unfortunately, refinement results from BUSTER (Bricogne et al., 2011) were found to be relatively rare and only two instances were found in our data set. However the statistics for these two proteins suggest 0.6–0.7 clashes per 100 residues that might be considered a reasonable target considering the results above. 3.3.2. Summary molecular geometry and refinement statistics Table 5 also provides a summary of the measures Table 3 (continued) PDB code Resolution (A˚ ) No. of residues Refinement program Bond- length r.m.s.d. (A˚ ) Bond- angle r.m.s.d. () Side-chain planarity r.m.s.d. (A˚ ) !-Angle standard deviation () R factor† Rfree† Clashes‡ Severe clashes§ Reference 3o86 1.60 709 PHENIX 0.010 1.3 0.004 5.8 0.173 0.198 17 0 Eidam et al. research papers (2010) 3oae 2.80 2105 CNS 0.011 1.5 0.006 1.4 0.272 0.288 238 40 Dasgupta et al. (2011) 3oag 2.30 669 REFMAC 0.009 1.2 0.003 7.8 0.208 0.262 13 2 Corminboeuf et al. (2010) 3oc2 1.97 495 PHENIX 0.007 1.1 0.002 5.4 0.181 0.223 21 3 Sainsbury et al. (2010) 3occ 1.70 1424 REFMAC 0.015 1.5 0.010 6.6 0.169 0.197 29 8 New York SGX Research Center for Structural Genomics (unpublished work) 3oep 1.75 485 REFMAC 0.006 1.1 0.003 5.4 0.191 0.215 0 0 Jacques et al. (2011) 3oi7 2.40 1028 REFMAC 0.012 1.3 0.003 5.9 0.237 0.269 19 8 Clasquin et al. (2011) 3oia 1.65 405 REFMAC 0.011 1.3 0.004 5.7 0.191 0.241 6 0 Lee et al. (2011) 3olz 2.75 743 PHENIX 0.006 0.9 0.002 5.0 0.211 0.265 26 1 Kumar & Mayer (2010) 3om1 1.68 740 PHENIX 0.006 1.0 0.002 5.0 0.193 0.224 14 1 Kumar & Mayer (2010) 3onw 2.38 702 REFMAC 0.009 1.1 0.002 5.1 0.251 0.280 13 1 Bosch et al. (2011) 3orv 1.91 1707 REFMAC 0.025 1.9 0.009 6.4 0.171 0.212 22 1 Abu Tarboush et al. (2010) 3oux 2.40 550 PHENIX 0.008 1.1 0.002 4.7 0.219 0.285 35 3 Sun & Weis (2011) 3p10 1.70 471 REFMAC 0.012 1.3 0.004 5.2 0.184 0.218 7 0 Begley et al. (2011) 3p14 2.51 1608 REFMAC 0.019 1.8 0.005 6.5 0.201 0.266 108 22 T. T. N. Doan, P. Prabhu, J. K. Lee, L. W. Wang, J. K. Kim, M. Jeya & Y. J. Ahn (unpublished work) 3p1a 1.70 281 REFMAC 0.015 1.5 0.006 5.1 0.169 0.211 4 0 Structural Genomics Consortium (unpublished work) 3p1m 2.54 1017 REFMAC 0.013 1.3 0.003 5.8 0.240 0.270 28 2 Structural Genomics Consortium (unpublished work) 3p2e 1.68 402 CNS 0.005 1.2 0.005 1.4 0.207 0.244 16 0 Husain et al. (2011) 3p32 1.90 306 REFMAC 0.015 1.3 0.004 5.6 0.226 0.254 3 0 Seattle Structural Genomics Center for Infectious Disease (unpublished work) 3p4i 2.35 760 REFMAC 0.016 1.5 0.003 5.7 0.200 0.244 11 0 Seattle Structural Genomics Center for Infectious Disease (unpublished work) 3p4l 1.80 198 CNS 0.010 1.6 0.008 2.2 0.205 0.240 7 2 Yang et al. (2011) 3p5o 1.60 127 REFMAC 0.007 1.0 0.003 4.9 0.179 0.207 1 0 Nicodeme et al. (2010) 3p5t 2.70 1653 REFMAC 0.010 1.3 0.003 6.1 0.225 0.272 179 33 Li, Tong et al. research papers (2011) 3p77 1.60 371 REFMAC 0.012 1.4 0.005 6.1 0.194 0.226 2 0 Hee et al. (2010) 3p7h 2.30 520 REFMAC 0.110 1.2 0.004 6.5 0.204 0.245 16 2 Chatwell et al. (2008) 3p8s 2.00 302 REFMAC 0.010 1.2 0.003 6.2 0.192 0.226 3 0 U. Sharma, N. Ahmed, M. V. Krishnasastry & C. G. Suresh (unpublished work) 3paj 2.00 599 REFMAC 0.009 1.2 0.007 2.0 0.213 0.259 20 2 Center for Structural Genomics of Infectious Diseases (unpublished work) 3pde 1.75 1149 REFMAC 0.013 1.6 0.004 5.0 0.177 0.209 17 0 New York SGX Research Center for Structural Genomics (unpublished work) 3pdt 1.80 251 REFMAC 0.011 1.3 0.003 5.9 0.202 0.247 4 0 Crawley et al. (2011) 3peh 2.75 523 REFMAC 0.013 1.7 0.007 2.5 0.236 0.279 8 1 Structural Genomics Consortium (unpublished work) 3pgj 2.49 1075 REFMAC 0.007 1.4 0.004 0.9 0.238 0.279 40 1 Center for Structural Genomics of Infectious Diseases (unpublished work) 3pgy 1.92 1609 REFMAC 0.017 1.5 0.006 5.7 0.190 0.227 29 2 Center for Structural Genomics of Infectious Diseases (unpublished work) 3ph7 2.50 1376 BUSTER 0.014 1.7 0.008 2.4 0.249 0.291 11 0 Artz et al. (2011) 3phe 2.20 2232 CNX 0.004 0.7 0.003 5.1 0.222 0.265 131 15 Kumar et al. (2011) 3pj9 2.10 549 REFMAC 0.017 1.5 0.005 6.2 0.206 0.221 16 2 Center for Structural Genomics of Infectious Diseases (unpublished work) 3pjp 1.60 389 PHENIX 0.006 1.0 0.003 5.6 0.214 0.251 5 0 Sun et al. (2010) 3pk0 1.74 1043 REFMAC 0.017 1.5 0.007 5.5 0.184 0.210 5 0 Seattle Structural Genomics Center for Infectious Disease (unpublished work) Total 66891 2639 349 Mean 0.014 1.4 0.005 5.1 0.207 0.243 No. per 100 residues 4.0 0.5 † As calculated in PrimeX without TLS scaling. ‡ A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater than 0.7 times that sum. § A severe clash occurs when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. } L.Tresaugues, M. Welin, C. H. Arrowsmith, H. Berglund, C. Bountra, R. Collins, A. M. Edwards, S. Flodin, A. Flores, S. Graslund, M. Hammarstrom, I. Johansson, T. Karlberg, S. Kol, T. Kotenyova, E. Kouznetsova, M. Moche, T. Nyman, C. Persson, H. Schuler, P. Schutz, M. I. Siponen, A. G. Thorsell, S. Table 4 944 Bell et al  PrimeX Acta Cryst (2012) D68 9 Table 4 Statistics for structures in the moderate-resolution data set as refined in PrimeX. PDB code Resolution (A˚ ) No. of residues Bond-length r.m.s.d. (A˚ ) Bond-angle r.m.s.d. () Side-chain planarity r.m.s.d. Acta Cryst. (2012). D68, 935–952 research papers Van der Berg, E. Wahlberg, J. Weigelt & P. Nordlund (unpublished work). Table 3 (continued) † As calculated in PrimeX without TLS scaling. ‡ A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater than 0.7 times that sum. § A severe clash occurs when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. } L.Tresaugues, M. Welin, C. H. Arrowsmith, H. Berglund, C. Bountra, R. Collins, A. M. Edwards, S. Flodin, A. Flores, S. Graslund, M. Hammarstrom, I. Johansson, T. Karlberg, S. Kol, T. Kotenyova, E. Kouznetsova, M. Moche, T. Nyman, C. Persson, H. Schuler, P. Schutz, M. I. Siponen, A. G. Thorsell, S. Van der Berg, E. Wahlberg, J. Weigelt & P. Nordlund (unpublished work). refinement programs. Unfortunately, refinement results from BUSTER (Bricogne et al., 2011) were found to be relatively rare and only two instances were found in our data set. However, the statistics for these two proteins suggest improved results from this program regarding close contacts. None of the other programs even approached the values of 0.6–0.7 clashes per 100 residues that might be considered a reasonable target considering the results above. 3.3.2. Summary molecular geometry and refinement statistics. Table 5 also provides a summary of the measures of molecular geometry over the three data sets in this study. The average bond-length r.m.s.d. for the PrimeX-refined 943 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 944 Bell et al.  PrimeX research papers Z score of 0.87 (0.49–1.23), values that are very similar to those of the PrimeX-refined structures. Individual bond-length r.m.s. Z scores are available as Supplementary Material1. The average side-chain group planarity r.m.s.d. for PrimeX was 0.006 A˚ , which is a fairly typical value for this quantity among the refinement programs. Side-chain planarity deviations were all small and not very different between these two data sets, nor did they differ much by refinement program (Table 5). The average !-angle standard deviation for PrimeX, 7.1, was larger than for any of the refinement programs used to produce the original moderate-resolution data set, but com- pared well with the value of 6.4 obtained for the ultrahigh- resolution set (Table 5). The values among all the refinement programs could be described as a range of values from 4.8 to 5.7, with two outliers near 2 for BUSTER and CNS/CNX. The number of examples of BUSTER-refined proteins was too small to draw a conclusion. However, CNS and CNX clearly often restrain the ! angle very tightly. This issue was originally observed by Priestle (2003). Note that the overall average for the ! angles did not represent the situation well, since two of the 12 structures in the CNS/CNX subgroup had standard deviations in the normal range (Table 3). These two unchar- acteristic CNS/CNX structures indicate that at least a few users of CNS/CNX have taken steps to loosen these peptide- bond planarity restraints. Ten of the 12 members of the CNS/ CNX subgroup had standard deviations for ! of 1.5 or less, implying flattened peptide bonds throughout these crystal structures. The !-angle standard deviation did not vary much among the other refinement programs. The average value for REFMAC did not stand out as the one from CNS/CNX does. Table 5 also allows comparison among the four programs originally used to refine the moderate-resolution data set. PHENIX and CNS clearly restrained bond lengths more tightly than did PrimeX. The bond-length r.m.s.d. for the REFMAC-refined set was not very different from the PrimeX- refined set. The average bond-angle r.m.s.d. for PrimeX was 2.2, which is somewhat larger than the average value of 1.4 over the original data set. The r.m.s. Z scores for bond angles changed from an average value of 0.71 (0.37–1.25) as deposited to an average value of 1.17 (1.00–1.49) after PrimeX refinement. The average bond-angle r.m.s.d. research papers research papers Table 4 (continued) Table 4 (continued) PDB code Resolution (A˚ ) No. of residues Bond-length r.m.s.d. (A˚ ) Bond-angle r.m.s.d. () Side-chain planarity r.m.s.d. (A˚ ) !-Angle standard deviation () R factor Rfree Clashes Severe clashes 3p10 1.70 471 0.018 2.1 0.006 6.3 0.173 0.222 4 0 3p14 2.51 1608 0.015 2.1 0.005 7.1 0.211 0.259 21 5 3p1a 1.70 281 0.023 2.0 0.009 5.8 0.162 0.212 2 0 3p1m 2.54 1017 0.013 2.1 0.002 6.9 0.231 0.273 8 0 3p2e 1.68 402 0.021 2.2 0.008 6.6 0.186 0.234 3 0 3p32 1.90 306 0.024 2.3 0.005 7.3 0.204 0.252 3 0 3p4i 2.35 760 0.019 2.1 0.004 7.0 0.195 0.241 2 0 3p4l 1.80 198 0.018 2.0 0.008 7.2 0.200 0.248 1 0 3p5o 1.60 127 0.023 2.1 0.008 6.1 0.156 0.209 1 0 3p5t 2.70 1653 0.016 2.0 0.004 7.4 0.230 0.285 4 0 3p77 1.60 371 0.021 2.3 0.008 6.8 0.173 0.221 2 0 3p7h 2.30 520 0.016 2.0 0.005 8.1 0.200 0.244 2 0 3p8s 2.00 302 0.018 2.2 0.006 7.7 0.165 0.221 2 0 3paj 2.00 599 0.018 2.2 0.006 6.2 0.179 0.251 6 0 3pde 1.75 1149 0.020 2.1 0.006 6.1 0.169 0.212 8 0 3pdt 1.80 251 0.018 2.0 0.005 7.6 0.182 0.237 2 0 3peh 2.75 523 0.019 2.6 0.006 8.4 0.234 0.288 10 0 3pgj 2.49 1075 0.014 1.9 0.003 7.0 0.249 0.287 3 0 3pgy 1.92 1609 0.017 2.0 0.005 6.7 0.173 0.225 8 0 3ph7 2.50 1376 0.016 2.0 0.006 6.4 0.248 0.304 8 0 3phe 2.20 2232 0.016 2.0 0.004 7.7 0.213 0.255 11 1 3pj9 2.10 549 0.021 2.2 0.004 7.3 0.185 0.220 10 0 3pjp 1.60 389 0.022 2.4 0.008 7.7 0.203 0.253 2 0 3pk0 1.74 1043 0.023 2.3 0.007 6.2 0.160 0.207 7 0 Total 66891 573 23 Mean 1.99 0.019 2.2 0.006 7.1 0.193 0.242 No. per 100 residues 0.9 0.03 of the PrimeX-refined structures. Individual bond-angle r.m.s. Z scores are available in the Supplementary Material1. proteins was 0.019 A˚ , compared with 0.015 A˚ for the original data set. The r.m.s. Z scores (Spronk et al., 2004) for bond lengths changed from an average value of 0.56 (0.15–1.27) as deposited to an average value of 0.89 (0.65–1.24) after PrimeX refinement. The ultrahigh-resolution set had a bond-length r.m.s.d. of 0.021 A˚ and a mean bond-length r.m.s. 1 Supplementary material has been deposited in the IUCr electronic archive (Reference: RR5017). Services for accessing this material are described at the back of the journal. Table 4 (A˚ ) !-Angle standard deviation () R factor Rfree Clashes Severe 2x3k 2.50 1145 0.019 2.3 0.005 8.0 0.212 0.267 19 0 2xda 1.85 150 0.025 2.5 0.007 6.9 0.175 0.215 3 0 2xn8 1.64 409 0.022 2.2 0.005 6.6 0.170 0.221 2 0 2xpp 1.74 161 0.016 1.9 0.005 5.2 0.197 0.251 3 0 2xs6 2.09 166 0.019 2.0 0.004 6.8 0.195 0.263 1 0 2xsn 2.68 1341 0.017 2.0 0.005 6.6 0.229 0.275 16 0 2xsq 1.72 179 0.022 2.1 0.009 6.4 0.158 0.195 0 0 2xsw 1.90 666 0.021 2.3 0.007 7.0 0.166 0.211 5 0 2xsx 1.70 869 0.023 2.1 0.007 6.4 0.162 0.203 4 0 2xu7 1.90 752 0.020 2.2 0.006 8.0 0.186 0.235 1 0 2xul 2.20 645 0.016 2.2 0.003 7.2 0.189 0.231 1 0 2xvs 1.80 166 0.022 2.3 0.008 6.9 0.177 0.252 1 0 2xvv 2.40 582 0.019 2.3 0.005 6.8 0.182 0.245 3 0 2xxj 1.96 1239 0.017 1.9 0.007 6.5 0.179 0.241 8 0 3acw 1.63 284 0.019 2.0 0.009 5.8 0.201 0.242 1 0 3aey 1.92 698 0.021 2.2 0.008 6.3 0.171 0.204 12 0 3ajx 1.60 828 0.022 2.2 0.006 5.7 0.178 0.211 1 0 3ale 2.50 1460 0.015 2.1 0.004 7.5 0.233 0.272 16 0 3am9 2.17 2584 0.017 2.1 0.005 7.1 0.175 0.241 23 0 3l9w 1.75 695 0.023 2.4 0.009 6.5 0.199 0.233 3 0 3lb4 1.56 274 0.018 2.3 0.012 5.1 0.244 0.277 1 0 3lfl 2.10 686 0.017 1.9 0.003 8.5 0.202 0.269 8 0 3lje 1.75 121 0.025 2.4 0.006 6.5 0.175 0.211 1 0 3ljq 1.90 570 0.020 2.2 0.006 7.2 0.144 0.200 5 0 3lju 1.70 373 0.021 2.2 0.007 6.9 0.192 0.237 1 0 3lpf 2.26 1180 0.017 2.3 0.005 10.2 0.280 0.325 30 1 3lre 2.20 589 0.016 2.1 0.005 7.0 0.218 0.273 2 0 3lrp 2.50 181 0.017 2.2 0.004 7.1 0.184 0.252 3 0 3lt3 2.10 404 0.019 2.4 0.005 7.5 0.188 0.282 2 0 3m0e 2.63 1729 0.015 1.9 0.004 6.8 0.236 0.273 18 0 3m0h 1.58 1685 0.022 2.2 0.008 5.8 0.144 0.178 9 0 3m4z 1.94 309 0.020 2.1 0.007 6.1 0.160 0.203 0 0 3m5o 1.60 410 0.015 2.0 0.007 7.0 0.206 0.247 2 2 3m67 1.80 257 0.026 2.6 0.005 7.9 0.168 0.226 4 0 3mbv 2.00 222 0.017 1.8 0.006 5.9 0.248 0.292 1 0 3mfa 1.63 194 0.021 2.3 0.012 8.6 0.181 0.225 1 0 3mif 2.00 310 0.021 2.4 0.008 9.3 0.194 0.242 3 0 3mk9 2.08 173 0.017 1.9 0.006 6.8 0.186 0.246 2 0 3mke 1.75 265 0.020 2.1 0.010 6.8 0.148 0.191 0 0 3mvi 1.60 698 0.023 2.2 0.008 6.3 0.162 0.205 3 0 3mxe 1.85 198 0.019 2.3 0.005 7.7 0.183 0.251 0 0 3n2v 1.55 158 0.023 2.4 0.008 7.2 0.178 0.227 0 0 3nfy 1.94 498 0.017 2.1 0.004 6.9 0.181 0.259 6 0 3ni0 1.60 182 0.017 1.9 0.003 5.2 0.213 0.254 0 0 3nk4 2.00 581 0.021 2.5 0.006 7.8 0.217 0.247 7 0 3nl6 2.61 1545 0.016 2.4 0.005 7.8 0.225 0.273 23 2 3nm8 2.00 570 0.018 2.2 0.007 7.4 0.191 0.262 2 0 3nmi 2.01 636 0.023 2.4 0.005 6.8 0.198 0.244 9 1 3nof 1.60 213 0.021 2.4 0.010 6.5 0.186 0.216 2 0 3nok 1.65 466 0.016 2.0 0.005 7.4 0.198 0.243 4 0 3nv6 2.20 404 0.015 2.0 0.004 6.6 0.196 0.260 0 0 3nxg 1.95 1291 0.018 2.2 0.006 7.5 0.166 0.213 8 0 3nxp 2.20 363 0.015 2.1 0.005 8.1 0.196 0.238 9 0 3o0a 1.77 425 0.019 2.2 0.007 7.2 0.187 0.248 2 2 3o3p 1.70 635 0.020 2.5 0.006 8.2 0.216 0.251 14 0 3o4h 1.82 2302 0.015 2.2 0.005 6.5 0.216 0.270 16 2 3o79 1.60 202 0.024 2.5 0.012 11.1 0.192 0.239 2 1 3o86 1.60 709 0.021 2.1 0.007 6.8 0.167 0.196 4 0 3oae 2.80 2105 0.017 2.5 0.005 9.4 0.237 0.294 39 0 3oag 2.30 669 0.015 2.1 0.005 8.3 0.210 0.261 1 0 3oc2 1.97 495 0.019 2.3 0.007 7.5 0.171 0.226 5 1 3occ 1.70 1424 0.021 2.1 0.007 7.1 0.156 0.192 15 5 3oep 1.75 485 0.020 2.2 0.008 6.5 0.168 0.210 4 0 3oi7 2.40 1028 0.015 1.9 0.005 6.9 0.241 0.280 7 0 3oia 1.65 405 0.022 2.3 0.008 7.0 0.171 0.237 5 0 3olz 2.75 743 0.014 2.0 0.003 6.8 0.238 0.264 10 0 3om1 1.68 740 0.022 2.4 0.007 6.9 0.183 0.223 6 0 3onw 2.38 702 0.018 2.2 0.005 7.4 0.225 0.263 6 0 3orv 1.91 1707 0.017 2.0 0.006 7.2 0.155 0.207 10 0 3oux 2.40 550 0.017 2.2 0.004 6.4 0.220 0.267 8 0 Table 4 Statistics for structures in the moderate-resolution data set as refined in PrimeX. Table 5 † Corrected for obvious errors in deposited structures, as shown in Tables 1 and 2. ‡ Corrected for clashes owing to errors in the st clashes and severe clashes per 100 residues are 0.9 and 0.03, respectively. At the same time, some users of REFMAC did very tightly restrain peptide bonds. The lowest standard deviation for ! was not from among the CNS/CNX-refined structures, but instead was produced by REFMAC (3pgj; 0.9). group relieved all close contacts and was confirmed by a small pair of negative and positive difference features in an Fo  Fc map (result not shown). The refinement program CNX did not correct this situation. A reasonable hypothesis for why it did not do so is that the interactions between the methionine methyl group and the other two residues, as represented through a united-atom model in CNX, were not unfavorable enough to cause a change in the positions of these atoms. Changes in overall structure quality owing to PrimeX refinement, as judged by the Ramachandran Z scores (Spronk et al., 2004), were generally small. Only one change was noted as significant by the program WHAT_CHECK. This change was from an original value of 3.16 for the protein 3nl6 as originally deposited to a value of 2.11 after PrimeX refine- ment. The mean Ramachandran Z score changed from 0.37 (range 3.16 to +3.69) as originally deposited to 0.51 (range 2.56 to +3.05) after PrimeX refinement. Individual Rama- chandran Z scores before and after PrimeX refinement are shown in the Supplementary Material. 3.4.2. Backbone change to relieve clash leads to additional ligand hydrogen bonds. In PDB entry 3nl6, atoms in the side chain of ValC209 clash with the side chain of ValC15 (Fig. 2). In producing the all-atom model derived from this structure through energy minimization, these interactions were suffi- ciently repulsive that the bond angles around C of ValC15 were distorted rather than allowing atoms to overlap to such an extreme extent. The close contact was relieved during PrimeX refinement using the ‘polish’ workflow by motion of residues C209 and C210 away from residue C15 and towards the bound thiamine phosphate (TPS), as shown in Fig. 2. This side-chain motion occurred with a change in the conformation of the main chain for residue C209. Table 5 This change in the back- bone position and a few other more subtle atomic shifts provided multiple additional hydrogen-bond interactions between the protein and the TPS molecule, a difference that has potentially major implications for the understanding of TPS binding. This large structural change during refinement was probably related to the resolution of the strain of close contacts in the model, but electrostatic gradients or other influences during refinement could also play a role. Assuming that the program was used as intended and in the absence of any indication to the contrary (Paul et al., 2010), phenix.refine seems to have tolerated these severe implied all-atom clashes during refinement. The average Rfree value over the moderate-resolution set was the same with or without the additional PrimeX all-atom refinement (0.243 versus 0.242; Tables 2 and 3). The average for all working R values was somewhat lower for the PrimeX- refined structures (0.193) versus the average from the original structures (0.207). research papers in the ultrahigh-resolution set was 2.5, which is greater than that produced by any of the other refinement programs, but closest to the value for PrimeX. The r.m.s. Z score for bond angles in the ultrahigh- resolution set was 1.12 (0.79–1.46), which is also similar to that 945 Acta Cryst. (2012). D68, 935–952 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 research papers Table 5 Summary of geometry and clash statistics. Structure set No. of structures Bond-length r.m.s.d. (A˚ ) Bond-angle r.m.s.d. () Side-chain planarity r.m.s.d. (A˚ ) !-Angle standard deviation () Clashes per 100 residues Severe clashes per 100 residues Ultrahigh-resolution set 18 0.021 2.5 0.011 6.4 0.6† 0.03† Moderate-resolution set + additional PrimeX refinement 94 0.019 2.2 0.006 7.1 0.7‡ 0.03‡ Moderate-resolution set as deposited 94 0.015 1.4 0.005 5.1 4.0 0.5 BUSTER-refined subset 2 0.014 1.7 0.008 2.6 0.9 0.07 CNS/CNX-refined subset 12 0.009 1.3 0.005 2.3 5.9 0.8 PHENIX-refined subset 14 0.008 1.1 0.003 5.4 4.7 0.5 REFMAC-refined subset 66 0.016 1.5 0.005 5.7 3.4 0.5 † Corrected for obvious errors in deposited structures, as shown in Tables 1 and 2. ‡ Corrected for clashes owing to errors in the structures (see text); the numbers of uncorrected clashes and severe clashes per 100 residues are 0.9 and 0.03, respectively. research papers field and electron-density gradients, which also required the motion of the glutamine to avoid the formation of a clash with the lysine. Whatever the causes, the result was a large co- ordinated movement of the lysine and glutamine side chains which was dramatic both in terms of the extent of the motion of the glutamine side chain and in the difference in the key interactions observed for the binding of ADP to this protein. is less easy to define. MacArthur & Thornton (1996) suggested from their study of proteins and small polypeptides that a standard deviation of 6 is appropriate. Bell et al.  PrimeX 947 Figure 1 MetC187 in PDB entry 3phe is shown in (a) as originally refined in CNX, with only a selection of H atoms added for clarity. Clashes between the terminal methyl group of the methionine and two other residues are shown as orange dashed lines. These close interactions were tolerated during the original refinement as a united-atom model. In (b) the location of the methyl group after PrimeX refinement is shown, where no clashes involving the methyl group were observed. The electron-density grid for this region is contoured at 1.0 from a 2Fo  Fc composite OMIT map. The molecular model as originally deposited contains several side chains, including GlnA758, that are misfitted and thus this structure might be considered by some to be a poor candidate for automated refinement. In this alternate view of the situation shown in Fig. 3, residue GlnA758 is positioned outside of the anticipated radius of convergence for refine- ment. However, one conclusion is clear: REFMAC was tolerant of the implied clash as described above either because it was designed to behave so or because a decision by the users (Crawley et al., 2011) caused REFMAC to behave in this way. PrimeX all-atom refinement is not tolerant of such inter- actions because of the highly unfavorable energetics calcu- lated for such an interaction and it does not allow users to modify its behavior to tolerate such interactions without extraordinary efforts. Even when considered in this context, the ability of the automated PrimeX polish workflow to improve the model in the manner described in Fig. 3 is encouraging. 4.1. Summary geometry statistics for PrimeX and other refinement programs While this study was primarily focused on close nonbonded contacts and refinement using an all-atom model, other issues regarding molecular geometry were also of interest and might best be discussed first. Only moderate-resolution structures deposited and released in 2010 were used in this study to ensure that the results reflected current practices in protein crystallography, especially with respect to geometric restraints. Use of the OPLS all-atom force field in PrimeX produced reasonable results with respect to summary geometry that were in line with other programs in terms of bond-length deviation and side-chain group planarity (Table 5). The results from the two other summary geometry descriptors monitored here deserve additional comment. The average of the bond-angle r.m.s.d.s for PrimeX (2.2) is greater than for any of the other programs that created the original moderate-resolution structure set (range 1.1–1.7; Table 5). However, the observation from the ultrahigh- resolution data set of an average bond-angle r.m.s.d. of 2.5 (range 1.4–3.1; Table 1) clearly suggests that this r.m.s.d. is reasonable. 3.4. Additional benefits from all-atom refinement The advantages of all-atom refinement of structures at moderate resolution extend well beyond the prevention and remediation of clashes. A few examples from the PrimeX refinements in this study illustrate how a detailed description of nonbonded contacts influenced and improved the results of refinement. 3.4.1. Repositioning of a methionine methyl group. Fig. 1 provides an example in which all-atom refinement used in PrimeX led to a significant improvement in the structural model. In PDB entry 3phe, clashes of the C" and associated H atoms of MetC187 with atoms from LeuC293 and TyrC296 suggest that at least one of these residues is in the wrong position. PrimeX refinement using the ‘polish’ workflow moved the methyl group as shown in Fig. 1 without manual intervention. The 2Fo  Fc electron-density map as shown did not give any clear indication of the correct position for this methyl group. However, the position as deposited was un- favourable and unlikely to be correct as judged from the observed clashes. The new position for the methionine methyl 3.4.3. Refinement of two side-chain positions provides a new view of ADP binding. In its original position in PDB entry 3pdt, as refined in REFMAC, a clash occurred between GlnA758 and PheA720 in the all-atom structure (Fig. 3). The change in structure after PrimeX refinement using the ‘polish’ workflow is hypothesized to have occurred through the following chain of events. The movement of the GlnA758 side chain was first driven by relief of this clash. Concurrently, the LysA722 side chain was moved towards the phosphate group of the ADP molecule under the influence of both the force 946 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 Acta Cryst. (2012). D68, 935–952 Figure 1 MetC187 in PDB entry 3phe is shown in (a) as originally refined in CNX, with only a selection of H atoms added for clarity. Clashes between the terminal methyl group of the methionine and two other residues are shown as orange dashed lines. These close interactions were tolerated during the original refinement as a united-atom model. In (b) the location of the methyl group after PrimeX refinement is shown, where no clashes involving the methyl group were observed. The electron-density grid for this region is contoured at 1.0 from a 2Fo  Fc composite OMIT map. Over-restraint of ! angles in CNS/CNX has been recog- nized for several years (Priestle, 2003). Considering the time that has passed since this publication, the number of structures from BUSTER, CNS/CNX and REFMAC observed with very low deviation of ! angles is hard to understand. While over- restraint is easy to recognize, the correct degree of variability 947 Bell et al.  PrimeX Acta Cryst. (2012). D68, 935–952 Figure 3 The region near ADP A811 in PDB entry 3pdt is shown in (a) as originally refined in REFMAC. All H atoms were added to this model and their positions were minimized while holding non-H atoms fixed, but only some of these H atoms are shown for clarity. Multiple clashes between the misplaced residue GlnA758 and PheA720 were apparently tolerated in the REFMAC refinement. The close contacts (orange dashed lines) were so severe that the energy minimization distorted the planarity of the aromatic system rather than allowing the higher energy interpenetration of atoms. The position of these two residues after refinement with PrimeX is shown in (b). In order to relieve the strain of the clash, the LysA722 side chain moved toward the ligand with a coordinated motion of the glutamine side chain into its correct position in strong electron density below the Lys residue. Hydrogen bonds are shown as purple dashed lines. The electron-density grid for this region is contoured at 1.0 from a 2Fo  Fc composite OMIT map. research papers values could be an increase in the number or severity of clashes. Forcing bond angles or torsion angles toward idealized values does carry a risk. If an interaction such as a nonbonded repulsion has driven a particular torsion or bond angle away from the idealized value, restraining it to be closer to the idealized value must make that other interaction more un- favorable. Thus, the result of strictly enforcing these ideal That the same Rfree was obtained with our force-field-based restraints as with Engh and Huber restraints suggests that these restraints are reasonably consistent with protein crystal structures. However, taken together, the decrease in the average R factor (Rwork), the relatively large r.m.s.d. for bond angles compared with the deposited structures and the Figure 2 The region near ValC209 in PDB entry 3nl6 is shown in (a) as originally produced with phenix.refine. All H atoms were added to this model and their positions were minimized while holding non-H atoms in fixed positions, but only some of these H atoms are shown for clarity. The extremely close contacts between the H atom attached to C of ValC15 and atoms of the ValC209 side chain (orange dashed lines) distorted the bond angles around C. The coordinates of residues C209 and C210 changed after PrimeX refinement as shown in (b), with a shift in the backbone conformation, relieving the close contacts and resulting in multiple additional hydrogen bonds to the ligand (purple dashed lines). The electron-density grid for this region is contoured at 1.0 from a g p p Figure 2 The region near ValC209 in PDB entry 3nl6 is shown in (a) as originally produced with phenix.refine. All H atoms were added to this model and their positions were minimized while holding non-H atoms in fixed positions, but only some of these H atoms are shown for clarity. The extremely close contacts between the H atom attached to C of ValC15 and atoms of the ValC209 side chain (orange dashed lines) distorted the bond angles around C. The coordinates of residues C209 and C210 changed after PrimeX refinement as shown in (b), with a shift in the backbone conformation, relieving the close contacts and resulting in multiple additional hydrogen bonds to the ligand (purple dashed lines). The electron-density grid for this region is contoured at 1.0 from a 2Fo  Fc composite OMIT map. research papers Figure 3 The region near ADP A811 in PDB entry 3pdt is shown in (a) as originally refined in REFMAC. All H atoms were added to this model and their positions were minimized while holding non-H atoms fixed, but only some of these H atoms are shown for clarity. Multiple clashes between the misplaced residue GlnA758 and PheA720 were apparently tolerated in the REFMAC refinement. The close contacts (orange dashed lines) were so severe that the energy minimization distorted the planarity of the aromatic system rather than allowing the higher energy interpenetration of atoms. The position of these two residues after refinement with PrimeX is shown in (b). In order to relieve the strain of the clash, the LysA722 side chain moved toward the ligand with a coordinated motion of the glutamine side chain into its correct position in strong electron density below the Lys residue. Hydrogen bonds are shown as purple dashed lines. The electron-density grid for this region is contoured at 1.0 from a 2Fo  Fc composite OMIT map. Figure 3 The region near ADP A811 in PDB entry 3pdt is shown in (a) as originally refined in REFMAC. All H atoms were added to this model and their positions were minimized while holding non-H atoms fixed, but only some of these H atoms are shown for clarity. Multiple clashes between the misplaced residue GlnA758 and PheA720 were apparently tolerated in the REFMAC refinement. The close contacts (orange dashed lines) were so severe that the energy minimization distorted the planarity of the aromatic system rather than allowing the higher energy interpenetration of atoms. The position of these two residues after refinement with PrimeX is shown in (b). In order to relieve the strain of the clash, the LysA722 side chain moved toward the ligand with a coordinated motion of the glutamine side chain into its correct position in strong electron density below the Lys residue. Hydrogen bonds are shown as purple dashed lines. The electron-density grid for this region is contoured at 1.0 from a 2Fo  Fc composite OMIT map. Fi 3 Fi 2 4.2. Advantages of all-atom refinement with PrimeX All-atom refinement of moderate-resolution protein crystal structures with PrimeX resulted in a more than fourfold decrease in the number of clashes and a 17-fold decrease in the number of severe clashes. This improvement in model quality was achieved without sacrificing the goodness of fit to the X-ray data as judged by the average Rfree values. Impor- tantly, these models also display good summary statistics, so that the protein models comply with reasonable molecular- geometry expectations. What else can be done to reduce the number of clashes in deposited structures? Perhaps the answer to this question resides in the standards for structure deposition in the PDB. A committee of the PDB is currently working on structure- validation tools for use associated with the deposition of coordinates (Read et al., 2011). A reason for optimism is that the work of the Richardson group was included in the report of the committee. From the point of view of many users of protein structures, the deposition of all-atom models derived from protein crystal structures should be required. Clashes determined from an all-atom model should be, at the very least, measured and documented for all protein models that are deposited, just as other outliers to molecular-geometry standards are now listed in the entry header. All-atom refinement with a force field allowed PrimeX refinement to fix errors that other refinement programs missed and provided a more accurate picture of critical protein features such as protein–ligand interactions, as illustrated in Figs. 1, 2 and 3. Resolution of clashes during refinement can help to ‘push’ the structure into the correct conformation, producing potentially remarkably large changes in confor- mation. They may also serve the role of preventing the structure from entering nonproductive conformations that are otherwise allowed in a less restrictive all-atom model. To achieve a higher standard for deposited protein struc- tures, additional tools that are sensitive to close contacts could help. PrimeX can contribute to this goal, and the automated polish workflow presented here was designed to achieve this goal with the minimum of human intervention. However, the workflow was designed with the assumption that the crystal structure coordinates on which it would operate would be essentially free of errors in the main-chain tracing or side- chain rotamer selection. 4.2. Advantages of all-atom refinement with PrimeX The prevalence of such errors in the data set examined here established the need for additional automated structure tools with the capability of making large changes in side-chain torsion angles or chain trace. Design of these workflows is in progress based on the tool set in the PrimeX refinement package (Bell et al., 2012). Although similar automated workflows exist for phenix.refine (Afonine et al., 2005) and indirectly for REFMAC (Murshudov et al., 2011) through the program SideAide in the PDB_REDO pipeline (Joosten et al., 2011), the frequency of clashes in structures refined by phenix.refine and REFMAC (Tables 3 and 5) raises the question whether these automated workflows can address the issue of all-atom clashes, no matter how capable and thorough these workflows are intended to be. While the program MolProbity (Davis et al., 2007; Chen, Arendall et al., 2010) is aimed at solving the right problem, these same results show that it is not being adequately employed to deal with the problem at hand. These results also contain an indication of the limits of usefulness of the polish workflow. Fully 85% of the structures that entered the workflow with 50 or more total clashes (Table 3) resulted in an increase in Rfree (Tables 3 and 4). A large number of clashes is a warning sign that the structure may contain errors that could have negative consequences after the application of this refinement process. Figure 2 The region near ValC209 in PDB entry 3nl6 is shown in (a) as originally produced with phenix.refine. All H atoms were added to this model and their positions were minimized while holding non-H atoms in fixed positions, but only some of these H atoms are shown for clarity. The extremely close contacts between the H atom attached to C of ValC15 and atoms of the ValC209 side chain (orange dashed lines) distorted the bond angles around C. The coordinates of residues C209 and C210 changed after PrimeX refinement as shown in (b), with a shift in the backbone conformation, relieving the close contacts and resulting in multiple additional hydrogen bonds to the ligand (purple dashed lines). The electron-density grid for this region is contoured at 1.0 from a 2Fo  Fc composite OMIT map. Bell et al.  PrimeX 948 Acta Cryst. (2012). D68, 935–952 research papers programs can be considered to be part of the solution to this problem. somewhat larger standard deviation for the ! angle above the optimal value conceived by MacArthur & Thornton (1996) could be interpreted as evidence that the restraints employed may require further tuning to decrease the risk of overfitting. This consideration will be examined in future publications. A role for CNS in curbing clashes is also currently available. CNS can be employed with a more complex energy model than is routinely used by crystallographers. As well as deploying an Engh and Huber-based restraint system, CNS is distributed with a force field that includes Lennard–Jones and electrostatic terms and that is regularly used for the determination of NMR structures (Linge et al., 2003). This force field has been employed to produce some very high quality NMR structures (see, for example, Nozinovic et al., 2010). Acta Cryst. (2012). D68, 935–952 5. Conclusion Ali, A., Reddy, G. S., Nalam, M. N., Anjum, S. G., Cao, H., Schiffer, C. A. & Rana, T. M. (2010). J. Med. Chem. 53, 7699–7708. This study documents the existence of numerous unnecessary close contacts, including many severe ones, implicit in united- atom models deposited in the PDB. Many of these close contacts can be readily removed, and doing so need not damage the agreement of the model with the observed X-ray diffraction data. Furthermore, attention to close contacts can bring to light errors in the placement of non-H atoms in protein crystal structure models. This latter point has also been made abundantly clear by over a decade of work by Jane Richardson, David Richardson and coworkers (Word et al., 1999, Davis et al., 2007; Chen, Arendall et al., 2010). ( ) Allen, F. H. (2002). Acta Cryst. B58, 380–388. Allen, F. H. (2002). Acta Cryst. B58, 380–388. Artz, J. D., Wernimont, A. K., Dunford, J. E., Schapira, M., Dong, A., Zhao, Y., Lew, J., Russell, R. G., Ebetino, F. 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Normally, scientists working in these areas are not in a position to evaluate the reliability of each protein crystal structure, nor are they able to judge whether the effects of remediation of crystal structures might result in different sorts of errors. 5. Conclusion If crystallographers, who are of course in the best position to do so, do not address these issues, then eventually other scientists will. The result will be that protein crystallographers will have less control over the form in which their experimental results are archived and deployed. The advent of remediated database alternatives to the PDB (Joosten et al., 2009) is partially an outgrowth of this problem and an indication that this anticipated conse- quence is already becoming a reality. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N. & Bourne, P. E. (2000). Nucleic Acids Res. 28, 235–242. 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Both the expected bond-length and bond-angle parameters of Engh & Huber (1991) and the parameterization of van der Waals radii by Rowland & Taylor (1996) are equally well grounded in high-resolution small-molecule crystallographic results. In protein crystallography the former geometric statistics are very strictly applied, while the latter receive much less attention. An understandable explanation for this contrast is the absence of H-atom coordinates in classic protein crystal structure models. However, if the requirements for the highest quality protein models possible are to be met, the considera- tion of nonbonded contacts in all-atom models must become more prominent. ( ) g Bru¨nger, A. T. (1989). Acta Cryst. A45, 42–50. Bru¨nger, A. T. (1992). X-PLOR Version 3.1. A System for X-ray Crystallography and NMR. New Haven: Yale University Press. Bru¨nger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. research papers lacking. In addition, clashes highlighted several clear errors in the coordinates of non-H atoms. Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. (2005). CCP4 Newsl. 42, contribution 8. Afonine, P. V., Grosse-Kunstleve, R. W., Chen, V. B., Headd, J. J., Moriarty, N. W., Richardson, J. S., Richardson, D. C., Urzhumtsev, ( ) Afonine, P. V., Grosse-Kunstleve, R. W., Chen, V. B., Headd, J. J., Moriarty, N. W., Richardson, J. S., Richardson, D. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2010). J. Appl. Cryst. 43, 669–676. A., Zwart, P. H. & Adams, P. D. (2010). J. Appl. Cryst. 43, 669–676. 4.3. Reducing clashes in deposited X-ray crystal structure models The refinement programs REFMAC and phenix.refine both have the capability to use riding H atoms during refinement. One might reasonably expect that the use of this feature would have a positive impact on the issue of clashes in all-atom models. Unfortunately, the extent to which the riding H atom option is actually employed in refinement is impossible to determine in many cases, even after consulting both the primary literature references and the PDB entry. The lack of definitive information on this issue makes it nearly impossible to determine from these experiments whether these programs are capable of reducing clashes to the levels deduced to be reasonable goals from the ultrahigh-resolution structures or from the PrimeX-refined structures. Comparing the frequency of clashes in Tables 3 and 4, one can only conclude that either (i) the riding H atom models and nonbonded contact restraints do not make as much difference as one might expect, (ii) the riding H atom option is very rarely used in these two programs or (iii) both are true. 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Uncovering spin-orbit coupling-independent hidden spin polarization of energy bands in antiferromagnets
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Uncovering spin-orbit coupling-independent hidden spin polarization of energy bands in antiferromagnets Lin-Ding Yuan1, Xiuwen Zhang 1, Carlos Mera Acosta2 & Alex Zunger1 Received: 17 November 2022 Accepted: 15 August 2023 Check for updates Received: 17 November 2022 Accepted: 15 August 2023 Check for updates Many textbook physical effects in crystals are enabled by some specific sym- metries. In contrast to such ‘apparent effects’, ‘hidden effect X’ refers to the general condition where the nominal global system symmetry would disallow the effect X, whereas the symmetry of local sectors within the crystal would enable effect X. Known examples include the hidden Rashba and/or hidden Dresselhaus spin polarization that require spin-orbit coupling, but unlike their apparent counterparts are demonstrated to exist in non-magnetic systems even in inversion-symmetric crystals. Here, we discuss hidden spin polariza- tion effect in collinear antiferromagnets without the requirement for spin- orbit coupling (SOC). Symmetry analysis suggests that antiferromagnets hosting such effect can be classified into six types depending on the global vs local symmetry. We identify which of the possible collinear antiferromagnetic compounds will harbor such hidden polarization and validate these symmetry enabling predictions with first-principles density functional calculations for several representative compounds. This will boost the theoretical and experimental efforts in finding new spin-polarized materials. Many traditional textbook physical effects in crystals are enabled by some specific symmetries, encoded in the crystal space group. Such are the symmetry conditions for the apparent electric polarization which defines various order parameters such as in ferroelectricity1, circular dichroism2, and pyroelectricity3. Another example of effects enabled by the recognized global system symmetry is the removal of spin degen- eracy of energy bands due to spin-orbit coupling (SOC) in non-magnetic crystals having broken inversion symmetry (such as the Rashba (R-1)4 and Dresselhaus (D-1)5 effects). When an effect is observed despite the needed enabling symmetry being absent, it is often assumed that the system contains some symmetry-altering imperfections. phenomena that are unexpected to exist based on the global sym- metry of the system. Examples of “Hidden Effect X” that is SOC-induced include (i) Rashba or Dresselhaus spin polarization, expected exclusively to occur in non-centrosymmetric crystals, but predicted6,7 and observed8–18 in centrosymmetric nonmagnetic crystals (denoted R-2 and D-2, respec- tively). Similar form of Hidden effect X are (ii) X = “anisotropic optical circular polarized luminescence” expected only in odd-layered transi- tion-metal dichalcogenides but observed19 also in even-layered crys- tals. Article https://doi.org/10.1038/s41467-023-40877-8 1Renewable and Sustainable Energy Institute, University of Colorado, Boulder, CO 80309, USA. 2Center for Natural and Human Sciences, Federal University of ABC, Santo Andre, São Paulo, Brazil. e-mail: Alex.Zunger@colorado.edu Uncovering spin-orbit coupling-independent hidden spin polarization of energy bands in antiferromagnets a SOC-independent hidden spin polarization schematically illustrated as two copies of spin split energy bands localized on sector-α and sector-β but globally mutually compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of spin split sector. Sectors in (a) are represented by color-shaded planes, the red and blue lines in the plane represent the spin-up and spin-down bands. The spin- splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presence or absence of the symmetry. a Hidden spin polarization in AFM polarization was pointed out recently in ref. 38. This involved utilizing first a few individual symmetry operations: U being a spin rotation of the SU(2) group acting on the spin 1/2 space that reverses the spin; T being spatial translation; Θ being time reversal, and I being the spatial inversion. These individual operations are then used for constructing two symmetry products: a SOC-free magnetic symmetry ΘIT, and a spin symmetry UT (where the former product can be simplified to ΘI by proper choice of inversion center). SOC-independent spin splitting38,39 would occur only when both symmetry products are simultaneously violated. Antiferromagnets with ΘIT symmetry28,41,42 will not show such spin splitting. Such symmetry conditions disen- tangle the SOC-independent splitting from the SOC-induced splitting by considering the symmetry at the zero SOC limit43–45, where spin and space are fully decoupled. Hidden spin polarization in AFM a Symmetry SS w/o SOC Magnetism Prototype Θ (✓) and (X) No AFM SST-1 Θ (✓) and (✓) No AFM SST-2 Θ (X) and (✓) No AFM SST-3 b Symmetry classification of the spin degenerate bulk b Given the symmetry conditions, it is thus possible to classify all different spin splitting prototypes38,39 for magnetic materials. There are three prototypes with no apparent spin splitting effect: (1) AFM compounds that violate UT but preserve ΘIT symmetry referred to as spin splitting prototype 1 (SST-1) antiferromagnets; (2) AFM com- pounds that preserve both UT and ΘIT symmetry referred to as SST-2 antiferromagnets; (3) AFM compounds that preserve UT but violate ΘIT symmetry referred to as SST-3 antiferromagnets. Enabling symmetry conditions for hidden SOC-independent spin polarization in antiferromagnets sp po a at o a t e o ag ets “Hidden spin polarization” is expected in collinear antiferromagnets when the bulk has zero net spin polarization, but its constituent sec- tors allow locally a spin splitting and spin polarization effect. Consider the combination of two possible prototypes constituting sector that gives hidden spin polarization locally but lead to three possible pro- totypes of the bulk symmetry (preserving either ΘIT or UT or both) that disallows apparent spin polarization, one can then classify six hidden spin polarization cases. Following the previous classification of spin splitting prototypes for apparent spin degeneracy and apparent spin splitting38,39, collinear antiferromagnetic materials with “hidden spin polarization” are those antiferromagnets whose bulk prototype being SST-I (I = 1, 2, 3) and constitute sector prototype being SST-J (J = 4, 5). Detailed discussions of the symmetry conditions for hidden spin polarization in collinear AFM are given in Supplementary Infor- mation Section A. non-magnetic state has an inversion. Prominently, the hidden spin polarization in these compounds facilitates the electrical reversal of their antiferromagnetic ordering25,29. (iv) X = “anomalous Hall effect” induced by SOC expected only in odd-layered ferromagnetic MnBi2Te4 systems but observed in even-layered antiferromagnetic MnBi2Te4 30 systems via a perturbative applied electric field. Here, we discuss a different form of hidden spin polarization effect (see Fig. 1a) whose corresponding apparent effect is indepen- dent of SOC31–40; And the hidden effect exists in antiferromagnetic materials where spin-up and spin-down bands are paired. This repre- sents a step further beyond the already known hidden Rashba and hidden Dresselhaus spin-polarization that unavoidably require a siz- able contribution from SOC. A careful analysis of the “global (bulk) vs local (sector)” symmetries suggests that antiferromagnets hosting the SOC-independent “hidden” spin polarization effect can be delineated into six types. We scrutinize a vast database of known collinear AFM materials and performed first-principles calculations on several selec- ted candidate compounds assuming zero SOC. We show that such hidden, SOC-independent effects reflect the intrinsic properties of the perfect crystal rather than an effect due to imperfections. The interest in this SOC-independent hidden spin polarization effect stems both from the evolving of the fundamental understanding of general hidden effects in solids, and from the ability to extend the pool of useful materials for potential spintronic applications. Enabling symmetry conditions for hidden SOC-independent spin polarization in antiferromagnets Figure 2 summarizes the six possible types of hidden spin polar- ization without SOC in antiferromagnets that are spin degenerate but contain spin split sectors (represented by color-shaped plane). Figure 2a–c illustrates the three cases where the spin degenerate antiferromagnets of SST-I (I = 1,2,3) can be decomposed into alter- nating ferromagnetic local sectors that locally violate both UT and ΘIT, thus allows spin splitting without SOC. FM materials that satisfy the conditions of violating both UT and ΘIT (always true) are denoted as SST-5 in Fig. 1. The three magnetic-induced hidden spin polarization cases can then be denoted as (a) bulk SST-1 sector SST-5; (b) bulk SST-2 sector SST-5, and (c) bulk SST-3 sector SST-5. Figure 2d–f illustrates the three cases where the spin degenerate AFM of SST-I (I = 1, 2, 3) can be decomposed into alternating antiferromagnetic local sectors that locally violate both UT and ΘIT, thus allows spin splitting without SOC. AFM materials that satisfy the condition are denoted as SST-4 in Fig. 1. Uncovering spin-orbit coupling-independent hidden spin polarization of energy bands in antiferromagnets In addition, there are two prototypes with apparent spin splitting effects: (4) AFM compounds that violate both UT and ΘIT symmetry referred to as SST- 4 antiferromagnets; (5) Ferromagnetic (FM) compounds that violate both UT and ΘIT symmetry referred to as SST-5 ferromagnets. The classification defined in bulk crystals38,39 can be generalized to sectors of a bulk based on the local sector symmetry. Figure 1b,c. summarizes the classification of “spin degenerate bulk” vs “spin-split sector”. This will later be applied to describe the symmetry conditions and to define the different prototypes for the hidden spin polarization effect in antiferromagnets. Symmetry SS w/o SOC Magnetism Prototype (X) and (X) Yes AFM SST-4 (X) and (X) Yes FM SST-5 c Symmetry classification of spin split sector Symmetry classification of spin split sector Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. a SOC-independent hidden spin polarization schematically illustrated as two copies of spin split energy bands localized on sector-α and sector-β but globally mutually compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of spin split sector. Sectors in (a) are represented by color-shaded planes, the red and blue lines in the plane represent the spin-up and spin-down bands. The spin- splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presence or absence of the symmetry. Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. a SOC-independent hidden spin polarization schematically illustrated as two copies of spin split energy bands localized on sector-α and sector-β but globally mutually compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of spin split sector. Sectors in (a) are represented by color-shaded planes, the red and blue lines in the plane represent the spin-up and spin-down bands. The spin- splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presence or absence of the symmetry. Uncovering spin-orbit coupling-independent hidden spin polarization of energy bands in antiferromagnets Such effects were originally dismissed as being due to some extrinsic sample imperfection20–22 but later on were shown to be an intrinsic property pertained to the individual layer23. (iii) X = “spin polarization” induced by SOC in antiferromagnetic systems. The effect is again expected only in non-centrosymmetric crystals (such as BiCoO3 24) but shown in centrosymmetric crystals (such as CuMnAs and Mn2Au25–28) where combined symmetry of inversion and time reversal disallows splitting. Here, “centrosymmetric” means the crystal in the In contrast, the “Hidden Effect X” in materials that are not sup- ported by the nominal enabling symmetry, yet effect X exists locally. The “Hidden effect X” reflects the intrinsic properties of the perfect crystal rather than imperfections that would disappear when the crystal becomes perfect. The understanding of such hidden intrinsic effects is important as it can demystify peculiar observations of Nature Communications| (2023) 14:5301 1 https://doi.org/10.1038/s41467-023-40877-8 Article Symmetry SS w/o SOC Magnetism Prototype Θ (✓) and (X) No AFM SST-1 Θ (✓) and (✓) No AFM SST-2 Θ (X) and (✓) No AFM SST-3 Symmetry SS w/o SOC Magnetism Prototype (X) and (X) Yes AFM SST-4 (X) and (X) Yes FM SST-5 c b a Symmetry classification of the spin degenerate bulk Symmetry classification of spin split sector Hidden spin polarization in AFM Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. a SOC-independent hidden spin polarization schematically illustrated as two copie of spin split energy bands localized on sector-α and sector-β but globally mutuall compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of spin split sector. Sectors in (a) are represented by color-shaded planes, the red an blue lines in the plane represent the spin-up and spin-down bands. The spin- splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presenc or absence of the symmetry. Symmetry SS w/o SOC Magnetism Prototype Θ (✓) and (X) No AFM SST-1 Θ (✓) and (✓) No AFM SST-2 Θ (X) and (✓) No AFM SST-3 Symmetry SS w/o SOC Magnetism Prototype (X) and (X) Yes AFM SST-4 (X) and (X) Yes FM SST-5 c b a Symmetry classification of the spin degenerate bulk Symmetry classification of spin split sector Hidden spin polarization in AFM Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. Compounds that have SOC-independent hidden spin polarization We now turn to discuss how the enabling symmetries are applied to individual sectors to give magnetic hidden spin polarization effects in real antiferromagnetic materials. Hidden spin polarization from individual ferromagnetic sectors. Figure 3 illustrates the hidden spin polarization effect in tetragonal CuMnAs54 (bulk belonging to SST-1 class with sectors belonging to SST- 5 class). The crystal is antiferromagnetically ordered with its magnetic moments collinearly aligned in the (010) direction. The magnetic space group (MSG) of the crystal is Pm’mn (MSG type III). The unit cell consists of two MnAs layers (α-sector and β-sector) that are ferro- magnetically ordered (Fig. 3a, red and blue color shaped polyhedral are used to indicate oppositely magnetized motifs centered on the magnetic sites). By considering the bulk antiferromagnets as a com- bination of two alternating non-centrosymmetric sectors (α-sector and β-sector), the material has been demonstrated as a useful platform for electrically switching25,29 the antiferromagnetic magnetization using the hidden spin polarization from the SOC segregated on each sector. Here, we point out a different SOC-independent scenario that might also be contributing to the observed electric switching in this material, i.e., the Zeeman effect within each ferromagnetic MnAs layer creates a local spin split state anchored on the layer. The two MnAs layers are connected by the ΘIT symmetry which restores the spin degeneracy of the bulk and results in a compensated net spin polarization (Fig. 3b). As shown by the reversed blue and red pattern which are used to map the relative magnitude of the spin up and spin down polarization, the hidden spin polarization is non-zero and is compensated by each other. Examples of hidden spin polarization in spin degenerate bulk As a first step, we will try to find real materials that falls into the six categories we defined. This can be done straightforwardly by applying the symmetry conditions to filter out candidate materials in existing antiferromagnetic databases. We conducted such filtering for MAGN- DATA database46 and identified a few antiferromagnetic materials of potential candidates for magnetic hidden spin polarization. Enabling symmetry conditions for SOC-independent apparent spin polarization in antiferromagnets Enabling symmetry conditions for SOC-independent apparent spin polarization in antiferromagnets Symmetry is essential to understand the energy bands’ degeneracy of a material. The symmetry conditions for apparent spin splitting or spin Nature Communications| (2023) 14:5301 2 Article https://doi.org/10.1038/s41467-023-40877-8 Bulk Sector SST-1 SST-2 SST-3 SST-5 Hidden spin polarization from individual FM sectors a b c SST-4 Hidden spin polarization from individual AFM sectors d e f Θ (✓) and (✓) Θ (X) and (✓) Θ (X) and (X) Θ (X) and (X) Θ (✓) and (X) Fig. 2 | Six types of SOC-independent magnetic hidden spin polarization in collinear antiferromagnets. These antiferromagnets have global symmetry that disallows spin splitting without SOC, but have lower local sector symmetry that allows spin splitting without SOC. Cases (a, b, c) is where hidden spin polarization arise from local ferromagnetic sectors and cases (d, e, f) is where the hidden spin polarization arise from local antiferromagnetic sectors. Shaded planes are used to indicate the individual sectors that have neither ΘIT nor UT symmetry and allow spin splitting in the absence of SOC; Parallel and antiparallel arrows of red and blue within the sector plane are used to indicate the ferromagnetic and anti- ferromagnetic ordering of the sector. Sector symmetry is indicated on top of each plane, and bulk symmetry is indicated by the arrow connecting the two sectors. c b b a c a d f e f d e Fig. 2 | Six types of SOC-independent magnetic hidden spin polarization in Fig. 2 | Six types of SOC-independent magnetic hidden spin polarization in collinear antiferromagnets. These antiferromagnets have global symmetry that disallows spin splitting without SOC, but have lower local sector symmetry that allows spin splitting without SOC. Cases (a, b, c) is where hidden spin polarization arise from local ferromagnetic sectors and cases (d, e, f) is where the hidden spin polarization arise from local antiferromagnetic sectors. Shaded planes are used to indicate the individual sectors that have neither ΘIT nor UT symmetry and allow spin splitting in the absence of SOC; Parallel and antiparallel arrows of red and blue within the sector plane are used to indicate the ferromagnetic and anti- ferromagnetic ordering of the sector. Sector symmetry is indicated on top of each plane, and bulk symmetry is indicated by the arrow connecting the two sectors. collinear antiferromagnets. Enabling symmetry conditions for SOC-independent apparent spin polarization in antiferromagnets These antiferromagnets have global symmetry that disallows spin splitting without SOC, but have lower local sector symmetry that allows spin splitting without SOC. Cases (a, b, c) is where hidden spin polarization arise from local ferromagnetic sectors and cases (d, e, f) is where the hidden spin polarization arise from local antiferromagnetic sectors. Shaded planes are used to The three AFM-induced hidden spin polarization cases can then be denoted as (d) bulk SST-1 sector SST-4; (e) bulk SST-2 sector SST-4, and (f) bulk SST-3 sector SST-4. We note that there are multiple ways to decompose the bulk system into sectors, e.g., the bulk SST-I (I = 1,2,3) might also be decomposed into sector SST-I (I = 1,2,3) (or equivalently SST-I (I = 1,2,3) sectors can be used to build the bulk SST-I (I = 1,2,3) materials), where the local spin polarization of each individual sector is still zero, therefore, are not the focus of this work. hypothetical or contradictory to enabling symmetry conditions for AFM spin splitting. Van der Waals compounds with spin splitting not only allow the potential practical controllability through external electric fields but also a platform to explore the coexistence of Van der Waals materials properties and AFM-induced spin splitting. The next step is to validate the predicted hidden spin polarization effect in some of these identified real materials. We studied the sector- projected spin textures on certain wavevector planes for three actual antiferromagnetic materials, CuMnAs54, Ca2MnO4 47 and FeBr2 56 using PBE + U method59 in the zero SOC limit. The results are presented below. Additional examples with DFT results are presented in Sup- plementary Information Section C. These examples proof the exis- tence of the hidden spin polarization effect. Nature Communications| (2023) 14:5301 https://doi.org/10.1038/s41467-023-40877-8 5a, show that such splitting is linearly proportional to the applied external electric field, but in opposite spin polarization ordering for the bottom conduction bands and the top valence bands. The linear field-dependent splitting suggests the split states are segregated on either layer (sector). Indeed, spatial distribution of the spin polarized states, Fig. 5b, c, shows the spin-up (red) state ΓCB1 is dominantly segregated on the α-sector, while the spin-down (blue) state ΓCB2 is dominantly segregated on the β-sector. Therefore, the hidden effect of two-fold degenerate energy states subspace (when Ez = 0) can be traced back to the individual FeBr2 layers. Because the applied electric field is small, the main characteristic of the observed spin polarization is inherited from the system without electric field. The layer-segregated states shown in Fig. 5b, c is thus a compelling evidence of the relationship between the global property of spin splitting induced by a global electric field and the local spin polarization. We note the hidden spin polarization effect from local “spin-split” sectors has also been recently exemplified and revealed via an electric field in some antiferromagnets41,63 where external electric field lifts the spin degeneracy. We also note that the layer Hall effect in the even-layered MnBi2Te4—in which electrons from the top and bottom layers spontaneously deflect in opposite direc- tions but globally compensate—has been observed with the help of an applied electric field30. These examples not only verify our under- standing of the hidden effect being intrinsic to the bulk but also sug- gest an external electric field as an effective knob for modulating the hidden effect. antiferromagnets made of spin split ferromagnetic sectors are also illustrated for CoBr2 56 (bulk belonging to SST-2 with sector belonging to SST-5) and Ca3Ru2O7 60 (bulk belonging to SST-3 with sector belonging to SST-5) in Supplementary Information Section C. We note that the corresponding hidden spin polarization pro- jected onto α-sector and β-sector, shown in Fig. 3c, are all aligned in the same direction with the magnetization. Thus, the spin remains a good quantum number. However, the magnitude of the projected spin polarization (mapped by color changing continuously from blue to red) may vary depending on the distribution of the degenerate states on the two sectors. For a pair of degenerate states, the sector projected spin polarization is the summed contribution from both states. https://doi.org/10.1038/s41467-023-40877-8 The linear field-dependent splitting suggests the split states are segregated on either layer (sector). Indeed, spatial distribution of the spin polarized states, Fig. 5b, c, shows the spin-up (red) state ΓCB1 is dominantly segregated on the α-sector, while the spin-down (blue) state ΓCB2 is dominantly segregated on the β-sector. Therefore, the hidden effect of two-fold degenerate energy states subspace (when Ez = 0) can be traced back to the individual FeBr2 layers. Because the applied electric field is small, the main characteristic of the observed spin polarization is inherited from the system without electric field. The layer-segregated states shown in Fig. 5b, c is thus a compelling evidence of the relationship between the global property of spin splitting induced by a global electric field and the local spin polarization. We note the hidden spin polarization effect from local “spin-split” sectors has also been recently exemplified and revealed via an electric field in some antiferromagnets41,63 where external electric field lifts the spin degeneracy. We also note that the layer Hall effect in the even-layered MnBi2Te4—in which electrons from the top and bottom layers spontaneously deflect in opposite direc- tions but globally compensate—has been observed with the help of an applied electric field30. These examples not only verify our under- standing of the hidden effect being intrinsic to the bulk but also sug- gest an external electric field as an effective knob for modulating the hidden effect. crystal is the ferromagnetically ordered FeBr2 layer (sector belonging to SST-5 class). The bilayer slab is built by stacking identical FeBr2 layer with alternating magnetic ordering. The two layers are connected by both ΘIT and UT symmetry, the bilayer hexagonal FeBr2 (MSG: PC-3c1) thus belongs to a bulk SST-2 class, featuring a spin degenerate energy band. However, the spin degenerate band structure of the SST-2 class FeBr2 (Fig. 5a, b) is lifted upon the application of an external electric field perpendicular to the layers (Ez) – a transition from SST-2 to SST-4. The spin splitting arises because of the external electric field Ez creates a non-equivalent potential on the sectors and breaks the ΘIT and UT symmetry of the bulk that connects the two layers. DFT calculations for different values of the applied field, inserted in Fig. https://doi.org/10.1038/s41467-023-40877-8 For example, the hidden spin polarization of the two spin degenerate states evenly distributed on both sector-α and sector- β(1/√2(|α↑+ |β↑ and 1/√2(|α↓|β↓〉) is (+0.5) +(−0.5) = 0 when projected onto sector-α or sector-β; while the hidden spin polarization of the two spin- degenerate states segregated on one of the sector (|α↑〉and |β↓〉) is 1 when projected onto sector−α and is −1 when projected onto sector. Hidden spin polarization from individual antiferromagnetic sectors. Fig. 4 illustrates the “hidden spin polarization” effect in anti- ferromagnetic tetragonal Ca2MnO4 47 (bulk belonging to SST-1 class with sector belonging to SST-4 class). The crystal is anti- ferromagnetically ordered with its magnetic moments collinearly aligned in the (001) direction. The MSG of the crystal is I41’/a’cd’ (MSG type III). The unit cell consists of two layers of MnO6 octahedral (α-sector and β-sector) that are antiferromagnetically ordered (Fig. 4a, red and blue color polyhedral are used to indicate oppositely mag- netized motifs centered on the magnetic sites). The “magnetic mechanism” [6] within each AFM-ordered sector then creates a local spin split state anchored on the layer. The two MnO4 layers are con- nected by the ΘIT symmetry which restores the spin degeneracy of the bulk and results in zero net spin polarization (Fig. 4b). However, the corresponding spin texture projected onto the α-sector and β-sector, shown in Fig. 4c, are persistently aligned in the same direction as its magnetization and are compensated to each other (as indicated by the reversed blue and red pattern which are used to map the relative magnitude of the spin up and spin down polarization). Examples of hidden spin polarization in spin degenerate bulk antiferromagnets made of spin split antiferromagnetic sectors are also illustrated for MnS2 61 (bulk belonging to SST-2 with sector belonging to SST-4) and La2NiO4 62 (bulk belonging to SST-3 with sector belonging to SST-4) in Supplementary Information Section C. https://doi.org/10.1038/s41467-023-40877-8 https://doi.org/10.1038/s41467-023-40877-8 Article Table 1 | Easily exfoliable 2D magnetic compounds with AFM configuration belonging to SST-4 class Formula SG Structure Prototype Eg (eV) Eb (meV/Å2) 3D SG SDB of 3D ID SDB FeSe P4/nmm FeSe 0.0 22.6 Cmme ICSD 290411 LaBr P3m1 ZrCl 0.6 11.7 R3m ICSD 23354 FeO2 Pmmm FeO2 0.0 16.3 Cmcm COD 9015156 PrOI P4mm PbClF 0.0 14.9 P4/nmm COD 1530611 FeOCl Pmmn PeOCl 0.0 14.2 Pmmn COD 1010645 VOBr Pmmn FeOCl 0.0 14.7 Pmmn ICSD 27010 The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The last three columns describe for the experimental parent structure: the 3D space group (3D SG), source database (SDB)58, and the ID in the source database (ID SDB). 1 | Easily exfoliable 2D magnetic compounds with AFM configuration belonging to SST-4 class Table 1 | Easily exfoliable 2D magnetic compounds with AFM configuration bel The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The last three columns des 3D space group (3D SG), source database (SDB)58, and the ID in the source database (ID SDB). type, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The last three columns describe for the experimental parent structure: th DB)58, and the ID in the source database (ID SDB). crystal is the ferromagnetically ordered FeBr2 layer (sector belonging to SST-5 class). The bilayer slab is built by stacking identical FeBr2 layer with alternating magnetic ordering. The two layers are connected by both ΘIT and UT symmetry, the bilayer hexagonal FeBr2 (MSG: PC-3c1) thus belongs to a bulk SST-2 class, featuring a spin degenerate energy band. However, the spin degenerate band structure of the SST-2 class FeBr2 (Fig. 5a, b) is lifted upon the application of an external electric field perpendicular to the layers (Ez) – a transition from SST-2 to SST-4. The spin splitting arises because of the external electric field Ez creates a non-equivalent potential on the sectors and breaks the ΘIT and UT symmetry of the bulk that connects the two layers. DFT calculations for different values of the applied field, inserted in Fig. 5a, show that such splitting is linearly proportional to the applied external electric field, but in opposite spin polarization ordering for the bottom conduction bands and the top valence bands. Compounds that have SOC-independent hidden spin polarization The identified candidates are: Ca2MnO4 47, CoSe2O5 48 and Fe2TeO6 49, K2CoP2O7 50 and LiFePO4 51 whose bulk prototype is SST-1 with sector prototype of SST-4; Sr2IrO4 52 whose bulk prototype is SST-2 with sector prototype of SST-4; SrCo2V2O8 53 whose bulk prototype is SST-3 with sector prototype of SST-4; CuMnAs54 and Mn2Au55 whole bulk proto- type is SST-1 with sector prototype of SST-5; FeCl2 and CoCl2 56 whose bulk prototype is SST-2 with sector prototype of SST-5; ErAuGe57 whose bulk prototype is SST-3 with sector prototype of SST-5. These materials formthe platform for the exploration of the magnetic hidden spin polarization effects. The opposite design philosophy (the bottom-to-top approach) is to construct layered bulk antiferromagnets with the hidden effect based on two-dimensional (2D) compounds that belong to SST-4 and SST-5 prototypes. By searching through the database of predicted naturally exfoliate 3D Van der Waals materials58, we find a list of 37 ferromagnetic 2D materials and 6 antiferromagnetic 2D monolayers that can be used as such building blocks (see Tables 1 and 2 for the list). Other predicted and synthesized layered 2D materials are either Nature Communications| (2023) 14:5301 3 The effect of SOC on the predicted hidden spin polarization The effect of SOC on the predicted hidden spin polarization The SOC-independent hidden spin polarization effect persists in the presence of SOC. This is because the effect being inherited from the unusual antiferromagnetic order rather than SOC38. Still, it is important to note the inclusion of SOC would modify the energy bands in both non-magnetic materials and magnetic materials43,64,65: (1) it reduces the degeneracy of certain bands which may cause additional spin splitting. (2) it mixes the spin polarized states of up and down (so spin is no longer a good quantum number), which results in momentum- dependent spin polarization that are not unidirectionally aligned; (3) it opens a gap for the crossing energy bands with opposite spin polarization. In compounds consist of low-Z elements the SOC- induced effect can be neglected. Revealing and tailoring the hidden spin polarization by external electric field. To demonstrate the symmetry connection between local sectors and the subsequent transition from hidden effect to apparent effect mediated by the breaking of the symmetryconnection, we apply in our calculations a perturbative symmetry-breaking exter- nal electric field on an antiferromagnetic compound with hidden spin polarization, hexagonal FeBr2 (DFT settings for applying the electric field is provided in Methods section). The basic building block of the Nature Communications| (2023) 14:5301 4 https://doi.org/10.1038/s41467-023-40877-8 Article Table 2 | Easily exfoliable 2D magnetic compounds with FM configuration belonging to SST-5 class invariant. By definition, antiferromagnets with primitive lattice trans- lations that reverse the microscopic magnetic moments are known as having black and white Bravais lattice that is classified as MSG type IV; Antiferromagnets without such translation T belongs to MSG type I and type III66. This suggests there is a one-to-one correspondence between the existence or absence of the UT in the spin space group and the MSG being type IV or type I/III. The effect of SOC on the predicted hidden spin polarization Use magnetic symmetry with SOC to describe the spin-splitting of energy bands without SOC In collinear antiferromagnetic compounds, the existence of UT in the Table 2 | Easily exfoliable 2D magnetic compounds with FM configuration belonging to SST-5 class Formula SG Structure Prototype Eg (eV) Eb (meV/Å2) 3D SG SDB of 3D ID SDB CoBr2 P3m1 CdI2 0.2 16.8 P3m1 COD 9016149 CoCl2 P3m1 CdI2 0.2 10.7 P3m1 COD 9014719 CoO2 P3m1 CdI2 0.0 22.6 P3m1 COD 1522027 FeBr2 P3m1 CdI2 0.0 15.5 P3m1 COD 9009102 FeI2 P3m1 CdI2 0.0 16.9 P3m1 COD 9009103 NiBr2 P3m1 CdI2 0.8 18.1 R3m COD 9008013 NiCl2 P3m1 CdI2 1.1 16.3 R3m COD 9009132 NiI2 P3m1 CdI2 0.3 21.5 R3m COD 9009133 VS2 P3m1 CdI2 0.0 27.7 P3m1 ICSD 651361 VSe2 P3m1 CdI2 0.0 25.4 P3m1 ICSD 86520 VTe2 P3m1 CdI2 0.0 27.1 P3m1 ICSD 603582 TmI2 P3m1 CdI2 0.0 10.5 P3m1 ICSD 43731 LaBr2 P6m2 MoS2 0.6 11.2 P63/ mmc ICSD 65481 FeTe P4/ nmm FeSe 0.0 26.6 P4/ nmm ICSD 169974 LaCl P3m1 ZrCl 0.0 11.0 R3m ICSD 24410 ScCl P3m1 ZrCl 0.0 13.8 R3m COD 4343683 TbBr P3m1 ZrCl 0.0 12.2 R3m ICSD 23353 YCl P3m1 ZrCl 0.0 17.6 R3m ICSD 30708 CuCl2 C2/m NbTe2 0.2 13.4 C2/m COD 9001506 EuOBr P4/ nmm PbClF 0.0 17.4 P4/ nmm ICSD 28531 EuOI P4/ nmm PbClF 0.0 14.8 P4/ nmm ICSD 27666 PrOBr P4/ nmm PbClF 0.0 24.1 P4/ nmm COD 2232654 NdOBr C2/m PbClF 0.2 21.8 P4/ nmm COD 9009172 SmOBr C2/m PbClF 0.2 18.4 P4/ nmm COD 1530050 TmOI C2/m PbClF 0.2 15.0 P4/ nmm COD 2310429 TbOBr Cmme PbClF 0.0 15.2 P4/ nmm ICSD 28532 CrOBr Pmmn FeOCl 0.5 14.8 Pmmn ICSD 27092 CrOCl Pmmn FeOCl 0.6 13.8 Pmmn ICSD 4086 CrSBr Pmmn FeOCl 0.4 19.5 Pmmn ICSD 69659 ErSCl Pmmn FeOCl 0.3 11.9 Pmmn ICSD 21009 ErSeI Pmmn FeOCl 0.0 11.6 Pmmn ICSD 50194 HoSI Pmmn FeOCl 0.5 10.9 Pmmn ICSD 425295 ErHCl P3m1 SmSI 0.0 10.9 R3m COD 1530725 SmSI P3m1 SmSI 0.0 11.4 R3m COD 1008317 YbOCl P3m1 SmSI 0.0 11.7 R3m ICSD 6077 CdOCl P3m1 BiTeI 0.3 25.6 P63mc COD 9016472 Co(OH)2 C2/m Mg(OH)2 0.0 18.3 P3m1 ICSD 88940 The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The effect of SOC on the predicted hidden spin polarization The last three columns describe for the experimental parent structure: the 3D space group (3D SG), source database (SDB)58, and the ID in the source data- base (ID SDB). The correspondence relation can be formally established by introducing an auxiliary MSG—a subgroup of the spin space group containing only elements of spatial and time reversal symmetries. This is referred to as “MSG without SOC” in the Appendix of ref. 38 or equivalently as “magnetic groups with pseudoscalar electron spin” in ref. 67. Following that, we can prove a chain relation as depicted in Eq. (1). ð1Þ ðcÞ ΘT in Standard MSG $ ðdÞ Standard MSG being type IV ð1Þ (a) The existence or not of a UT symmetry in the SSG corresponds to (b) the existence or not of a ΘT symmetry in the auxiliarygroup. This is because the ΘU symmetry preserves any collinear magnetic ordering and is a symmetry of any collinear antiferromagnets43–45. Meanwhile, (b) the existence or not of a ΘT symmetry in the auxiliary MSG (without SOC) corresponds to (c) the existence or not of a ΘT symmetry in the standard MSG (with SOC). Antiferromagnetic materials whose (c) MSG preserve (or violate) ΘT symmetry is classified as (d) MSG type IV (or MSG type I/III)66. The established correspondence relation thus justifies the use of MSG (with SOC)—avoiding the use of the “less familiar” spin symmetry64—to predict whether the spin splitting effect without SOC will occur. This also allows the use of the tabulated magnetic structure symmetry information provided in material database46 to sort out candidate materials39. For the prediction of the degeneracy of the full bands without SOC, a comprehensive analyze of the spin symmetry group and its irreducible representation is necessary43,64,65. Experimental detectability Experimental detectability Analogous to the detection of SOC-induced hidden spin polarization in nonmagnetic compounds (also known as R-2 and D-2 effects) [12], a hidden property can be observed when a probe can resolve the local sectors where the property is not compensated. Specific to hidden spin polarization, the spatial segregation of the spin polarization states allows in principle the detection of the hidden effect in Hidden versus apparent spin polarization in noncollinear antiferromagnets While the current paper focuses on the hidden spin polarization in collinear antiferromagnetic compounds, we note that the hidden effect can also exist in noncollinear antiferromagnetic compounds. When a bulk noncollinear antiferromagnetic compound has ΘIT sym- metry, the energy bands are spin degenerate. If the system can be further divided into separate sectors thatlocally violate ΘIT, then there could exist hidden spin polarization pertaining to the individual sec- tors. However, one should note that the symmetry condition of having UT for preserving spin degeneracy in noncollinear antiferromagnetic compounds39 is not valid anymore, this is because (1) when the spin arrangement is non-coplanar, the MSG type IV does not guarantee the existence of UT; Moreover, (2) when the spin arrangement is coplanar, MSG type IV guarantees the existence of UT, but the existence of such UT does not always guarantee spin degeneracy. Specifically, when the spin states are not aligned in the same plane of the coplanar plane, the UT symmetry will not reverse the spin states as it works in the collinear magnetic systems. These properties of noncollinear antiferromagnets offer new knobs to tune the hidden versus apparent spin polarization via tilting the local magnetic motifs. The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The last three columns describe for the experimental parent structure: the 3D space group (3D SG), source database (SDB)58, and the ID in the source data- base (ID SDB). Use magnetic symmetry with SOC to describe the spin-splitting of energy bands without SOC The up and down spins are mapped to the color from blue to red. The crystal and magnetic structure for tetragonal Ca2MnO4 used in our DFT calculations are taken from ref. 47. degenerate band structure of Ca2MnO4; (c) Hidden spin polarization from each individual sector of the lowest two conduction bands (C1 and C2) on ΓXR k-plane. The up and down spins are mapped to the color from blue to red. The crystal and magnetic structure for tetragonal Ca2MnO4 used in our DFT calculations are taken from ref. 47. Fig. 4 | Hidden spin polarization from the individual antiferromagnetic sector in bulk tetragonal Ca2MnO4 (bulk belonging to SST-1 class with sector belonging to SST-4 class). a Crystal structure of antiferromagnetic tetragonal Ca2MnO4 composed of two antiferromagnetic sectors with opposite magnetic ordering (the magnetic ordering is indicated by red and blue polyhedra) in the unit cell. The two layers are referred to as sector-α and sector-β, respectively; (b) Spin degenerate band structure of Ca2MnO4; (c) Hidden spin polarization from each individual sector of the lowest two conduction bands (C1 and C2) on ΓXR k-plane. The up and down spins are mapped to the color from blue to red. The crystal and magnetic structure for tetragonal Ca2MnO4 used in our DFT calculations are taken from ref. 47. spin polarization in AFM, since the unit cell can always be built in terms of two or more sectors, electric field is a practically direct way of inducing and controlling the existence of spin splitting (as well as its magnitude) via modulating the symmetry relationship between the sectors. For example, in the spin degenerate bulk antiferro- magnets made of a pair of spin-split antiferromagnetic sectors (e.g., FeSe discussed in Supplementary Information Section D) or ferro- magnetic sectors (e.g., FeBr2 discussed in the Results Section), external electric field would break the ΘIT and UT symmetry between the spin split sectors, which then implies a transition from hidden effect to apparent effect. In fact, the electric field applied couples with the electron spin through the magnetoelectric effect68, which is only allowed under specific symmetry conditions41. Additionally, transport properties that are even functions of the sectors can take non-vanishing values in a hidden system. For example, non- reciprocal nonlinear current respond to an applied electric field is recently demonstrated in antiferromagnetic tetragonal CuMnAs69. This serves as a guide in search for systems exhibiting this particular antiferromagnets. Use magnetic symmetry with SOC to describe the spin-splitting of energy bands without SOC In collinear antiferromagnetic compounds, the existence of UT in the spin space group (SSG, symmetry group of the system without SOC) means there is a spatial translation T that connects the atomic sites with opposite magnetic moments and keeps the crystal structure Nature Communications| (2023) 14:5301 5 Article https://doi.org/10.1038/s41467-023-40877-8 Fig. 3 | Hidden spin polarization from individual ferromagnetic sectors in bulk tetragonal CuMnAs (bulk belonging to SST-1 class with sector belonging to SST-5 class). a Crystal structure of antiferromagnetic CuMnAs composed of two ferromagnetic layers with opposite magnetization (indicated by red and blue polyhedra) in the unit cell. The Cu atoms are dismissed. The two layers are referred to as sector-α and sector-β, respectively; (b) Spin degenerate band structure of CuMnAs; (c) Hidden spin polarization from each individual sector of the highest two valence bands (V1 and V2) on ΓXS k-plane. The up and down spins are mapped to the color from blue to red. The crystal and magnetic structure for tetragonal CuMnAs used in our DFT calculations are taken from ref. 54. Fig. 3 | Hidden spin polarization from individual ferromagnetic sectors in bulk tetragonal CuMnAs (bulk belonging to SST-1 class with sector belonging to SST-5 class). a Crystal structure of antiferromagnetic CuMnAs composed of two ferromagnetic layers with opposite magnetization (indicated by red and blue polyhedra) in the unit cell. The Cu atoms are dismissed. The two layers are referred to as sector-α and sector-β, respectively; (b) Spin degenerate band structure of CuMnAs; (c) Hidden spin polarization from each individual sector of the highest two valence bands (V1 and V2) on ΓXS k-plane. The up and down spins are mapped to the color from blue to red. The crystal and magnetic structure for tetragonal CuMnAs used in our DFT calculations are taken from ref. 54. Fig. 4 | Hidden spin polarization from the individual antiferromagnetic sector in bulk tetragonal Ca2MnO4 (bulk belonging to SST-1 class with sector belonging to SST-4 class). a Crystal structure of antiferromagnetic tetragonal Ca2MnO4 composed of two antiferromagnetic sectors with opposite magnetic ordering (the magnetic ordering is indicated by red and blue polyhedra) in the unit cell. The two layers are referred to as sector-α and sector-β, respectively; (b) Spin degenerate band structure of Ca2MnO4; (c) Hidden spin polarization from each individual sector of the lowest two conduction bands (C1 and C2) on ΓXR k-plane. Use magnetic symmetry with SOC to describe the spin-splitting of energy bands without SOC Since this effect is intrinsic to the bulk it can be distinguished from the surface effect as the latter sensitively depends on the effective penetration depth of the probing beam [43]. Albeit, for the hidden spin polarization from individual AFM sectors, to detect the AFM spin polarization of the individual sectors, one needs to choose the surface configuration that respects the symmetries of the indivi- dual sector that ensure the anti-ferromagnetism of the sector, e.g., mirror plane symmetries perpendicular to the surface plane that connect the spin up and spin down magnetic moments of the AFM sector. Especially, systems with the degenerate states segregated on the different sectors would result in a minimally compensated hidden spin polarization, thus contributing to a robust signal when selectively probing the individual sector, thus being ideal platforms for the detection of the hidden effect. Electric and magnetic field control of the hidden effect One of the most desirable features of spin-related phenomena is the possibility of electric and magnetic control. In the case of the hidden Electric and magnetic field control of the hidden effect Data availability The VASP configuration and output files thatsupport the finding of this study have been deposited in figshare with the identifier [data DOI:10.6084/m9.figshare.22693042]. Other data related to this research are available from the corresponding author upon reasonable request. References 1. Litvin, D. B. Ferroelectric space groups. Acta Crystallogr. Sect. A 42, 44–47, https://doi.org/10.1107/S0108767386099920 (1986). 1. Litvin, D. B. Ferroelectric space groups. Acta Crystallogr. Sect. A 42, 44–47, https://doi.org/10.1107/S0108767386099920 (1986). Electric and magnetic field control of the hidden effect One of the most desirable features of spin-related phenomena is the possibility of electric and magnetic control. In the case of the hidden Nature Communications| (2023) 14:5301 Nature Communications| (2023) 14:5301 6 Article https://doi.org/10.1038/s41467-023-40877-8 Fig. 5 | Revealing the hidden spin polarization in hexagonal FeBr2 using an external electric field. a spin split band structure of FeBr2 with a 10 meV/Å z-oriented external electric field. Red and blue lines represent the spin-up and spin- down polarized bands. Insert depicts the spin splitting between the bottom two conduction bands at Γ (denoted as ΓCB1 and ΓCB2) as a function of the external electric field; (b) wavefunction plot for ΓCB1; and (c) wavefunction plot for ΓCB2. The crystal and magnetic structure for triagonal FeBr2 are taken from ref. 56. and was tailored into a bilayer slab for the calculations with external electric field. Fig. 5 | Revealing the hidden spin polarization in hexagonal FeBr2 using an external electric field. a spin split band structure of FeBr2 with a 10 meV/Å z-oriented external electric field. Red and blue lines represent the spin-up and spin- down polarized bands. Insert depicts the spin splitting between the bottom two conduction bands at Γ (denoted as ΓCB1 and ΓCB2) as a function of the external electric field; (b) wavefunction plot for ΓCB1; and (c) wavefunction plot for ΓCB2. The crystal and magnetic structure for triagonal FeBr2 are taken from ref. 56. and was tailored into a bilayer slab for the calculations with external electric field. spatially well separated. This results in weak inter-sector coupling, and consequently physically significant hidden spin polarization effect. response behavior. Furthermore, the bulk antiferromagnets formed by ferromagnetic layers with alternatively aligned magnetic moments along the direction perpendicular to the ferromagnetic layers (thus hosting hidden spin polarization) could have very dif- ferent magnetoresistance from the bulk ferromagnets formed by the same ferromagnetic layers but with uniformly aligned magnetic moments. Therefore, switching between the AFM and FM states by external magnetic field could lead to significant change of magne- toresistance, mimicking the tunneling magnetoresistance effect70. These perspectives offer electric and/or magnetic means to control the spin-related properties in antiferromagnets. Methods DFT setup l i 6 Zhang X Liu Q Luo J W Freeman A J & Zunger A Hidden spin 4. Rashba, E. & Sheka, V. Symmetry of energy bands in crystals of wurtzite type II. Symmetry of bands with spin-orbit interaction included. Fiz. Tverd. Tela, Collected Pap. 2, 62–76 (1959). 5. Dresselhaus, G. Spin-orbit coupling effects in Zinc blende struc- tures. Phys. Rev. 100, 580–586 (1955). 6. Zhang,X., Liu, Q., Luo, J.-W., Freeman, A. J. & Zunger, A. Hidden spin polarization in inversion-symmetric bulk crystals. Nat. Phys. 10, 387–393, https://doi.org/10.1038/nphys2933 (2014). 6. Zhang,X., Liu, Q., Luo, J.-W., Freeman, A. J. & Zunger, A. Hidden spin polarization in inversion-symmetric bulk crystals. Nat. Phys. 10, 387–393, https://doi.org/10.1038/nphys2933 (2014). 7. Yuan, L. et al. Uncovering and tailoring hidden Rashba spin–orbit splitting in centrosymmetric crystals. Nat. Commun. 10, 906, https://doi.org/10.1038/s41467-019-08836-4 (2019). 7. Yuan, L. et al. Uncovering and tailoring hidden Rashba spin–orbit splitting in centrosymmetric crystals. Nat. Commun. 10, 906, https://doi.org/10.1038/s41467-019-08836-4 (2019). 8. 8. Riley, J. M. et al. Direct observation of spin-polarized bulk bands in an inversion-symmetric semiconductor. Nat. Phys. 10, 835–839, https://doi.org/10.1038/nphys3105 (2014). How is the hidden spin polarization calculated 9. Santos-Cottin, D. et al. Rashba coupling amplification by a stag- gered crystal field. Nat. Commun. 7, 11258 (2016). We evaluated the hidden spin polarization on sector-η by projecting the calculated degenerate wavefunctions |ϕ1〉, |ϕ2〉onto the atomic orbital basis |ilm〉and sum over the sites i within sector-η in the primitive unit cell, Sη = P s = 1,2 P i2η P lmhϕsj^Sjilmihilmjϕsi. This expression sums contribution from both degenerate bands (s = 1,2). 10. Razzoli, E. et al. Selective probing of hidden spin-polarized states in inversion-symmetric bulk MoS2. Phys. Rev. Lett. 118, 086402, https://doi.org/10.1103/PhysRevLett.118.086402 (2017). 11. Yao, W. et al. Direct observation of spin-layer locking by local Rashba effect in monolayer semiconducting PtSe2 film. Nat. Com- mun. 8, 14216, https://doi.org/10.1038/ncomms14216 (2017). 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We adopt the GGA + U method76 to account for the on-site Coulomb interactions of localized 3d orbitals involved in the calculations. We used U = 3.9 eV, J = 0 eV on Mn-3d orbits for Ca2MnO4, U = 5.3 eV, J = 0 eV on Fe-3d orbits for insulating FeBr2. These Hubbard U values are derived in ref. 77 using the approach outlined in ref. 78. For CuMnAs, a metal, we used a smaller U value on Mn-3d orbits (U = 1.9 eV, J = 0 eV39). We follow the approach proposed by Neugebauer and Scheffler79 to apply a uniform electric field to the bilayer slab in the calculations. This approach treats the artificial per- iodicity of the slab by adding a planar dipole sheet in the middle of the vacuum region. 3. Nespolo, M., Aroyo, M. I. & Souvignier, B. Crystallographic shelves: space-group hierarchy explained. J. Appl Crystallogr. 51, 1481–1491, https://doi.org/10.1107/S1600576718012724 (2018). 3. Nespolo, M., Aroyo, M. I. & Souvignier, B. Crystallographic shelves: space-group hierarchy explained. J. 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L.-D.Y., X.Z., C.M.A., and A.Z. analyzed the results and wrote the manuscript. 65. Liu, P., Li, J., Han, J., Wan, X. & Liu, Q. Spin-group symmetry in magnetic materials with negligible spin-orbit coupling. Phys. Rev. X 12, 021016, https://doi.org/10.1103/PhysRevX.12.021016 (2022). 66. Zamorzaev, A. Generalization of Fedorov groups. Kristallografiya 2, 15–20 (1957). 67. Turek, I. Altermagnetism and magnetic groups with pseudoscalar electron spin. Phys. Rev. B 106, 094432, https://doi.org/10.1103/ PhysRevB.106.094432 (2022). Competing interests Competing interests The authors declare no competing interests. 68. Fiebig, M. Revival of the magnetoelectric effect. J. Phys. D: Appl. Phys. 38, R123, https://doi.org/10.1088/0022-3727/38/8/R01 (2005). 9 Nature Communications| (2023) 14:5301 9 Article Additional information Additional information Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY
Vestnik Universiteta imeni O.E. Kutafina (MGÛA)/Vestnik Universiteta imeni O. E. Kutafina
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V. S. ELISEEV, V. S. ELISEEV, Professor at the Department of Environmental Law of the Kutafin Moscow State Law University (MSAL), Dr. Sci. (Law), Associate Professor v.s.eliseev@mail.ru 125993, Russia, Moscow, ul. Sadovaya-Kudrinskaya, 9 V. S. ELISEEV, Professor at the Department of Environmental Law of the Kutafin Moscow State Law University (MSAL), Dr. Sci. (Law), Associate Professor v.s.eliseev@mail.ru 125993, Russia, Moscow, ul. Sadovaya-Kudrinskaya, 9 Елисеев В. С. Место теории экономического права в системе правового регулирования экономики Елисеев В. С. Место теории экономического права в системе правового регулирования экономики Елисеев В. С. 175 175 МЕСТО ТЕОРИИ ЭКОНОМИЧЕСКОГО ПРАВА В СИСТЕМЕ ПРАВОВОГО РЕГУЛИРОВАНИЯ ЭКОНОМИКИ Аннотация. Статья анализирует место теории экономического права в системе правового регулирования экономических отношений, показывает развитие экономического законодательства в истори- ческом контексте, сопоставляет теорию экономического права с хозяйственным, гражданским, предпринимательским, коммерческим и экономическим правом. Автор обосновывает вывод о том, что теория экономического права является составной частью теории права. Обосновывается необходимость использования теории эко- номического права в ее перспективе, которая позволит согласовы- вать экономическое законодательство. Теория рассматривает в качестве основы правового воздействия экономические институты как первичные области формирования правовых режимов, имеющих комплексный межотраслевой характер. Вячеслав Сергеевич ЕЛИСЕЕВ, Вячеслав Сергеевич ЕЛИСЕЕВ, профессор кафедры экологического и природоресурсного права Университета имени О.Е. Кутафина (МГЮА), доктор юридических наук, доцент v.s.eliseev@mail.ru 125993, Россия, г. Москва, ул. Садовая-Кудринская, д. 9 Сергеевич ЕЛИСЕЕВ, профессор кафедры экологического и природоресурсного права Университета имени О.Е. Кутафина (МГЮА), Ключевые слова: экономическое право, теория экономического права, правовое регулирование экономических отношений, концепция (тео- рия) правового регулирования экономических отношений, развитие экономического законодательства. доктор юридических наук, доцент v.s.eliseev@mail.ru 125993, Россия, г. Москва, ул. Садовая-Кудринская, д. 9 DOI: 10.17803/2311-5998.2020.71.7.175-184 1 См., например: Лаптев В. В. Хозяйственное право — право предпринимательской деятель- ности // Государство и право. 1993. № 1. С. 33—38 ; Дозорцев В. А. Один кодекс или два? (Нужен ли хозяйственный кодекс наряду с гражданским?) // Правовые проблемы рыночной экономики в Российской Федерации : Труды Института законодательства и сравнительного правоведения при Правительстве Российской Федерации. Вып. 57. М., 1994. С. 115—143. 2 Победоносцев К. П. Курс гражданского права // Сочинения : в 2 т. СПб. : Типография Мин. пут. сообщения (А. Бенке), 1896. Ч. 1—3. 4 Источники русского торгового права : сборник извлечений из Свода законов, уставов, полисных условий и договоров. М., 1914. 5 Ершова И. В. Проблемы правового режима государственного имущества в хозяйствен- ном обороте: теоретические основы и пути совершенствования. М. : Юриспруденция, 2001. С. 10—11. 1 См., например: Лаптев В. В. Хозяйственное право — право предпринимательской деятель- ности // Государство и право. 1993. № 1. С. 33—38 ; Дозорцев В. А. Один кодекс или два? (Нужен ли хозяйственный кодекс наряду с гражданским?) // Правовые проблемы рыночной экономики в Российской Федерации : Труды Института законодательства и сравнительного правоведения при Правительстве Российской Федерации. Вып. 57. М., 1994. С. 115—143. 2 Победоносцев К. П. Курс гражданского права // Сочинения : в 2 т. СПб. : Типография Мин. пут. сообщения (А. Бенке), 1896. Ч. 1—3. 3 Шершеневич Г. Ф. Учебник торгового права [по изд. 1914 г.]. М. : Спарк, 1994. 4 Источники русского торгового права : сборник извлечений из Свода законов, уставов, полисных условий и договоров. М., 1914. 5 Ершова И. В. Проблемы правового режима государственного имущества в хозяйствен- ном обороте: теоретические основы и пути совершенствования. М. : Юриспруденция, 2001. С. 10—11. 3 Шершеневич Г. Ф. Учебник торгового права [по изд. 1914 г.]. М. : Спарк, 1994. 6 Аскназий С. И. Очерки хозяйственного права в СССР. Л., 1926. 7 Таль Л. Понятие и сущность хозяйственного права // Право и жизнь. 1924. Кн. 9. С. 22. 8 Гинс Г. Поиски нового права // Новая экономика и новое право. Харбин, 1938. С. 26, 38. 9 Либерман С. Последний этап нэпа и хозяйственное право // Проблемы марксизма. 1931. № 5—6. 10 Вольф В. Ю. Основы хозяйственного права. М., 1928 ; Стучка П. И. Курс советского гражданского права. Т. 1. М.-Л., 1931. 11 Стучка П. И. Указ. соч. 12 Стучка П. И., Амфитеатров Г. Н. Основы гражданского законодательства СССР. М., 1931. С. 5. 12 Стучка П. И., Амфитеатров Г. Н. Основы гражданского законодательства СССР. М., 1931. С. 5. PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY Abstract. The article analyzes the place of the "Theory of economic law" in the system of legal regulation of economic relations. It shows the histori- cal development of economic legislation. The "Theory of economic law" is compared with economic, civil, business, commercial and economic law. The author believes that the " Theory of economic law "is an integral part of the "Theory of law". The article substantiates the need to use the "Theory of economic law" in its perspective, which will allow to coordinate economic legislation. The Theory considers economic institutions as the basis of le- © В. С. Елисеев, 2020 7/2020 176 gal influence. These institutions are the primary areas in the formation of legal regimes. Legal regimes have a complex cross-sectoral character. Keywords: economic law, theory of economic law, legal regulation of eco- nomic relations, concept (theory) of legal regulation of economic relations, development of economic legislation. В  настоящее время вновь возрастает острота дискуссий между представи- телями различных отраслей права об их месте в системе правового регу- лирования экономических отношений, актуальность которых приобретает новый оттенок в силу появления «экономического права», вызвавшего в свой адрес очередную волну критики, как конструктивной, так и политизированной. В На этом фоне появляются рассуждения о теории экономического права, для понимания места которой в системе правового регулирования экономики необ- ходимо обратиться к историческому аспекту развития правовых школ, направ- ленных на правовое обеспечение экономических отношений с различных пози- ций, на основе концепций гражданского, хозяйственного, предпринимательского, коммерческого и экономического права1. В первую очередь следует коснуться дореволюционного периода развития права в России, основу которого составляли работы таких известных предста- вителей гражданского права, как К. П. Победоносцев2, Г. Ф. Шершеневич3 и др. Из наиболее показательных документов того времени следует выделить, в частности, Устав о промышленности, Устав торговый, Устав о векселях и подоб- ные законы4, которые, по сути, были комплексными нормативными документами, что объясняется тем, что в дореволюционной России существовал достаточно разветвленный государственный сектор экономики, место которого в общем рос- сийском экономическом пространстве было значительно. В научной литературе отмечается, что государству достаточно часто приходилось выкупать частные проекты с тем, чтобы «достраивать и перестраивать» начатые стройки, как, в частности, это имело место в отношении строительства объектов железнодо- рожной инфраструктуры5. Что касается стратегических объектов, как КВЖД, то их строительство без участия государства было невозможно в принципе. 7/2020 7/2020 Елисеев В. С. Место теории экономического права в системе правового регулирования экономики Елисеев В. С. 11 Стучка П. И. Указ. соч. 13 Курс советского хозяйственного права / под ред. Е. Б. Пашуканиса и Л. Я. Гинцбурга. М., 1935. Т. 1. 14 Мартемьянов В. С. Юридическая трагедия // Юридическая газета. 1992. № 1, 2. 15 Вышинский А. Я. Основные задачи науки советского социалистического права // Совет- ское государство и право. 1939. № 1. С. 22. 16 Братусь С. Н., Алексеев С. С. О разработке правовых вопросов управления народным хозяйством // Правоведение. 1963. № 4. С.45. 17 Венедиктов А. В. Государственная социалистическая собственность. М.-Л. : АН СССР, 1948. 18 Лаптев В. В. Предмет и система хозяйственного права. М. : Юрид. лит., 1969. 19 Мамутов В. К. Совершенствование правового регулирования хозяйственной деятель- ности. Киев, 1982. PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY Место теории экономического права в системе правового регулирования экономи 177 Февральские и особенно октябрьские события 1917 г. изменили ситуацию в ее основе: произошла национализация и был сформирован государственный и колхозно-кооперативный сектор экономики; гражданское право уступило место публичному правовому регулированию. На первое место выходит хозяйственное право. При этом исследователи отмечают, что термин «хозяйственное право» частенько подменял термин «гражданское право», иногда мало чем отличаясь от последнего6. И хотя наука больше шла по пути практического творчества, был накоплен огромный опыт хозяйствования, планирования, формирования социалистической (государственной) инфраструктуры. Главной особенностью научных взглядов того периода была чрезмерная надежда на «революционное правосознание», что являлось большим недостатком для формирования права7. Вместе с тем стало очевидно, что хозяйственное право выходит за рамки частного, а также административного права, являясь самостоятельным комплекс- ным образованием8, — речь шла в первую очередь о государственном секторе экономики. В данный период сформировались три основных взгляда на хозяйственное право: во-первых, оно охватывало все области народного хозяйства (как в тот период обозначали экономику страны); во-вторых, хозяйственное право регу- лировало только государственный сектор экономики (социалистические произ- водственные отношения)9; в-третьих, нельзя не отметить гражданско-правовую концепцию отрицания роли хозяйственного права как самостоятельной отрасли10. Следует отметить возникновение в 20‑х гг. прошлого столетия «двухсектор- ной» теории, разработанной П. И. Стучкой11, появившейся вместе с так называ- емой новой экономической политикой молодого социалистического государства. Суть данного взгляда сводилась к признанию параллельного существования частного сектора экономики, регламентируемого на основе гражданского права, и государственного сектора экономики, регулируемого хозяйственным правом. Главной конструктивной мыслью данной теории являлось разделение правового регулирования для различных секторов экономики. Логическим венцом теории П. И. Стучки были предложения о принятии двух самостоятельных кодексов12. Однако этот взгляд был принципиально отвергнут на I Всесоюзном съезде марксистов-государственников в 1931 г. Продолжением марксистской политики государства является появление школы единого хозяйственного права, связанной с именами Л. Я. Гинцбурга и Е. Б. Па- 7/2020 178 шуканиса13. По сути, это была попытка одним документов в виде Хозяйственного кодекса объединить государственный и частный сектора экономики на основе комплексного правового регулирования экономических отношений. Авторы сде- лали заключение, что административные и гражданско-правовые нормы права должны гармонично сочетаться, а не противоречить друг другу, на что данная школа и была направлена. Однако под влиянием представителей цивилистики в сложный период 1937— 1938 гг. авторам была поставлена в вину попытка уничтожения гражданского права, их объявили врагами народа и репрессировали14. В середине 1930‑х гг. появляется «дуалистическая концепция», сформиро- ванная под влиянием А. Я. Вышинского: вертикальные экономические отноше- ния предлагалось регулировать административным правом, а горизонтальные отношения — гражданским правом. 13 Курс советского хозяйственного права / под ред. Е. Б. Пашуканиса и Л. Я. Гинцбурга. М., 1935. Т. 1. 19 Мамутов В. К. Совершенствование правового регулирования хозяйственной деятель- ности. Киев, 1982. PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY Сама же концепция единого хозяйственного права была подвергнута острой критике15. После Великой Отечественной войны появляется концепция хозяйственно- административного права, сформированная в начале 1960‑х гг. С. Н. Братусем и С. С. Алексеевым. Согласно данному подходу при административно-правовом регулировании после «погружения вглубь» хозяйственных отношений образуется «мертвая зона», которая должна регулироваться хозяйственно-административ- ным правом — относительно самостоятельной подотраслью юридической науки16. В дальнейшем С. С. Алексеев от этой позиции отказался и перешел на позиции гражданско-правового регулирования экономических отношений. Для послевоенного периода большое значение имела разработка А. В. Вене- диктовым конструкции права оперативного управления17, которая и в настоящее время не потеряла своей актуальности для правового регулирования экономи- ческих отношений с участием государственных организаций. Наиболее значимым событием является разработка в конце 1950‑х гг. про- шлого века В. В. Лаптевым18 и В. К. Мамутовым19 третьей школы хозяйственного права. Согласно данной концепции, правовое регулирование экономики имеет монистический характер — здесь в основе принцип единства хозяйственно-право- вого регулирования. Вместе с тем имеет место двойственность частно-публично- го характера: правовое регулирование вертикальных экономических отношений предполагает административный характер, а правовое регулирование горизон- 7/2020 7/2020 Елисеев В. С. Место теории экономического права в системе правового регулирования экономики 179 179 тальных отношений — гражданско-правовой. В целом авторы придерживались мнения о единстве хозяйственных отношений (их однородности)20. тальных отношений — гражданско-правовой. В целом авторы придерживались мнения о единстве хозяйственных отношений (их однородности)20. Как отмечал В. К. Мамутов, школа последовательно рассматривала хозяй- ственное право в качестве самостоятельной отрасли права, но эта отрасль не является «рафинированной» — она имеет «взаимопроникающие начала с дру- гими отраслями права»21. Авторы полагали, что необходимо принять Хозяйственный кодекс, проект ко- торого в СССР был разработан, но не был принят. Что касается населения, то отношения с его участием относятся к предмету гражданского права. Следует отметить, что после распада Советского Союза В. К. Мамутовым, возглавившим украинскую школу хозяйственного права, эта цель была достиг- нута — принят Хозяйственный кодекс Украины22. Что касается России, то уси- лия В. В. Лаптева и его сподвижников со стороны органов власти поддержки не нашли. В науке выделяется четвертая школы хозяйственного (предпринимательского) права, которая связывается с именем В. С. Мартемьянова23. 26 Предпринимательское (хозяйственное) право / под ред. В. В. Лаптева, С. С. Занковского. М. : Волтерс Клувер, 2006. 560 с. 25 Предпринимательское право России: итоги, тенденции и пути развития : монография / Е. П. Губин, В. А. Вайпан, И. С. Шиткина [и др. ] ; отв. ред. Е. П. Губин. М. : Юстицинформ, 2019. 664 с. 20 Теоретические проблемы хозяйственного права. М. : Наука, 1975. С. 28. 21 Отграничение хозяйственного права от других отраслей права и их взаимодействие // Хозяйственное право : учебник / В. К. Мамутов, Г. Л. Знаменский, К. С.Халиулин [и др.] ; под ред. В. К. Мамутова. Киев : Юринком Интер, 2002. С. 34—38. 22 Господарський кодекс України // Відомості Верховної Ради. 2003. № 18. Ст. 144. 23 Мартемьянов В. С. Хозяйственное право : курс лекций : в 2 т. М. : Бек, 1994. 24 См.: Ершова И. В., Енькова Е. Е., Тарасенко О. А., Трофимова Е. В. Государственное регулирование предпринимательской деятельности : учебник / отв. ред. И. В. Ершова. М. : Проспект, 2020. 224 с. 25 Предпринимательское право России: итоги, тенденции и пути развития : монография / Е. П. Губин, В. А. Вайпан, И. С. Шиткина [и др. ] ; отв. ред. Е. П. Губин. М. : Юстицинформ, 2019. 664 с. 26 Предпринимательское (хозяйственное) право / под ред. В. В. Лаптева, С. С. Занковского. М. : Волтерс Клувер, 2006. 560 с. 20 Теоретические проблемы хозяйственного права. М. : Наука, 1975. С. 28. 24 См.: Ершова И. В., Енькова Е. Е., Тарасенко О. А., Трофимова Е. В. Государственное регулирование предпринимательской деятельности : учебник / отв. ред. И. В. Ершова. М. : Проспект, 2020. 224 с. PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY На наш взгляд, кор- ректнее именовать ее школой предпринимательского права, поскольку, во-первых, термин «хозяйственное право» фактически выбыл из оборота и используется лишь как «дань традиции», но не более того; во-вторых, все-таки принадлеж- ность хозяйственных отношений нужно связывать с государственным сектором экономики, а не со всей экономикой в целом, а представители данной школы при- держиваются именно общего охвата экономических отношений; в-третьих, ряд представителей данной школы придерживаются теории предпринимательского права как составной части гражданского права. В настоящее время школа предпринимательского права получила развитие под влиянием кафедры предпринимательского и корпоративного права Универ- ситета имени О.Е. Кутафина (МГЮА)24 (заведующий кафедрой — И. В. Ершова), кафедры предпринимательского права МГУ имени М. В. Ломоносова25 (заведу- ющий кафедрой — Е. П. Губин), сектора предпринимательского и корпоративного права Института государства и права Российской академии наук26 (руководитель 7/2020 180 сектора — С. С. Занковский). В этом ряду нельзя также не отметить А. Г. Быкова27, В. К. Андреева28, Н. М. Коршунова29, И. В. Дойникова, В. С. Белых30. На самостоятельном существовании коммерческого права настаивают Б. И. Пугинский и его последователи, прежде всего с кафедры коммерческого права МГУ имени М. В. Ломоносова. Они разделяют идеи дуализма гражданского права и отстаивают идею самостоятельности коммерческого права, выступаю- щего подотраслью гражданского права31. Вместе с тем все многообразие концепций хозяйственного, предприниматель- ского и коммерческого права существует при подавляющем господстве граждан- ско-правовой концепции предпринимательского права. Согласно такому подходу предпринимательское право, равно как и коммерческое, теряет свою самосто- ятельность и рассматривается как часть гражданского права. Что касается «хо- зяйственного права», то такое наименование рассматривается как «пережиток прошлого»32. Появление экономического права тоже не имеет полной определенности: в первую очередь необходимо отметить предельно широкий подход, когда эконо- мическое право рассматривается как обобщенная учебная дисциплина, собрав- шая различные институты права, в той или иной степени непосредственно отно- сящиеся к регулированию экономических отношений33, подобно использованию экономистами термина «хозяйственное право»34. При таком подходе вести речь 29 Предпринимательское право : учебник для студентов вузов, обучающихся по специаль- ности 030501 «Юриспруденция» / Н. Д. Эриашвили [и др.] ; под ред. Н. М. Коршунова, Н. Д. Эриашвили, П. В. Алексия. 6-е изд., перераб. и доп. М. : Юнити-Дана ; Закон и право, 2017. 535 с. 31 Коммерческое право: актуальные проблемы и перспективы развития : сб. статей к юби- лею доктора юрид. наук, проф. Б. И. Пугинского / сост. Е. А. Абросимова, С. Ю. Филип- пова. М. : Статут, 2011. 286 с. 32 См., например: Братусь С. Н. Предмет и система советского гражданского права. М., 1963. С. 30 Предпринимательское право: современный взгляд : монография / МГУ имени М. В. Ло- моносова ; Е. А. Абросимова, В. К. Андреев, Е. Г. Афанасьев [и др.] ; отв. ред. С. А. Ка- релина, П. Г. Лахно. М. : Юстицинформ, 2019. 600 с. 27 Быков А. Г. Предпринимательское право: проблемы формирования и развития // Вестник Моск. ун-та. Серия «Право». 1993. № 6. 31 Коммерческое право: актуальные проблемы и перспективы развития : сб. статей к юби- лею доктора юрид. наук, проф. Б. И. Пугинского / сост. Е. А. Абросимова, С. Ю. Филип- пова. М. : Статут, 2011. 286 с. 27 Быков А. Г. Предпринимательское право: проблемы формирования и развития // Вестник Моск. ун-та. Серия «Право». 1993. № 6. 28 Право и экономическое развитие: проблемы государственного регулирования экономики : монография / В. К. Андреев, Л. В. Андреева, К. М. Арсланов [и д.] ; под ред. В. А. Вай- пана, М. А. Егоровой. М. : Юстицинформ, 2017. 432 с. 29 Предпринимательское право : учебник для студентов вузов, обучающихся по специаль- ности 030501 «Юриспруденция» / Н. Д. Эриашвили [и др.] ; под ред. Н. М. Коршунова, Н. Д. Эриашвили, П. В. Алексия. 6-е изд., перераб. и доп. М. : Юнити-Дана ; Закон и право, 2017. 535 с. 30 Предпринимательское право: современный взгляд : монография / МГУ имени М В Ло- 28 Право и экономическое развитие: проблемы государственного регулирования экономики : монография / В. К. Андреев, Л. В. Андреева, К. М. Арсланов [и д.] ; под ред. В. А. Вай- пана, М. А. Егоровой. М. : Юстицинформ, 2017. 432 с. 35 Ершов В. В., Ашмарина Е. М., Корнев В. Н. Экономическое право Российской Федера- ции : монография. М. : РГУП, 2017. 284 с. 36 Альбов А. П., Ручкина Г. Ф. Экономическое право как новое направление развития правовой науки // Ученые труды Российской академии адвокатуры и нотариата. 2015. № 3 (38). С. 97. 37 Альбов А. П., Ручкина Г. Ф. Указ. соч. С. 99. 38 Ашмарина Е. М. Экономическое право как микромодель системы российского права // Вопросы экономики и права. 2015. № 84. С. 7—13. 39 Мирошник С. В. Экономическое право в системе российского права // Вопросы эконо- мики и права. 2016. № 1. С. 12—14. 40 Лаптев В. А. «Мегаотрасли» — экономическое, корпоративное и энергетическое право или юридические фикции? // Экономика. Право. Общество. 2017. № 3. С. 28—33. 41 Веленто И. И., Елисеев В. С. Теория экономического права : курс лекций. Гродно : ГрГУ, 2004. 417 с. PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY 11 ; Толстой Ю. К. Понятие права собственности // Проблемы гражданского и административного права / под ред. Б. Б. Черепахина, О. С. Иоффе, Ю. К. Толстого. Л., 1962. С. 143 ; Дозорцев В. А. Один кодекс или два? (Нужен ли хозяйственный кодекс наряду с гражданским?) // Правовые проблемы рыночной экономики в Российской Фе- дерации : Труды Института законодательства и сравнительного правоведения при Пра- вительстве Российской Федерации. М., 1994. Вып. 57. С. 115—143. 33 Экономическое право : хрестоматия : в 3 т. СПб. : Питер, 2004. 34 Круглова Н. Ю. Хозяйственное право : учебное пособие для бакалавров. 6-е изд., пере- раб. и доп. М. : Юрайт, 2015. 883 с. 7/2020 7/2020 Елисеев В. С. Место теории экономического права в системе правового регулирования экономики 181 181 о новой отрасли права не приходится, поскольку в указанном «сборнике» сосре- доточены разделы различных отраслей права, в том числе административного, гражданского, предпринимательского, финансового, аграрного, налогового и т.д. о новой отрасли права не приходится, поскольку в указанном «сборнике» сосре- доточены разделы различных отраслей права, в том числе административного, гражданского, предпринимательского, финансового, аграрного, налогового и т.д. Глобальный подход к экономическому праву предлагают ученые Российско- го государственного университета правосудия (Е. М. Ашмарина, В. В. Ершов, В. Н. Корнев и др.)35, рассматривая его в качестве мегаотрасли, которая «пред- ставляет собой рецепцию и аккумуляцию основ всех отраслей первого порядка по признаку всеобъемлющего характера своего предмета» в «единый правовой комплекс — экономическое право Российской Федерации». Оно должно «устано- вить связь между различными отраслями права, регулирующими экономические отношения, государственной политикой и экономикой»36 и «призвано объединить в себя все правовые нормы, регулирующие общественные отношения в сфере экономики, которые будут встроены в мировоззрение и культуру народа, на ко- тором и будет происходить поступательное развитие экономики»37. Е. М. Ашмариной предлагается примерная структура экономического права: Общая часть: «экономические основы экономического права», «экономическое право РФ»; Особенная часть: «правовое регулирование отношений в сфере фи- нансов», «правовое обеспечение денежного обращения, кредита и банковской деятельности», «учетные системы и их правовое регулирование»38 и т.п. Наиболее часто встречающиеся аргументы критиков экономического права: «Экономическое право не отвечает классическим критериям и не может быть признано самостоятельной отраслью права», поскольку оно «регулирует раз- нообразные экономические отношения, сочетает в себе как публичные, так и частные начала»39. В. А. Лаптев отмечает, что по факту «мы опять возвращаемся к давно суще- ствующему понятию “хозяйственное” либо “предпринимательское право”, что ставит под сомнение открытие новой отрасли — экономического права»40. 41 Веленто И. И., Елисеев В. С. Теория экономического права : курс лекций. Гродно : ГрГУ, 2004. 417 с. PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY Устранению приведенных недостатков может способствовать позиция, постро- енная на обращении к теории экономического права. Данная позиция разработана автором настоящей статьи совместно с И. И. Веленто в 1994 г., если считать за основу дату выпуска первой книги с указанным названием на базе Гродненского государственного университета имени Я. Купалы (Республика Беларусь)41. 7/2020 182 182 Первоначально авторы определяли место экономического права как главной правовой отрасли, регулирующей экономические отношения, в основу построения которой предлагался так называемый Главный кодекс42. Однако впоследствии от данной идеи они отказались, сделав вывод, что такое обобщение — предмет исключительно правовой теории, именно на этом (теоретическом) уровне не- обходимо вести речь о гармонизации различных направлений экономического законодательства. Вместе с тем, чтобы обобщить все отрасли права, в той или иной степени регулирующие экономические отношения, актуальным является применение категории «экономическое право». Собственно, категория «экономические отношения» ранее не приветство- валась в праве: вместо нее применялся термин «хозяйственные отношения», который впоследствии был заменен на «имущественные отношения». С разви- тием рыночных отношений наступило понимание того, что определенные отрас- ли права регулируют экономические отношения и табу с данного термина было снято. С этих позиций категория «экономические отношения» («экономическое поведение»), по сути, заимствованная из экономической теории, наиболее точно отражает связь экономики и ее правового регулирования (правового обеспечения). В настоящее время вышла в свет кардинально обновленная книга: «Теория экономического права: теория отраслей права, обеспечивающих экономические отношения»43. Теория не ставит своей целью формирование новой мегаотрасли права — экономического права, как это предлагают представители Российского государ- ственного университета правосудия. Главная ее идея заключается в том, что в ней отражены объективные закономерности правового регулирования (правового обеспечения) экономических отношений, которые едины для всех отраслей права, в той или иной степени регулирующих экономические отношения. Причем за ос- нову правового регулирования берутся функционально обособленные институты (сформированные области) экономических отношений. По указанной причине те- ория экономического права рассматривается всего лишь как раздел теории права. В свою очередь, экономический институт выступает с позиции права комплекс- ным образованием, в состав которого входят, во-первых, отношения, подлежащие правовой регламентации регулятивными нормами права; во-вторых, юридически нейтральные отношения, в которых не возникают юридические обязательства, хотя экономические притязания присутствуют (как, например, в игорном бизнесе); в-третьих, негативное экономическое поведение, которое подлежит сдержива- нию и подавлению охранительными нормами права, — все указанное входит в логически оформленную единую область (институт) экономических отношений и соответствующий институт правового регулирования. Теория не ставит своей целью формирование новой мегаотрасли права — экономического права, как это предлагают представители Российского государ- ственного университета правосудия. 42 Елисеев В. С. Проблемы кодификации в регулировании экономических отношений в Российской Федерации и Республике Беларусь // Российско-белорусская наука XXI века и проблемы законодательства : материалы Междунар. науч.-практ. конф. Калуга, 2002 / В. С. Елисеев (отв. ред.). Калуга : Гриф, 2002. С. 51—63. 43 Елисеев В. С., Веленто И. И. Теория экономического права: теория отраслей права, обе- спечивающих экономические отношения : учебное пособие. М. : Проспект, 2019. 416 с. 42 Елисеев В. С. Проблемы кодификации в регулировании экономических отношений в Российской Федерации и Республике Беларусь // Российско-белорусская наука XXI века и проблемы законодательства : материалы Междунар. науч.-практ. конф. Калуга, 2002 / В. С. Елисеев (отв. ред.). Калуга : Гриф, 2002. С. 51—63. 43 Елисеев В. С., Веленто И. И. Теория экономического права: теория отраслей права, обе- спечивающих экономические отношения : учебное пособие. М. : Проспект, 2019. 416 с. PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY Главная ее идея заключается в том, что в ней отражены объективные закономерности правового регулирования (правового обеспечения) экономических отношений, которые едины для всех отраслей права, в той или иной степени регулирующих экономические отношения. Причем за ос- нову правового регулирования берутся функционально обособленные институты (сформированные области) экономических отношений. По указанной причине те- ория экономического права рассматривается всего лишь как раздел теории права. В свою очередь, экономический институт выступает с позиции права комплекс- ным образованием, в состав которого входят, во-первых, отношения, подлежащие правовой регламентации регулятивными нормами права; во-вторых, юридически нейтральные отношения, в которых не возникают юридические обязательства, хотя экономические притязания присутствуют (как, например, в игорном бизнесе); в-третьих, негативное экономическое поведение, которое подлежит сдержива- нию и подавлению охранительными нормами права, — все указанное входит в логически оформленную единую область (институт) экономических отношений и соответствующий институт правового регулирования. 7/2020 7/2020 7/2020 Елисеев В. С. Место теории экономического права в системе правового регулирования экономики Елисеев В. С. Место теории экономического права в системе правового регулирования экономики 183 183 Окончательным оформленным правовым образованием, регулирующим эко- номический институт, с позиции теории экономического права выступает правовой режим — окончательно сформированная комплексная совокупность норм права (правовой институт), охватывающая определенную область экономических отно- шений. Правовой режим состоит из норм права различной природы: императив- ных и диспозитивных, регулятивных и охранительных. Деление права на отрасли рассматривается исключительно с позиции юридической необходимости, кото- рая позволяет правовому регулированию наиболее полно охватить различные стороны экономического института. С этих позиций отрасль права, построенная на основе метода правового регулирования, не должна становиться кантовской «вещью в себе», тем более не должна стремиться занять главенствующее по- ложение, а должна учитывать свое воздействие наравне с другими правовыми нормами, принадлежащими различным отраслям права, которые выступают со- ставной частью правового режима. Теория экономического права имеет следующую структуру: Общая часть «Теоретические основы правового обеспечения экономических отношений» включает следующие разделы (параграфы): «О предмете право- вого обеспечения экономических отношений», «О методах правового регулиро- вания экономических отношений», «Правовой режим как фундамент правового регулирования экономических отношений», «Особенности формирования охра- нительного законодательства», «Эффективность правового обеспечения эконо- мических отношений», «О проблемах развития правовых наук, обеспечивающих экономические отношения». Особенная часть «Теории правового обеспечения отдельных областей эко- номических отношений» включает параграфы: «Теория правового обеспечения рынков», «Теория правового обеспечения экономического саморегулирования и государственного регулирования экономики», «Теория права собственности», «Теория субъекта хозяйствования», «Теория правового обеспечения государ- ственного сектора экономики», «Общая теория обязательств», «Теория договор- ных обязательств», «Теория налоговых обязательств» и «Теория обязательств государственной поддержки», «Теории составных обязательств». PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY Наконец, Специальная (аналитическая) часть включает на данном этапе два параграфа: «Реформирование экономического законодательства СССР» и «Ре- формирование экономического законодательства Российской Федерации». Очевидно, что в перспективе предполагается развитие структуры теории эко- номического права. БИБЛИОГРАФИЯ 1. Альбов А. П., Ручкина Г. Ф. Экономическое право как новое направление развития правовой науки // Ученые труды Российской академии адвокатуры и нотариата. — 2015. — № 3 (38). — С. 93—99. 2. Братусь С. Н., Алексеев С. С. О разработке правовых вопросов управления народным хозяйством // Правоведение. — 1963. — № 4. 3. Веленто И. И., Елисеев В. С. Теория экономического права : курс лекций. — Гродно : ГрГУ, 2004. — 417 с. 7/2020 184 4. Вышинский А. Я. Основные задачи науки советского социалистического пра- ва // Советское государство и право. — 1939. — № 1. 5. Дозорцев В. А. Один кодекс или два? (Нужен ли хозяйственный кодекс наря- ду с гражданским?) // Правовые проблемы рыночной экономики в Российской Федерации : Труды Института законодательства и сравнительного правове- дения при Правительстве Российской Федерации. — Вып. 57. — М., 1994. — С. 115—143. 6. Елисеев В. С., Веленто И. И. Теория экономического права: теория отраслей права, обеспечивающих экономические отношения : учебное пособие. — М. : Проспект, 2019. — 416 с. 7. Ершов В. В., Ашмарина Е. М., Корнев В. Н. Экономическое право Российской Федерации : монография. — М. : РГУП, 2017. — 284 с. 8. Ершова И. В. Проблемы правового режима государственного имущества в хозяйственном обороте: теоретические основы и пути совершенствования. — М. : Юриспруденция, 2001. 9. Курс советского хозяйственного права / под ред. Е. Б. Пашуканиса и Л. Я. Гинц- бурга. — М., 1935. — Т. 1. 10. Лаптев В. А. «Мегаотрасли» — экономическое, корпоративное и энергети- ческое право или юридические фикции? // Экономика. Право. Общество. — 2017. — № 3. — С. 28—33. 11. Лаптев В. В. Предмет и система хозяйственного права. — М. : Юрид. лит., 1969. 12. Мартемьянов В. С. Хозяйственное право : курс лекций : в 2 т. — М. : Бек, 1994. 13. Мартемьянов В. С. Юридическая трагедия // Юридическая газета. — 1992. — № 1, 2. 14. Мирошник С. В. Экономическое право в системе российского права // Вопро- сы экономики и права. — 2016. — № 1. — С. 12—14. 15. Отграничение хозяйственного права от других отраслей права и их взаимо- действие // Хозяйственное право : учебник / В. К. Мамутов, Г. Л. Знаменский, К. С.Халиулин [и др.] ; под ред. В. К. Мамутова. — Киев : Юринком Интер, 2002. 16. Стучка П. И., Амфитеатров Г. Н. Основы гражданского законодательства СССР. — М., 1931. 17. Экономическое право : хрестоматия : в 3 т. БИБЛИОГРАФИЯ — СПб. : Питер, 2004. 7/2020 7/2020
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The Monogenean Which Lost Its Clamps
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To cite this version: Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck, Eric P. Hoberg. The Monogenean Which Lost Its Clamps. PLoS ONE, 2013, 8 (11), pp.e79155. ￿10.1371/jour- nal.pone.0079155￿. ￿hal-00930013￿ The Monogenean Which Lost Its Clamps Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck, Eric P. Hoberg To cite this version: Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck, Eric P. Hoberg. The Monogenean Which Lost Its Clamps. PLoS ONE, 2013, 8 (11), pp.e79155. ￿10.1371/jour- nal.pone.0079155￿. ￿hal-00930013￿ The Monogenean Which Lost Its Clamps Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck, Eric P. Hoberg Abstract PLoS ONE 8(11) rnal.pone.0079155 Editor: Gordon Langsley, Institut national de la sante´ et de la recherche me´dicale - Institut Cochin, France itor: Gordon Langsley, Institut national de la sante´ et de la recherche me´dicale - Institut Cochin, France Received June 21, 2013; Accepted September 25, 2013; Published November 22, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This project was supported by the ‘‘Service de Syste´matique Mole´culaire’’ of MNHN (CNRS UMS 2700), the network ‘‘Bibliothe`que du Vivant’’ funded by the CNRS, the MNHN, the INRA and the CEA (Genoscope), and a grant (Action The´matique du Muse´um (www.mnhn.fr) ‘‘Formes possibles, Formes re´alise´es’’) awarded by MNHN to JLJ and CR. (These grants have no numbers). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: justine@mnhn.fr The Monogenean Which Lost Its Clamps Jean-Lou Justine1*, Chahrazed Rahmouni1, Delphine Gey2, Charlotte Schoelinck ne1*, Chahrazed Rahmouni1, Delphine Gey2, Charlotte Schoelinck1,3, Eric P. Hober Jean-Lou Justine1*, Chahrazed Rahmouni1, Delphine Gey2, Charlotte Schoelinck1,3, Eric P. Hoberg4 1 UMR 7138 ‘‘Syste´matique, Adaptation, E´volution’’, Muse´um National d’Histoire Naturelle, CP 51, Paris, France, 2 UMS 2700 Service de Syste´matique mole´culaire, Muse´um National d’Histoire Naturelle, Paris, France, 3 Molecular Biology, Aquatic Animal Health, Fisheries and Oceans Canada, Moncton, Canada, 4 United States National Parasite Collection, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America Abstract Ectoparasites face a daily challenge: to remain attached to their hosts. Polyopisthocotylean monogeneans usually attach to the surface of fish gills using highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 60 years ago, the clamps were considered to be absent but few specimens were available and this observation was later questioned. In addition, genera within the family Protomicrocotylidae have either clamps of the ‘‘gastrocotylid’’ or the ‘‘microcotylid’’ types; this puzzled systematists because these clamp types are characteristic of distinct, major groups. Discovery of another, new, species of the genus Lethacotyle, has allowed us to explore the nature of the attachment structures in protomicrocotylids. Lethacotyle vera n. sp. is described from the gills of the carangid Caranx papuensis off New Caledonia. It is distinguished from Lethacotyle fijiensis, the only other species of the genus, by the length of the male copulatory spines. Sequences of 28S rDNA were used to build a tree, in which Lethacotyle vera grouped with other protomicrocotylids. The identity of the host fish was confirmed with COI barcodes. We observed that protomicrocotylids have specialized structures associated with their attachment organ, such as lateral flaps and transverse striations, which are not known in other monogeneans. We thus hypothesized that the clamps in protomicrocotylids were sequentially lost during evolution, coinciding with the development of other attachment structures. To test the hypothesis, we calculated the surfaces of clamps and body in 120 species of gastrocotylinean monogeneans, based on published descriptions. The ratio of clamp surface: body surface was the lowest in protomicrocotylids. We conclude that clamps in protomicrocotylids are vestigial organs, and that occurrence of ‘‘gastrocotylid’’ and simpler ‘‘microcotylid’’ clamps within the same family are steps in an evolutionary sequence, leading to the absence of these attributes in species of Lethacotyle. hmouni C, Gey D, Schoelinck C, Hoberg EP (2013) The Monogenean Which Lost Its Clamps. PLoS ONE 8(11): e79155. doi:10.1371/ ation: Justine J-L, Rahmouni C, Gey D, Schoelinck C, Hoberg EP (2013) The Monogenean Which Lost Its Clamps. HAL Id: hal-00930013 https://hal.science/hal-00930013v1 Submitted on 16 Aug 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. PLOS ONE | www.plosone.org Hosts Five specimens of Caranx papuensis Alleyne & MacLeay, 1877 were obtained in Noume´a City, New Caledonia, from amateur fishermen fishing from the piers of the harbour, or were bought at the fishmarket, from commercial fishmongers. The latter host specimens came from professional fisherman who specialize on mackerels, fish close to Noume´a City and bring back their catch within hours from the nearby fishing-grounds. Fish specimens are detailed in Table 1 with registration number, date, locality, length, weight and availability of photographs. Accurate identification of marine fish is often a problem in the South Pacific [20–24], and Figure 1. The single specimen of Lethacotyle available for study before this paper. The slide containing the single specimen of Lethacotyle available for study before this paper: holotype of Lethacotyle fijiensis Manter & Prince, 1953 (urn:lsid:zoobank.org:act:DA367684- AAC2-44D0-A8E8-64894AFA647A), slide USNPC 48718. Our study is another example of the importance of Museum collections for modern research [86,87]. doi:10.1371/journal.pone.0079155.g001 Parasites Monogeneans were collected alive or recently dead, flattened in cold ethanol, and routinely processed, including staining with carmine and mounting on a microscopic slide in Canada balsam [25]. Drawings were made using an Olympus BH2 microscope equipped with a drawing tube and differential interference contrast (DIC) optics. Measurements were made from pencil drawings with the help of a custom-made transparent rule, previously calibrated with a stage micrometer. Drawings were scanned and redrawn on a computer with Adobe Illustrator. All measurements are given in micrometres unless otherwise indicat- ed. In the text and Tables, ‘‘juvenile’’ designates specimens with incomplete development of genital organs, especially of charac- teristic sclerotised organs. Further, during our current studies of Lethacotyle including comparisons among related monogeneans, we noted that clamps in species of protomicrocotylids were relatively small in compar- ison to the body. Thus, although individual clamps were not especially small, all clamps together occupied a small surface area of the body in comparison to other species of polyopisthocotylean monogeneans. Our observations suggest that clamps are reduced, or vestigial, in this family, an assertion based on the ubiquitous distribution of these attributes among basal polyopisthocotyleans and the putative phylogenetic relationships for the Protomicroco- tylidae Johnston & Tiegs, 1922 [4,6]. To test this hypothesis, we explored the phylogenetic placement of the Protomicrocotylidae and we compared the ratio for surface of clamps: surface of body in 120 monogenean species belonging to the Gastrocotylinea Lebedev, 1972. We found that the protomicrocotylids had the lowest ratio. Finally, we discuss the evolutionary significance of the absence of clamps in Lethacotyle spp., a unique feature among polyopisthocotylean monogeneans. The Monogenean Which Lost Its Clamps The Monogenean Which Lost Its Clamps Thus, in all, our current knowledge of Lethacotyle, in spite of its uniqueness and interest, is based on the observation of four specimens, three adults and one juvenile, in which only one has been kept in a museum and is available to study (Figure 1). No work has been published on Lethacotyle during the past 40 years and the doubts concerning the absence of clamps, expressed in the original description [15] and subsequent comments [16,19], have remained problematic. photographs of the fish were used to determine species identity by several ichthyologists. In addition, fish tissues were collected, stored in 95% or 100% ethanol, and processed for molecular identification. Specimens of Caranx sexfasciatus Quoy & Gaimard, 1825 from the same locality were examined and provided specimens of the monogenean Neomicrocotyle sp. used for compar- ison of morphology and molecules. We collected off New Caledonia, another South Pacific island, a series of specimens of a previously unrecognized species of Lethacotyle. Specimens were collected in perfect condition for morphological study and were submitted to modern molecular analysis; the new species is described herein. Museum specimens The following museum slides were examined: Bilaterocotyle novaeguineae Rohde, 1977, paratype, USNPC 74800 (1 slide) (current status: Bilaterocotyloides novaeguineae (Rohde, 1977) Lebedev, 1986); Neomicrocotyle sp. from Caranx sexfasciatus off New Caledonia, MNHN JNC3242; Protomicrocotyle celebesensis Yamaguti, 1953, MNHN HEL80, HEL81; Protomicrocotyle mannarensis Ramalingam, 1960, USNPC 74798, BMNH 1978.6.15.6; Protomicrocotyle manteri Bravo-Hollis, 1966, paratype, USNPC 75514; Protomicrocotyle mirabilis (MacCallum, 1918) Johnston & Tiegs, 1922, BMNH 2002.8.12.3-4, BMNH 2007.7.25.34, 2007.7.25.30-33 (2 slides); Protomicrocotyle pacifica Meserve, 1938, USNPC 100122 (3 slides) (current status: Neomicrocotyle pacifica (Meserve, 1938) Yamaguti, 1968 [26]); Protomicrocotyle sp., BMNH 1985.11.8.48-47, BMNH 1985.11.8.48-52 (2 slides). The following slides could not be shipped but photographs were taken by curators: Protomicrocotyle celebesensis, MPM 22909 (SY6739); Neomicrocotyle carangis Yama- guti, 1968, holotype, USNPC 63672, and MPM 15660 (B2421- 2423); Lethacotyle fijiensis, holotype, USNPC 48718 (Figure 1); Protomicrocotyle pacifica, holotype, USNPC 9166. Names in the above list are those from the original labels, sometimes updated with correct taxonomy and current usage. Patricia Pilitt (USNPC) and Eileen Harris (BMNH) are thanked for arranging specimen loans. Introduction body of the monogenean is deformable and allows it to feed from blood sucked from the gill [9]. Monogeneans are Platyhelminthes, mostly ectoparasites on fish. Although the monophyly of the Monogenea is dubious [1,2], there is no doubt that each of the two components of the monogeneans, namely the Polyopisthocotylea and the Monopisthocotylea, are each monophyletic and members of the Neodermata, the parasitic and terminal group of Platyhelminthes, together with the Cestoda and the Trematoda [3–5]. Members of both monogenean groups deal with a major issue of parasitic life, attachment to the host, by a posterior organ named the haptor (or opisthaptor) which possesses specialized attachment structures [6–8]. Although all known polyopisthocotyleans have suckers or clamps, a single exception is represented by the species Lethacotyle fijiensis Manter & Prince, 1953 [15]. This worm is a parasite on the gills of an unnamed carangid fish off Fiji, a South Pacific island. The species was described, however, from only two specimens (among which only one is still in a museum collection) and the authors mentioned that there was a possibility that the clamps could have been lost - this is not an unusual phenomenon when specimens are not collected in optimal conditions. Hargis (1957) [16] also expressed doubt over the accuracy of the original description and considered that the complete absence of clamps was ‘‘unique and puzzling.’’ Later Ramalingam (1966, 1968) [17,18] found other specimens of Lethacotyle Manter & Prince, 1953 on a carangid off the Andaman Islands, and confirmed the absence of clamps in adult and juvenile worms. However, Ramalingam’s papers [17,18] were largely ignored, i.e. by Llewellyn (1971) [19] who commented that ‘‘such extraordinary occurrences deserve re- examination.’’ In the Polyopisthocotylea (the name means ‘‘many sucker-cups at the rear’’ [9]) the posterior haptor includes suckers or clamps [6], and the latter are considered one of the major morphological synapomorphies of the group [10]. These clamps, ranging in number from a few to hundreds, are highly specialized structures, often armed with sclerotised elements [6,11–13]. Clamps attach to the host’s surface (generally the gill of a marine fish) and thus allow the worm to resist the flow of water running through the gill chamber and to maintain position on its host [14]. The anterior November 2013 | Volume 8 | Issue 11 | e79155 1 PLOS ONE | www.plosone.org Molecular sequences Fish DNA was extracted from tissue samples of three specimens (Table 1) using NucleoSpin 96 tissue kit (Macherey-Nagel) following the manufacturer’s instructions. The 59 region of the cytochrome oxidase I (COI) mitochondrial gene was amplified using the primers FishF1 (59-TCAACCAACCACAAAGA- CATTGGCAC-39) and FishR1 (59-TAGACTTCTGGGTGGC- CAAAGAATCA-39) [27]. Species identification was confirmed using the BOLD identification engine [28]. One monogenean was cut in two parts: the anterior part, including the key sclerotised reproductive organs, was mounted, using routine methods, on a microscopic slide [25] as for whole worms, and the posterior part was used for DNA extraction. Thanks to this method, perfect traceability was insured between morphological and molecular methods (i.e. both were performed on the same monogenean individual); in addition, for host-parasite traceability, the individual host fish of the same individual monogenean was used for sequencing (Table 1). DNA was also extracted from another, whole individual monogenean and provided the same sequence. p q For monogeneans, as little tissue was available, DNA was extracted using NucleoSpin 96 tissue kit with a modified protocol: the NucleoSpin 96 Tissue Binding Plate was replaced by the Plasmid Binding Plate (Macherey-Nagel) and elution was per- formed in 60 mL. A 28S rDNA fragment of 700 bp was amplified using the universals primers C19 (59-ACCCGCTGAATTTAAG- CAT-39) and D2 (39-TCCGTGTTTCAAGACGG-59) [29]. PCR reactions were performed in final volume of 20 ml, containing: 1 ng of DNA, 16 CoralLoad PCR buffer, 3 mM MgCl2, 66 mM of each dNTP, 0.15 mM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). Thermocycles consisted in an initial denaturation step at 94uC for 49, followed by 38 cycles of denaturation at 94uC for 300, annealing at 60uC, for 300, and extension at 72uC for 19. The final extension was conducted at 72uC for 79. PCR products were visualized on a 1.5% agarose gel, purified and directly sequenced in both directions on 3730xl DNA Analyzer 96-capillary sequencers (Applied Biosystems) at Geno- scope (E´vry, France). Sequences were edited and assembled using CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA). Sequences were deposited in GenBank under the accession numbers KF378583–KF378585 (fish) and KF378588– KF378589 (monogeneans). The Monogenean Which Lost Its Clamps The Monogenean Which Lost Its Clamps of Caranx papuensis examined, specimens of Lethacotyle vera n. sp., and results. Locality Fork Length (mm) Weight (g) Photo Fish Sequences Parasites Parasite Sequences Noume´a harbour 372 843 Yes - 3 specimens on slides; 2 adults, 1 juvenile - Noume´a harbour 413 1,250 No - 4 adult specimens on slides - Noume´a fish market 275 350 Yes COI: KF378585 1 adult specimen on slide - Noume´a fish market 345 749 Yes COI: KF378583 3 specimens: 1 juvenile on slide JNC3188A1; 1 adult, cut, anterior on slide JNC3188A2c, sequenced; 1 adult JNC3188A3c, sequenced Specimen JNC3188A2c: 28S: KF378588 Noume´a fish market .500 unknown Yes COI: KF378584 1 adult specimen on slide - Table 1. Specimens of Caranx papuensis examined, specimens of Lethacotyle vera n. sp., and results. JNC Date Locality Fork Length (mm) Weight (g) Photo Fish Sequences Parasites Parasite Sequences JNC1185 05-07-2004 Noume´a harbour 372 843 Yes - 3 specimens on slides; 2 adults, 1 juvenile - JNC1189 06-07-2004 Noume´a harbour 413 1,250 No - 4 adult specimens on slides - JNC1988 04-10-2006 Noume´a fish market 275 350 Yes COI: KF378585 1 adult specimen on slide - JNC3188 17-06-2010 Noume´a fish market 345 749 Yes COI: KF378583 3 specimens: 1 juvenile on slide JNC3188A1; 1 adult, cut, anterior on slide JNC3188A2c, sequenced; 1 adult JNC3188A3c, sequenced Specimen JNC3188A2c: 28S: KF378588 JNC3209 16-07-2010 Noume´a fish market .500 unknown Yes COI: KF378584 1 adult specimen on slide - www.plosone.org Nomenclatural acts The electronic edition of this article conforms to the require- ments of the amended International Code of Zoological Nomen- clature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix ‘‘http://zoobank.org/’’. The LSID for this publication is: urn:lsid: zoobank.org:pub:596C3FF5-CD24-4733-95FD-CC060A7FF0EE. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS. Figure 1. The single specimen of Lethacotyle available for study before this paper. The slide containing the single specimen of Lethacotyle available for study before this paper: holotype of Lethacotyle fijiensis Manter & Prince, 1953 (urn:lsid:zoobank.org:act:DA367684- AAC2-44D0-A8E8-64894AFA647A), slide USNPC 48718. Our study is another example of the importance of Museum collections for modern research [86,87]. d i 10 1371/j l 0079155 001 doi:10.1371/journal.pone.0079155.g001 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 2 Phylogenetic analysis of polyopisthocotylean monogeneans g The data matrix was built from the published alignment of Olson & Littlewood [30] (available from http://ebi.edu.au/ftp/ databases/embl/align/ALIGN_000150.dat), restricted to the Polyopisthocotylea excluding Polystomatidae and Sphyranuridae (this corresponds to the group designated as Oligonchoinea in [30]; for equivalences of monogenean terminology, see Table 1 in [4]) to which were added two newly obtained sequences of 28S: one from Lethacotyle vera n. sp. (KF378588), and one from an unidentified species of Neomicrocotyle Ramalingam, 1960 (KF378589) from Caranx sexfasciatus. There is general agreement that the Oligonchoinea are monophyletic and that the Hexabo- thriidae are basal among them [10,30–32], so the two hexabo- thriids of the original alignment were chosen as an outgroup. Phylogenetic reconstruction was computed using the GTR+I+C model, selected as the best-fitting model of nucleotide evolution for 28S marker with ModelTest [33], in conjunction with PAUP 4.0b10 [34], following the AIC criterion. Trees were inferred using two probabilistic approaches: maximum likelihood with a non- parametric bootstrap (BP) using RaxML [35] and Bayesian PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79155 3 The Monogenean Which Lost Its Clamps Figure 2. Photograph of the holotype of Lethacotyle fijiensis Manter & Prince, 1953. Lethacotyle fijiensis Manter & Prince, 1953 (urn:lsid:zoobank.org:act:DA367684-AAC2-44D0-A8E8-64894AFA647A). Holotype, slide USNPC 48718. A, body. B, posterior part of body, different focus. C, D, spines of male copulatory organ, two different focuses. E, sclerotised vagina. Original photographs taken by Patricia Pilitt, USNPC. doi:10.1371/journal.pone.0079155.g002 Inference (BI) using MrBayes version 3.1.2 [36]. Maximum likelihood (ML) analyses were carried out online on the CIPRES Science Gateway (The CIPRES Portals. URL: http://www.phylo. org/sub_sections/portal) with RAxML-HPC BlackBox (7.2.7) [35]. BI analyses were performed using 1,000,000 generations with sampling every 100 generations and four Metropolis-coupled Markov chains Monte Carlo (MCMCMC) and other parameters by default. Two independent analyses were conducted to check for convergence of the results. The parameter estimates and convergence were checked using Tracer version 1.4 [37]. The first 25% of sampled trees were discarded prior to constructing a 50% majority rule consensus tree. Posterior probabilities (PP - Bayesian analysis) and Bootstrap values (BP - Maximum likelihood analysis) were used as indicators of node credibility and we used PP$0.95 and BP$75% as significant values. Morphology of available specimens Museum specimens (or sometimes photographs of specimens) of species of protomicrocotylids, belonging to the genera Lethacotyle, Protomicrocotyle, Neomicrocotyle, and Bilaterocotyle were examined for the presence of clamps and other structures on the haptor. The number of clamps was found to be consistent with the published descriptions of species; particularly, we found no specimen with an incomplete number of clamps (i.e. only 5 clamps when 6 were Ethics statement Fish used for collection of parasites were dead at the time we acquired them for study, having been commercially caught, and available for purchase at the Noume´a fish market; no permits were required for the described study, which complied with all relevant regulations. described for the species). The single specimen of Lethacotyle fijiensis has no clamp (Figure 2), as emphasized in its original description [15]; the same is true for all specimens of our new species (formally described below). Analysis of relative importance of clamps in gastrocotylinean monogeneans We compared the structure and the taxonomic distribution of clamps across the major group, the Gastrocotylinea Lebedev, 1972 [38], which contains the protomicrocotylids [39]. This is one of the largest groups of polyopisthocotylean monogeneans, which is characterized by a common, complex clamp structure known as ‘‘gastrocotylid’’ [19,40] (but see below for changes of this structure in some protomicrocotylids). p y Figures in the global literature were extracted from published PDF files or scanned from printed papers with a table top scanner with a 600 dpi resolution. The outlines of the body and of individual clamps were drawn with Adobe Illustrator and then filled in black. Drawings were exported in JPG format and area measurements (whole body including clamps and total of all clamps) were taken with ImageJ [41] on digital files. We checked against WoRMS [42] (date: 14 May 2013), the list of species of Gastrocotylinea for which we could obtain illustrations of sufficient quality. Our database includes 120 species; 9 of these species were not in WoRMS; the remaining 111 species represented 78% of the 142 species included in WoRMS. The 120 figures are available in a Supplementary File. The statistical significance of differences between families was tested with Mann & Whitney U test. Figure 2. Photograph of the holotype of Lethacotyle fijiensis Manter & Prince, 1953. Lethacotyle fijiensis Manter & Prince, 1953 (urn:lsid:zoobank.org:act:DA367684-AAC2-44D0-A8E8-64894AFA647A). Holotype, slide USNPC 48718. A, body. B, posterior part of body, different focus. C, D, spines of male copulatory organ, two different focuses. E, sclerotised vagina. Original photographs taken by Patricia Pilitt, USNPC. doi:10 1371/journal pone 0079155 g002 Abbreviations Two types of clamps were found in specimens of protomicro- cotylids (Figure 3), i.e. ‘‘gastrocotylid’’ type (with additional sclerite) and ‘‘microcotylid’’ type (without the sclerite). Parasitological collections: BMNH, NHMUK: British Museum (Natural History), London, UK; MNHN, Muse´um National d’Histoire Naturelle, Paris, France; MPM, Meguro Parasitological Museum, Tokyo, Japan; USNPC, United States National Parasite Collection, Beltsville, USA. In addition, we looked for striations on the haptor in specimens, or sought for the mention of striations in the descriptions. Table 2 shows that haptoral striations were often, but not always, mentioned in the descriptions of protomicrocotylids. Such striations are apparently not recorded (or observed) in other members of the Gastrocotylinea (and in polyopisthocotylean monogeneans as well), with the possible exception of a pseudodiclidophorid [43]. Relative importance of clamps in gastrocotylinean monogeneans Examples of line drawings are shown in Figure 4; all 120 drawings are in the supplementary file. Data are in Table 3. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 4 The Monogenean Which Lost Its Clamps Figure 3. Clamps in various genera of Protomicrocotylidae. Examples of clamps in various genera of Protomicrocotylidae. A, Bilaterocotyloides novaeguineae (Rohde, 1977) Lebedev, 1986 (USNPC 74800). B, Protomicrocotyle sp. (MNHN JNC1163A5). C, Neomicrocotyle sp. (MNHN JNC3242A4). Black: additional sclerite, characteristic of the ‘‘gastrocotylid’’ clamp. Bilaterocotyloides and Protomicrocotyle have clamps of the ‘‘gastrocotylid’’ type, Neomicrocotyle has clamps of the ‘‘microcotylid’’ type, and Lethacotyle has no clamp. doi:10.1371/journal.pone.0079155.g003 Figure 3. Clamps in various genera of Protomicrocotylidae. Examples of clamps in various genera of Protomicrocotylidae. A, Bilaterocotyloides novaeguineae (Rohde, 1977) Lebedev, 1986 (USNPC 74800). B, Protomicrocotyle sp. (MNHN JNC1163A5). C, Neomicrocotyle sp. (MNHN JNC3242A4). Black: additional sclerite, characteristic of the ‘‘gastrocotylid’’ clamp. Bilaterocotyloides and Protomicrocotyle have clamps of the ‘‘gastrocotylid’’ type, Neomicrocotyle has clamps of the ‘‘microcotylid’’ type, and Lethacotyle has no clamp. doi:10.1371/journal.pone.0079155.g003 as C. papuensis based on comparisons to the 12 available specimens in the database. Results of the comparison are presented in Figure 5 (data shown for all 120 species) and Figure 6 (data grouped by families). Among the 25 species with the smallest clamp: body ratios, 21 (84%) are protomicrocotylids (Figure 5). The clamp: body ratio in proto- microcotylids is the smallest of all families (Figure 6); ratios are smaller in protomicrocotylids than in each of the other families, and the differences are significant, except for the pseudodiclido- phorids (Table 4). Results of the comparison are presented in Figure 5 (data shown for all 120 species) and Figure 6 (data grouped by families). Among the 25 species with the smallest clamp: body ratios, 21 (84%) are protomicrocotylids (Figure 5). The clamp: body ratio in proto- microcotylids is the smallest of all families (Figure 6); ratios are smaller in protomicrocotylids than in each of the other families, and the differences are significant, except for the pseudodiclido- phorids (Table 4). Type-locality: Off Noume´a, New Caledonia. Type-locality: Off Noume´a, New Caledonia. Type-material: Holotype MNHH JNC3209A1, collected 16-07- 2010, Noume´a fish market. Paratypes: MNHN, JNC1185, JNC1189, JNC1988, JNC3188 (whole specimens); NHMUK, 1 slide, 2013.10.8.1; USNPC, 1 slide, 107263. One paratype cut in two parts, anterior part on slide MNHN JNC3188A2c, posterior part used for sequencing. doi:10.1371/journal.pone.0079155.t002 Description of the new species Body and clamp surfaces: examples of line data extraction in each of the 8 families of the Gastrocotylinea. All species drawn to same body length. A, Gotocotylidae, Goto Bychowskicotylidae, Tonkinopsis transfretanus. C, Gastrocotylidae, Allopseudaxinoides euthynni. D, Neothoracocotylidae, Pr Allodiscocotylidae, Metacamopia indica. F, Pseudodiclidophoridae, Allopseudodiclidophora opelu. G, Chauhaneidae, Cotyloatlantic Protomicrocotylidae, Lethacotyle vera n. sp (no clamps). Details in Table 3. doi:10.1371/journal.pone.0079155.g004 The Monogenean Whi Figure 4. Body and clamp surfaces: examples of line drawings in 8 families. Body and clamp surfaces: examples of line drawings used data extraction in each of the 8 families of the Gastrocotylinea. All species drawn to same body length. A, Gotocotylidae, Gotocotyla niphoni Bychowskicotylidae, Tonkinopsis transfretanus. C, Gastrocotylidae, Allopseudaxinoides euthynni. D, Neothoracocotylidae, Pricea minimae Allodiscocotylidae, Metacamopia indica. F, Pseudodiclidophoridae, Allopseudodiclidophora opelu. G, Chauhaneidae, Cotyloatlantica mediterranea Protomicrocotylidae, Lethacotyle vera n. sp (no clamps). Details in Table 3. doi:10.1371/journal.pone.0079155.g004 The Monogenean Which Lost Its Clam Figure 4. Body and clamp surfaces: examples of line drawings in 8 families. Body and clamp surfaces: examples of line drawings used for data extraction in each of the 8 families of the Gastrocotylinea. All species drawn to same body length. A, Gotocotylidae, Gotocotyla niphonii. B, Bychowskicotylidae, Tonkinopsis transfretanus. C, Gastrocotylidae, Allopseudaxinoides euthynni. D, Neothoracocotylidae, Pricea minimae. E, Allodiscocotylidae, Metacamopia indica. F, Pseudodiclidophoridae, Allopseudodiclidophora opelu. G, Chauhaneidae, Cotyloatlantica mediterranea. H, Protomicrocotylidae, Lethacotyle vera n. sp (no clamps). Details in Table 3. doi:10.1371/journal.pone.0079155.g004 are herein shown in Figure 2. Other protomicrocotylids: see Materials and Methods. are herein shown in Figure 2. Other protomicrocotylids: see Materials and Methods. at two thirds from centre of lappet; median anchors on posterior edge of lappet; hooks just external to corresponding median anchors. Lateral anchor with inner root partly divided medially (Figure 7G), outer root simple, strongly recurved point; median anchor with flattened triangular root and strongly recurved point (Figure 7H); hook with elongate, straight shaft (Figure 7I). Transverse striations on posterior part of haptor, including whole surface of terminal lappet and most terminal part of haptor, but not lateral flaps (Figure 7F). Pattern of striation similar on ventral and dorsal sides, 20–25 striations on terminal lappet, regularly parallel, 15–20 striations on body, less regular. November 2013 | Volume 8 | Issue 11 | e79155 Description of the new species p q g Prevalence: 5/5 (100%); intensity 1–4 (Table 1). Lethacotyle vera Justine, Rahmouni, Gey, Schoelinck & Hoberg n. sp. urn:lsid:zoobank.org:act:0B7ABE99-07AF- 4088-97F3-1A154DBA614D Etymology: vera, Latin for true, meaning that Lethacotyle, a genus differentiated by absence of clamps, was based on true observa- tions. Type-host: Caranx papuensis Alleyne & MacLeay. Comparative material examined. Lethacotyle fijiensis Manter & Prince, 1953, holotype, USNPC 48718; the holotype slide (Figure 1) could not be shipped but photographs were taken and Molecular identification of hosts: The blast search processed on BOLD engine [28] for the fish specimens MNHN JNC1988, JNC3188, JNC3209 (Table 1), confirmed the species identification Table 2. Striations and other structures mentioned in protomicrocotylids. Species Observation Reference Subfamily Protomicrocotylinae Lethacotyle vera n. sp. Figure 2 This paper Lethacotyle fijiensis ‘‘Dorsal surface of haptor with fine transverse striations’’ p. 105 [15] Lethacotyle sp. from Andaman I. (as L. fijiensis) Description of flaps pp. 108–109 (see discussion of present article); [18] Protomicrocotyle mirabilis «Les faces ventrales et dorsales du hapteur et de la languette poste´rieure posse`dent des stries transversales» (p. 320); [55] Protomicrocotyle mirabilis (as Acanthodiscus mirabile) ‘‘body towards posterior disc transversally striated and spiny along dorsal surface’’ (p. 93); Figure 49 [64] Protocotyle celebensis ‘‘The caudal lobe is distinctly striated transversely like the posterior end of the body proper, giving a serrate appearance in profile’’; Fig. 45 [65] Bilaterocotyle chirocentrosus Transversal striations not described, but well visible on Figs. 14, 15 [66] Neomicrocotyle indicus ‘‘The posterior portion of the body and the dumb-bell shaped haptor show transverse striations which give a spiny appearance to the surface of the worm’’; Fig. 1 [67] Bilaterocotyle lucknowensis Fig. 7.52 (left Fig. and Fig. G) [68] Bilaterocotyle mamaevi ‘‘Lappet two discs, each lappet lamellated’’. Fig. 7.53 (left Fig. and Fig. G) [68] Subfamily Vallisiopsiinae Youngiopsis australis Fig. 42D [39] Vallisiopsis contorta ‘‘La partie e´largie raye´e du corps’’ ; Fig. 1 [40] doi:10.1371/journal.pone.0079155.t002 PLOS ONE | www.plosone.org 5 November 2013 | Volume 8 | Issue 11 | e79155 Table 2. Striations and other structures mentioned in protomicrocotylids. PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5 The Monogenean Which Lost Its Clamps are herein shown in Figure 2. Other protomicrocotylids: see at two thirds from centre of lappet; median anch Figure 4. Body and clamp surfaces: examples of line drawings in 8 families. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 7 November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species Vaginal pore ventral, at midlength of MCO bulb level, on either side of body midline but opposite to that of haptoral pads. Vagina comprising anterior smooth part and posterior sclerotised part. Sclerotised part (Figure 7D) cone-shaped, with pointed extremity posterior; internal longitudinal crests with irregular spines; posterior end a small sclerotised conical canal. Smooth canal between sclerotised part and rest of female organs not seen. Taxonomic discussion of the new species Classification of Lethacotyle. A diversity of taxonomic opinions illustrates the problematic nature and difficulty for classification of Lethacotyle and more generally for the Proto- microcotylidae. For example, Lethacotyle was classified within the family Discocotylidae Price, 1936, subfamily Vallisiinae Price, 1943 in the original description [15]; in Protomicrocotylidae Poche, 1926, Lethacotylinae Unnithan, 1962 by Unnithan (1962) [44] and in Protomicrocotylidae Johnson & Tiegs, 1922, Lethacotylinae Yamaguti, 1963, independently by Unnithan (1962) [44] and with a different definition of the subfamily, by Yamaguti (1963) [8]; in Gastrocotylidae Price, 1943, Valisiinae Price, 1943 by Hargis, 1957 [16]; and in Protomicrocotylidae (Johnston & Tiegs, 1922), Protomicrocotylinae Johnston & Tiegs, 1922 in the monograph by Lebedev (1986) [39]. The taxonomic confusion about the authority for the family Protomicrocotylidae in this list will not be commented upon here, but the challenge for classification clearly originates in the structure of the clamps (or their absence). The classification of polyopisthocotylean monoge- neans is mainly based on clamp structure, but protomicrocotylids are unique in that this structure changes relative to each genus within the family: Protomicrocotyle has clamps of the gastrocotylid type, but Neomicrocotyle has clamps of the microcotylid type. In addition, the asymmetrical morphology of the haptor in proto- microcotylids has been considered as ‘‘extraordinary’’ [19]. Unfortunately, the genus Lethacotyle was not included in discussions of modern phylogenies of monogeneans [10,45]. Vitellarium in two lateral fields, never dense; anterior extremity at level of seminal vesicle; posterior extremity at level of haptoral anterior pad, i.e. anteriormost and posteriormost part of intestine not coextensive with vitellarium. Vitellarium in two lateral fields, never dense; anterior extremity at level of seminal vesicle; posterior extremity at level of haptoral anterior pad, i.e. anteriormost and posteriormost part of intestine not coextensive with vitellarium. Egg elongate, with long anterior and posterior filaments (Figure 7E). In utero, egg length 220–225, width 82–95, filament length 412–467 (n = 2). Egg elongate, with long anterior and posterior filaments (Figure 7E). Description (Figures 7–8) Body elongate, fusiform (Figure 7A). Tegument of body proper smooth; tegument of posterior part of haptor with parallel transverse striations. Haptor devoid of clamps, slightly asymmetrical, comprising lateral pads and terminal lappet. Lateral pads two, anterior short, posterior long. Terminal lappet transversally elongate ovate, symmetrical, armed with 3 pairs of ventral sclerites (1 pair of hooks, 2 pairs of anchors). Lateral anchors located approximately Mouth subterminal, ventral. Prohaptoral suckers ovoid, asep- tate, lying diagonally in posterolateral wall of buccal cavity. PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 6 The Monogenean Which Lost Its Clamps The Monogenean Which Lost Its Cla November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 7 The Monogenean Which Lost Its Clamps Figure 5. Ratio between clamp surface and body surface in species of gastrocotylinean monogeneans. Ratio between clamp surface and body surface in species of gastrocotylinean monogeneans. Ratios are ordered in decreasing sequence. Red: protomicrocotylids; blue: species of other families. doi:10.1371/journal.pone.0079155.g005 Figure 5. Ratio between clamp surface and body surface in species of gastrocotylinean monogeneans. Ratio between clamp surface and body surface in species of gastrocotylinean monogeneans. Ratios are ordered in decreasing sequence. Red: protomicrocotylids; blue: species of other families. doi:10.1371/journal.pone.0079155.g005 Pharynx subovate, muscular, median and immediately posterior to prohaptoral suckers. Oesophagus long, devoid of diverticula, bifurcating to 2 intestinal caeca at level of genital atrium. Intestinal caecum in each lateral field of body proper, extending into haptor to anteriormost part of lappet; lateral intestinal diverticula numerous, branched, often indistinct; short diverticula in anterior haptor, no diverticula in lappet. Phylogenetic position of the new species A phylogenetic analysis of 28S sequences (Figure 9) show that the new species forms a clade (PP = 0.99, BPML = 95) with Neomicrocotyle pacifica (from Caranx hippos (Linnaeus, 1766) off Mexico [30]) and Neomicrocotyle sp. (our specimens from Caranx sexfasciatus off New Caledonia), the two other protomicrocotylids of the dataset. Germarium intercaecal, with posterior immature mass, anteri- orly directed branch, posteriorly directed looped mature branch (Figure 8E). Small coiled canal with visible wall from extremity of germarium to posterior part of ootype. Ootype elongate, median, with basal Mehlis’ glands, continued anteriorly as uterus. Uterus linear, thin walled, extending up to genital atrium; superposed to seminal vesicle along part of its path. Median vitelline duct ventrally superposed to ootype (in holotype), anterior paired vitelline branches visible only on short distance. Differential diagnosis Lethacotyle vera n. sp. is similar to the single species described in the genus, L. fijiensis, based on the following characters (Table 5): body shape, and especially haptoral shape; total absence of clamps; body dimensions (mean 4340, 2300–5720) vs 3156–3759 in L. fijiensis [15]; presence of a circle of spines in MCO and shape of individual spines; number of spines 23 (17–27) vs 24–25 in L. fijiensis; shape of cone-like sclerotised vagina; shape and position of sclerotised haptoral parts. Genital atrium unarmed, median. Testes ovoid, pregermarial, intercaecal, in 2 bilateral rows along body midline. Vas deferens expanding just anterior to anteriormost testis into seminal vesicle filled with sperm; seminal vesicle continued anteriorly by wide canal to male copulatory organ (MCO); vas efferentia and prostate not visible. MCO an elongate bulb, with muscular wall and internal coiled canal, armed with anterior spines; mass of bulb sometimes protruding anterior to spines. Spines arranged in a tight circle (‘‘genital corona’’), with tips directed outward and extending into genital atrium. Spines elongate, with blunt ends, elongate root, and thumb located at anterior third. General arrangement of spines of genital corona slightly variable with specimens (Figures 7B, 7C, 8C, 8D), but morphology of spines similar in all adult specimens. It differs in MCO spine length (mean 5065.7, 35–66) vs 24 in L. fijiensis and shape of sclerotised vagina longitudinal crests, with irregular spines along length vs with minute terminal spines in L. fijiensis. The length of MCO spines in the holotype of L. fijiensis was ascertained by scaled photographs. Note that in specimens of ‘‘L. fijiensis’’ described by Ramalingam [17,18] the length of the MCO spines was reported as 15 (vs 24 in original description [15]) and thus this might represent another species (see below); L. vera n. sp. is distinct from this putative species by the length of MCO spines. November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species In utero, egg length 220–225, width 82–95, filament length 412–467 (n = 2). Juvenile specimens (Figures 8A, 8B). Two juvenile specimens briefly described for differential maturation of organs and sclerotised parts. One specimen (slide MNHN JNC3188A1, Figure 8A), 1300 in length, 320 in width: body almost symmetrical, haptoral pads barely visible; MCO spines incom- plete, 22–24 in length, no thumbs on spines (Figure 8B); vagina a denser zone, no sclerotisation; germarium visible, testis zone an indistinct mass; haptoral hooks not well oriented. One specimen (slide MNHN JNC1185A3, not figured), 1700 in length, 350 in width, body symmetrical, MCO a dense mass without sclerotisation, all other genital organs indistinct; haptoral sclerotised parts well visible, morphology of lateral and median anchors similar to adult. Note that the longest juvenile specimen is apparently the less mature according to less differentiated sclerotised parts. Our molecular phylogenetic analysis shows that Lethacotyle vera n. sp. groups with two species of Neomicrocotyle and thus confirms that November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 8 The Monogenean Which Lost Its Clamps Table 3. Surface of clamps and body in species of gastrocotylinean monogeneans. Species Family Body surface Clamp surface Ratio Reference, page (mm2) (mm2) % Allodiscocotyla chorinemi Yamaguti, 1953 Allodiscocotylidae 221,079 23,301 10.54 [8] p. 547 Allodiscocotyla diacanthi Unnithan, 1962 Allodiscocotylidae 891,326 53,724 6.03 [8] p. 547 Allodiscocotyla lae Yamaguti, 1968 Allodiscocotylidae 525,572 13,728 2.61 [69] p. 251 Camopia rachycentri Lebedev, 1970 Allodiscocotylidae 12,492,318 150,559 1.21 [39] p. 152 Hargicola oligoplites (Hargis, 1957) Lebedev, 1970 as Vallisia oligoplites Hargis, 1957 Allodiscocotylidae 4,944,569 248,805 5.03 [16] p. 7 Metacamopia chorinemi (Yamaguti, 1953) Lebedev, 1984 as Vallisia chorinemi Yamaguti, 1953 Allodiscocotylidae 1,278,926 16,737 1.31 [65] p. 65 Metacamopia indica (Unnithan, 1962) Lebedev, 1972 Allodiscocotylidae 1,488,158 13,866 0.93 [39] p. 154 Metacamopia oligoplites Takemoto, Amato & Luque, 1996 Allodiscocotylidae 3,273,506 62,243 1.90 [70] p. 167 Vallisia riojai Caballero & Bravo-Hollis, 1963 Allodiscocotylidae 971,416 10,768 1.11 [71] p. 175 Vallisia striata Parona & Perugia, 1890 Allodiscocotylidae 18,448,597 373,475 2.02 [72] p. 19 Bychowskicotyle plectorhynchi Lebedev, 1969 Bychowskicotylidae 857,260 65,536 7.64 [39] p. 100 Gaterina talaensis Lebedev, 1969 Bychowskicotylidae 619,337 33,586 5.42 [39] p. 101 Tonkinopsis transfretanus Lebedev, 1972 Bychowskicotylidae 1,236,435 59,827 4.84 [39] p. 104 Yamaguticotyla jucunda (Lebedev, 1969) Lebedev, 1984 Bychowskicotylidae 1,189,653 50,151 4.22 [39] p. 103 Yamaguticotyla truncata (Goto, 1894) Bychowskicotylidae 4,924,003 87,494 1.78 [39] p. November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species Cont. Species Family Body surface Clamp surface Ratio Reference, page (mm2) (mm2) % Engraulicola thrissocles (Tripathi, 1959) Lebedev, 1971 Gastrocotylidae 1,014,407 69,075 6.81 [39] p. 70 Engrauliphila grex Unnithan, 1967 Gastrocotylidae 208,190 24,488 11.76 [77] p. 218 Engrauliscobina triaptella Unnithan, 1967 Gastrocotylidae 1,465,577 91,211 6.22 [77] p. 221 Engraulixenus malabaricus Unnithan, 1967 Gastrocotylidae 556,699 56,562 10.16 [77] p. 215 Eyelavera typica Unnithan, 1968 Gastrocotylidae 9,500,350 844,481 8.89 [39] p. 74 Gastrocotyle indica Subhapradha, 1951 Gastrocotylidae 281,048 27,274 9.70 [68] p. 361 Gastrocotyle kurra Unnithan, 1968 Gastrocotylidae 2,864,859 266,456 9.30 [68] p. 362 Gastrocotyloides dillonhargisi Lebedev, 1980 Gastrocotylidae 1,273,059 233,778 18.36 [39] p. 72 Irinaxine miniata Ghichenok, 1980 Gastrocotylidae 741,990 59,441 8.01 [39] p. 60 Pellonicola arabiana Khan & Karyakarte, 1977 Gastrocotylidae 1,059,163 66,909 6.32 [68] p. 367 Pellonicola elongatus Unnithan, 1967 Gastrocotylidae 353,696 27,079 7.66 [77] p. 225 Pellonicola lanceolatus Kritsky & Bilqees, 1973 Gastrocotylidae 1,785,588 102,492 5.74 [78] p. 198 Pseudaxine bivaginalis Dillon & Hargis, 1965 Gastrocotylidae 1,137,648 44,201 3.89 [79] p. 276 Pseudaxine kurra Unnithan, 1968 Gastrocotylidae 1,909,954 37,999 1.99 [75] p. 268 Pseudaxinoides caballeroi Lebedev, 1977 Gastrocotylidae 2,096,832 93,128 4.44 [39] p. 57 Quadrivalvula asymmetrica Ghichenok, 1980 Gastrocotylidae 3,430,344 994,784 29.00 [39] p. 77 Sibitrema poonui Yamaguti, 1966 Gastrocotylidae 15,402,407 235,741 1.53 [74] p. 430 Cathucotyle cathuaui Lebedev, 1968 Gotocotylidae 1,303,159 261,873 20.10 [80] p. 450 Cathucotyle filipinensis Hayward & Rohde, 1999 Gotocotylidae 2,971,409 413,969 13.93 [80] p. 453 Cathucotyle sinensis Hayward & Rohde, 1999 Gotocotylidae 22,961,031 1,562,400 6.80 [80] p. 455 Gotocotyla acanthura (Parona & Perugia, 1896) Meserve, 1938 Gotocotylidae 3,209,235 466,446 14.53 [80] p. 431 Gotocotyla africanensis Hayward & Rohde, 1999 Gotocotylidae 2,445,683 250,315 10.23 [80] p. 438 Gotocotyla bivaginalis (Ramalingam, 1961) Rohde, 1976 Gotocotylidae 7,414,296 274,996 3.71 [80] p. 440 Gotocotyla elagatis Meserve, 1938 as Gotocotyla meservei Yamaguti, 1953 Gotocotylidae 1,314,267 179,701 13.67 [65] p. 56 Gotocotyla heapae Hayward & Rohde, 1999 Gotocotylidae 1,291,877 69,182 5.36 [80] p. 443 Gotocotyla niphonii Hayward & Rohde, 1999 Gotocotylidae 1,932,305 171,386 8.87 [80] p. 445 Gotocotyla queenslandici Hayward & Rohde, 1999 Gotocotylidae 1,321,566 258,123 19.53 [80] p. 447 Neogotocotyla rohdii Hadi & Bilqees, 2010 Gotocotylidae 2,991,506 347,296 11.61 [81] p. 22 Mexicotyle mexicana (Meserve, 1938) Lebedev, 1984 Neothoracocotylidae 2,566,693 133,237 5.19 [39] p. 90 Neothoracocotyle acanthocybii (Meserve, 1938) Hargis, 1956 Neothoracocotylidae 126,796 12,940 10.21 [39] p. 88 Pricea fotedari Gupta & Sharma, 1979 Neothoracocotylidae 2,219,395 250,628 11.29 [68] p. Taxonomic discussion of the new species 60 Ahpua piscicola Caballero & Bravo-Hollis, 1973 Chauhaneidae 10,832 404 3.73 [73] p. 39 Allopseudopisthogyne constricta Yamaguti, 1965 Chauhaneidae 4,293,278 170,812 3.98 [74] p. 75 Caniongiella australis (Young, 1968) Lebedev, 1976 Chauhaneidae 624,748 7,033 1.1 [39] p. 127 Caniongiella bychowskyi Lebedev, 1976 Chauhaneidae 1,204,269 26,531 2.20 [39] p. 126 Chauhanea madrasensis Ramalingam, 1953 Chauhaneidae 2,631,446 172,925 6.57 [39] p. 120 Cotyloatlantica mediterranea (Euzet & Trilles, 1960) as Chauhanea mediterranea Euzet & Trilles, 1960 Chauhaneidae 3,309,353 305,081 9.22 [40] p. 190 Gemmaecaputia corrugata Tripathi, 1959 Chauhaneidae 404,195 13,015 3.22 [8] p. 487 Metopisthogyne sphyraenae Yamaguti, 1966 Chauhaneidae 3,415,824 337,042 9.87 [74] p. 426 Oaxacotyle oaxacensis (Caballero & Bravo, 1964) Lebedev, 1984 Chauhaneidae 397,303 56,968 14.34 [39] p. 138 Opisthogyne keralae Unnithan, 1962 Chauhaneidae 262,328 19,843 7.56 [44] p. 318 Paracaniongiella brinkmanni (Unnithan, 1962) Lebedev, 1976 Chauhaneidae 236,647 8,027 3.39 [68] p. 359 Paragemmaecaputia crassicauda Ramalingam, 1960 Chauhaneidae 189,399 9,836 5.19 [68] p. 357 Pentatres sphyraenae Euzet & Razarihelisoa, 1959 Chauhaneidae 497,849 29,309 5.89 [39] p. 132 Pseudochauhanea elongata Kritsky, Bilqees & Leiby, 1972 Chauhaneidae 593,000 16,000 2.70 [39] p. 122 Pseudochauhanea macrorchis Lin, Liu & Zhang in Zhang, Yang & Liu, 2001 Chauhaneidae 1,037,805 41,361 3.99 [75] p. 261 Pseudochauhanea mexicana Lamothe, 1967 Chauhaneidae 2,354,237 123,241 5.23 [39] p. 120 Pseudochauhanea sphyraenae Yamaguti, 1965 Chauhaneidae 2,795,654 110,955 3.97 [69] p. 251 Pseudomazocraes monsivaisae Caballero & Bravo Hollis, 1955 Chauhaneidae 810,544,311 37,813,692 4.67 [76] p. 108 Pseudomazocraes selene Hargis, 1957 Chauhaneidae 771,077 49,995 6.48 [16] p. 7 Pseudopisthogyne lepidocybii Yamaguti, 1965 Chauhaneidae 2,106,993 189,587 9.00 [57] p. 75 Pseudopisthogynopsis lepidocybii Yamaguti, 1965 Chauhaneidae 8,160,745 821,312 10.06 [39] p. 117 Salinacotyle mexicana (Caballero & Bravo-Hollis, 1963) Lebedev, 1984 Chauhaneidae 1,425,544 103,400 7.25 [39] p. 138 Allopseudaxine macrova (Unnithan, 1957) Yamaguti, 1963 Gastrocotylidae 4,858,980 388,611 8.00 [8] p. 265 Allopseudaxine yaito Yamaguti, 1968 Gastrocotylidae 14,764,474 485,975 3.29 [69] p. 251 Allopseudaxinoides euthynni Yamaguti, 1965 Gastrocotylidae 11,587,179 720,066 6.21 [57] p. 84 Amphipolycotyle chloroscombrus Hargis, 1957 Gastrocotylidae 209,204 22,656 10.83 [16] p. 5 Areotestis sibi Yamaguti, 1965 Gastrocotylidae 33,887,590 629,933 1.86 [57] p. 79 Churavera macrova Unnithan, 1968 Gastrocotylidae 2,844,619 67,889 2.39 [68] p. 368 Cypselurobranchitrema spilonotopteri Yamaguti, 1966 Gastrocotylidae 204,576 13,906 6.80 [74] p. 432 Engraulicola forcepopensis George, 1960 Gastrocotylidae 303,387 13,117 4.32 [68] p. 366 Engraulicola micropharyngella Unnithan, 1967 Gastrocotylidae 293,283 12,295 4.19 [77] p. 212 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9 The Monogenean Which Lost Its Clamps Table 3. November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species 383 Pricea microcotylae Chauhan, 1945 Neothoracocotylidae 31,523 3,995 12.67 [66] p. 148 Pricea minimae Chauhan, 1945 Neothoracocotylidae 796,183 170,955 21.47 [66] p. 146 Pricea solandri Gupta & Channa, 1977 Neothoracocotylidae 29,180 4,158 14.25 [68] p. 382 Pseudothoracocotyla ovalis (Tripathi, 1956) Yamaguti, 1963 Neothoracocotylidae 1,104,406 122,155 11.06 [82] p. 164 Pseudothoracocotyla whittingtoni Hayward & Rohde, 1999 Neothoracocotylidae 6,151,442 2,086,478 33.92 [82] p. 167 Scomberocotyle scomberomori (Koratha, 1955) Hargis, 1956 Neothoracocotylidae 2,837,224 293,771 10.35 [39] p. 89 Thoracocotyle crocea MacCallum, 1913 as Paradawesia bychowskyi Bravo & Lamothe, 1976 Neothoracocotylidae 3,604,259 390,338 10.83 [39] p. 94 Pricea multae Chauhan, 1945 Neothoracocotylidae 3,561,945 371,996 10.44 [83] p. 173 Scomberomorocotyle munroi Rohde & Hayward, 1999 Neothoracocotylidae 640,210 74,389 11.62 [84] p. 5 Chauhanocotyle rottleri Khoche & Dad, 1975 Protomicrocotylidae 912,981 13,257 1.45 [68] p. 356 Bilaterocotyle chirocentrosus Chauhan, 1945 Protomicrocotylidae 670,131 15,726 2.35 [66] p. 138 Bilaterocotyle lucknowensis (Agrawal & Sharma, 1986) Pandey & Agrawal, 2008 Protomicrocotylidae 92,963 2,413 2.60 [68] p. 350 Bilaterocotyle multitesticularis Khan & Karyakarte, 1982 Protomicrocotylidae 1,180,417 9,192 0.78 [68] p. 349 Bilaterocotyle polynemusi Gupta & Krishna, 1980 Protomicrocotylidae 1,358,610 15,508 1.14 [68] p. 347 Bilaterocotyle spindalis Deo & Karyakarte, 1980 Protomicrocotylidae 1,659,994 45,651 2.75 [68] p. 348 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 The Monogenean Which Lost Its Clamps Table 3. Cont. Species Family Body surface Clamp surface Ratio Reference, page (mm2) (mm2) % Bilaterocotyloides carangis Ramalingam, 1961 Protomicrocotylidae 1,135,204 13,434 1.18 [39] p. 114 Bilaterocotyloides madrasensis Radha, 1966 Protomicrocotylidae 441,228 4,984 1.13 [39] p. 116 Bilaterocotyle mamaevi Agrawal, 1988 Protomicrocotylidae 27,004 485 1.80 [68] p. 352 Bilaterocotyloides novaeguineae (Rohde, 1977) Lebedev, 1986 Protomicrocotylidae 442,980 11,411 2.58 [39] p. 114 Bilaterocotyloides spinulosus Liu in Zhang, Yang & Liu, 2001 Protomicrocotylidae 2,197,940 11,976 0.54 [75] p. 247 Lethacotyle fijiensis Manter & Price, 1953 Protomicrocotylidae 2,788,607 0 0 [39] p. 117 Lethacotyle vera n. sp. Protomicrocotylidae 2,562,639 0 0 This paper Neomicrocotyle carangis Yamaguti, 1968 Protomicrocotylidae 4,287,184 15,571 0.36 [39] p. 110 Neomicrocotyle indica Ramalingam, 1960 Protomicrocotylidae 49,651 232 0.47 [67] p. 375 Neomicrocotyle sp. JNC 3242A7 Protomicrocotylidae 2,663,686 15,327 0.58 This paper Neomicrocotyle unnithani Yamaguti, 1968 Protomicrocotylidae 2,019,641 13,440 0.67 [44] p. 344 Protomicrocotyle celebesensis Yamaguti, 1953 Protomicrocotylidae 1,791,383 11,869 0.66 [65] p. 56 Protomicrocotyle ivoriensis Wahl, 1972 Protomicrocotylidae 2,939,959 7,682 0.26 [55] p. 324 Protomicrocotyle madrasensis Ramalingam, 1960 Protomicrocotylidae 736,440 5,113 0.69 [67] p. Taxonomic discussion of the new species 375 Protomicrocotyle mannarensis Ramalingam, 1960 Protomicrocotylidae 1,934,754 10,205 0.53 [67] p. 377 Protomicrocotyle manteri Bravo-Hollis, 1966 Protomicrocotylidae 1,608,092 10,245 0.64 [39] p. 106 Protomicrocotyle minuta Ramalingam, 1960 Protomicrocotylidae 334,808 6,589 1.97 [67] p. 377 Protomicrocotyle mirabilis (MacCallum, 1918) Johnston & Tiegs, 1922 Protomicrocotylidae 231,559 7,854 3.39 [55] p. 321 Protomicrocotyle nayaritensis Bravo-Hollis, 1979 Protomicrocotylidae 7,317,320 13,238 0.18 [85] p. 190 Vallisiopsis contorta Subhapradha, 1951 Protomicrocotylidae 29,848 281 0.94 [39] p. 17 Vallisiopsis sphyraenae Yamaguti, 1968 Protomicrocotylidae 6,746,717 48,813 0.72 [69] p. 251 Youngiopsis australis (Young, 1968) Lebedev, 1972 Protomicrocotylidae 1,561,104 16,863 1.08 [39] p. 117 Allopseudodiclidophora opelu Yamaguti, 1965 Pseudodiclidophoridae 5,244,324 20,471 0.39 [57] p. 73 Gephyrocotyle ixoracorona Unnithan, 1966 Pseudodiclidophoridae 482,140 14,380 2.98 [68] p. 340 Pseudodiclidophora decapteri Yamaguti, 1965 Pseudodiclidophoridae 1,163,641 16,908 1.45 [57] p. 70 Quilonella ventrosa Lebedev & Parukhin, 1970 Pseudodiclidophoridae 954,583 13,250 1.39 [39] p. 81 Sawquirahcotyle indica Lebedev, 1976 Pseudodiclidophoridae 2,316,402 45,956 1.98 [39] p. 85 Winkenthughesia australis Robinson, 1961 Pseudodiclidophoridae 8,558,914 244,842 2.86 [43] p. 261 The outlines of body and clamps were redrawn on computer from original publications or from our own drawings, and the surface was calculated using ImageJ. Names of species follow WoRMS [42]; if different, name used in publication also indicated. All computerized line drawings available as Supplementary Material. Data ordered in alphabetical order of families and species. doi:10 1371/journal pone 0079155 t003 Table 3. Cont. The outlines of body and clamps were redrawn on computer from original publications or from our own drawings, and the surface was calculated using ImageJ. Names of species follow WoRMS [42]; if different, name used in publication also indicated. All computerized line drawings available as Supplementary Material. Data ordered in alphabetical order of families and species. doi:10.1371/journal.pone.0079155.t003 the genus Lethacotyle belongs to the Protomicrocotylidae, in spite of the absence of clamps. The host of the Lethacotyle species described by Ramalingam is ‘‘C. sexfasciatus’’ but the author mentioned that the carangids were 15 cm [18] and 5.2–26.5 cm [17] in length. Maturity of C. sexfasciatus is attained at 40 cm, common length is 60 cm, and maximum published weight is 18 kg [48]. Species identification of carangids, when they are adult, is often difficult, but the validity of species identification of the small specimens examined by Ramalingam is certainly dubious. Taxonomic discussion of the new species An hypothesis could be proposed, in which Lethacotyle would be a primitive species without clamps, with more derived species having clamps; our analyses clearly falsify this hypothesis, and demonstrate that the Protomicrocotylidae is not among the basal groups among the polyopisthocotyleans and the Gastrocotylinea. Species in Lethacotyle Therefore, we consider that: (a) the host of L. fijiensis in Fiji is an unknown carangid (due to insufficient host identification by Manter & Prince [15]); (b) the host of the Lethacotyle species described by Ramalingam is an unknown carangid, due to identification from immature fish specimens [17,18], and we see no reason why it should be the same species as Manter & Prince’s host fish. It might be C. sexfasciatus, as claimed by the author; however, we examined several C. sexfasciatus from off Australia and New Caledonia, and found no species of Lethacotyle [49]; (c) it is likely, based on collections from widely separated areas (Andaman Islands vs Fiji, which are separated by 9,000 km), the probability of different host species, and differences in measurements of the Manter & Prince (1953) described L. fijiensis from two specimens from ‘‘yellow jack’’ [15]; the identification of the host fish is vague, as often with Manter’s work (other cases: [46,47]), and almost useless (many carangids are partly yellow). Only one monogenean specimen, the holotype of L. fijiensis, is kept in the USNPC collections (Figure 1). Ramalingam [17,18] described a species of Lethacotyle from ‘‘Caranx sexfasciatus’’ from off the Andaman Islands, and claimed it was the same species as L. fijiensis. No deposition of specimens in a curated collection or museum is mentioned in the papers. The MCO spines as described by Ramalingam are 15 mm in length. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 11 The Monogenean Which Lost Its Clamps Figure 6. Ratio between clamp surface and body surface in families of gastrocotylinean monogeneans. Ratio between clamp surface and body surface in families of gastrocotylinean monogeneans. Ratios are ordered in decreasing order of mean. Protomicrocotylids have the lowest mean and lowest minimum. For significance see Table 4. doi:10.1371/journal.pone.0079155.g006 Figure 6. Ratio between clamp surface and body surface in families of gastrocotylinean monogeneans. Ratio between clamp surface and body surface in families of gastrocotylinean monogeneans. Ratios are ordered in decreasing order of mean. Protomicrocotylids have the lowest mean and lowest minimum. For significance see Table 4. doi:10.1371/journal.pone.0079155.g006 MCO spines (Table 5), that the species described by Ramalingam is distinct from both L. fijiensis and L. vera n. sp.; (d) and thus, Lethacotyle probably comprises, at least, three species. Families are in decreasing order of ratio. P values correspond to Mann & Whitney U tests between each family and the Protomicrocotylidae; all families have a significantly greater ratio than the Protomicrocotylidae, except the Pseudodiclidophoridae. doi:10 1371/journal pone 0079155 t004 079155.t004 PLOS ONE | www.plosone.org 12 November 2013 | Volume 8 | Issue 11 | e79155 Species in Lethacotyle Clamps in protomicrocotylids vs other monogeneans Our results (Figures 5, 6) show that the clamp surface is significantly smaller in species of the protomicrocotylids in comparison to other gastrocotylinean monogeneans. In addition, our description of L. vera n. sp. confirms that clamps are completely absent in members of the genus Lethacotyle. Clamps are an important and characteristic part of the anatomy of polyopistho- cotylean monogeneans, and are clearly the main organ used for attachment to the host [6,8,11–13]. Protomicrocotylids, no less than other monogeneans, need to maintain attachment to their host. In a fluid environment maintenance of position on the Our species is the first referred to Lethacotyle with a precise host identification. We have examined a number of other carangids from several genera off New Caledonia [47,49–53] and found L. vera n. sp. only on C. papuensis, suggesting that species of Lethacotyle are specific to Caranx species. It is likely that the ‘‘yellow jack’’ of Manter & Prince (1953) [15] and the carangid of Ramalingam [17,18], both identified with suboptimal precision, were species of Caranx, but, as explained above, not necessarily conspecific. Table 4. Significant differences of clamp surface: body surface ratios in families of gastrocotylinean monogeneans. Families n Minimum Maximum Mean P value (Total = 118) (%) (%) (%) Neothoracocotylidae 12 5.19 33.92 13.61 0.000483 Gotocotylidae 9 3.71 20.10 11.47 0.000483 Gastrocotylidae 26 1.53 29 7.60 0.001699 Chauhaneidae 22 1.13 14.34 5.89 0.007222 Bychowskicotylidae 5 1.78 7.64 4.78 0.001699 Allodiscocotylidae 10 0.93 10.54 3.27 0.004136 Pseudodiclidophoridae 6 0.39 2.98 1.84 0.209316 Protomicrocotylidae 28 0 3.39 1.12 - Families are in decreasing order of ratio. P values correspond to Mann & Whitney U tests between each family and the Protomicrocotylidae; all families have a significantly greater ratio than the Protomicrocotylidae, except the Pseudodiclidophoridae. doi:10.1371/journal.pone.0079155.t004 Table 4. Significant differences of clamp surface: body surface ratios in families of gastrocotylinean monogeneans. ferences of clamp surface: body surface ratios in families of gastrocotylinean monogeneans. Table 4. Significant differences of clamp surface: body surface ratios in families of gastrocotylinean Families are in decreasing order of ratio. P values correspond to Mann & Whitney U tests between each family and the Protomicrocotylidae; all families have a significantly greater ratio than the Protomicrocotylidae, except the Pseudodiclidophoridae. doi:10.1371/journal.pone.0079155.t004 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 12 The Monogenean Which Lost Its Clamps Figure 7. November 2013 | Volume 8 | Issue 11 | e79155 Species in Lethacotyle E, outline of ovary (paratype JNC1189A1). doi:10.1371/journal.pone.0079155.g008 Figure 8. Lethacotyle vera n. sp. Juvenile and other details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3- 1A154DBA614D). A, juvenile (specimen MNHN JNC3188A1). B, spines of MCO in juvenile. C, spines of MCO in paratype MNHN JNC3188A2c (posterior part of body processed for molecular study); D, spines of MCO in paratype MNHN JNC1189A2. E, outline of ovary (paratype JNC1189A1). doi:10.1371/journal.pone.0079155.g008 posterior lappet is perpendicular to the longitudinal axis of the worm, and firmly applied against the gill surface. coming into contact with the filaments may either press against them thus helping to hold on to them or after getting a hold around the filaments may adpress them against the body and thus effect a hold on to the gills’’. He concluded ‘‘this mode of effecting attachment to the gills by means of outgrowths of body surface is unique in Monogenea. An adventious growth of the body surface as seen in this case is rather unique and possibly nothing parallel is known among the animal kingdom’’. It is apparent that development of a complex of organs associated with the haptor, and a reduction in the size and complexity of the clamps is associated with evolution of the Protomicrocotylidae. In this group, development of organs for the attachment on the host, including lateral flaps and posterior tegumental striations, or a combination of these two structures, apparently renders clamps of little significance for attachment. It is not clear which came first (reduction of the clamps or development of a complex of tegumental organs for attachment), and comprehensive phylogenetic analysis of all members of the family would be needed to resolve this question [56]. Given the overall phylogenetic placement of the family, and relative to other Gastrocotylinea, clamps must be considered vestigial organs in most protomicrocotylids (genera Protomicrocotyle, Neomicrocotyle, Bilaterocotyle and Bilaterocotyloides) and are absent in species of Lethacotyle. The existence of two major types of clamp structures (gastrocotylid type in Protomicrocotyle, microcotylid type in Neomi- crocotyle) which puzzled systematists [8,16,19], is consistent with a secondary loss of the accessory sclerites in Neomicrocotyle, trans- Unfortunately, we cannot confirm Ramalingam’s observations and hypotheses, having not observed living worms. Striations are visible on the posterior lappet of L. vera n. sp., and also on other protomicrocotylids (Table 5). Such transverse striations are rather unique among monogeneans. Species in Lethacotyle Lethacotyle vera n. sp. Adult and details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3-1A154DBA614 A, whole body; B, spines of male copulatory organ (MCO). C, spines of MCO in other specimen (paratype MNHN JNC1189A3). D, sclerotized vagina egg, in utero. F, striations on posterior part of body; G, H, I, hooks (paratype MNHN JNC1185A3). A, B, D, F: holotype, MNHN JNC3209A1. doi:10.1371/journal.pone.0079155.g007 Figure 7. Lethacotyle vera n. sp. Adult and details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3-1A154DBA614D). A, whole body; B, spines of male copulatory organ (MCO). C, spines of MCO in other specimen (paratype MNHN JNC1189A3). D, sclerotized vagina. E, egg, in utero. F, striations on posterior part of body; G, H, I, hooks (paratype MNHN JNC1185A3). A, B, D, F: holotype, MNHN JNC3209A1. doi:10.1371/journal.pone.0079155.g007 the haptor, and the striations on the posterior haptoral lappet. Hooks are relatively small in protomicrocotylids and are thus not considered of importance in attachment. external surfaces of the host represents a challenge, and one potentially heightened for protomicrocotylids that possess minis- cule clamps, and for species of Lethacotyle, in which clamps are completely absent. Ramalingam [18], apparently from a study of living specimens (although this is not clearly stated in his paper) described the flaps of the haptor and reported that ‘‘the gap between the flaps in the anteroposterior axis can be narrowed by the contraction of the body in this region as well as by the extensile power of the flaps thus bringing their free ends in contact with each other or may lead to overlapping condition’’. He explained that the flaps ‘‘on We hypothesize that other structures play a role in host attachment in protomicrocotylids, as habitat selected by these monogeneans (the fish gill) does not differ substantially from that characteristic of other gastrocotylineans which have fully devel- oped clamps. Among protomicrocotylids, fixation may be attained by the combined action of the haptoral hooks, the lateral flaps of November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 13 PLOS ONE | www.plosone.org The Monogenean Which Lost Its Clamps Figure 8. Lethacotyle vera n. sp. Juvenile and other details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3- 1A154DBA614D). A, juvenile (specimen MNHN JNC3188A1). B, spines of MCO in juvenile. C, spines of MCO in paratype MNHN JNC3188A2c (posterior part of body processed for molecular study); D, spines of MCO in paratype MNHN JNC1189A2. Species in Lethacotyle Some information about the precise habitat of protomicrocotylids are available; Rohde [54] stated that Protomicrocotyle sp. was only found on the posterior surface of the internal filaments of the first gill of Caranx melampygus Cuvier, 1833. Wahl described the position of specimens of Protomicrocotyle ivoriensis Wahl, 1972 and P. mirabilis according to their asymmetry and noted that the posterior lappet was intercalated between two gill lamellae ([55], p. 329). Indeed, transverse striations are probably efficient for attachment, by increasing friction, only when the November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 14 The Monogenean Which Lost Its Clamps Table 5. Measurements of Lethacotyle species. Lethacotyle L. vera n. sp. L. vera n. sp. L. vera n. sp. L. fijiensis ‘‘L. fijiensis’’ ‘‘L. Species in Lethacotyle doi:10.1371/journal.pone.0079155.t005 form: mean (minimum–maximum), except for a few measurements with sample .30, for which measurements are in the form: mean maximum). 005 forming the more complex gastrocotylid clamp into a simpler microcotylid-like clamp. phylogenetically disparate polyopisthocotyleans. Among some pseudodiclidophorids, evolution towards a reduced role of clamps has occurred without attaining the secondarily simplified micro- cotylid-like structure nor the total absence observed within the protomicrocotylids. Establishing phylogenetic context is a primary foundation necessary to differentiate between secondary loss (as proposed for these lineages of monogeneans) in contrast to plesiomorphic absence [56]. Additionally, the phylogenetic framework is critical for establishing the temporal association and sequence of evolutionary modification in complex attributes. The Pseudodiclidophoridae also have a small clamp: body ratio, slightly higher than but not significantly different from the protomicrocotylids (Figure 6, Table 4). As our study concerns mainly the protomicrocotylids, we provide here only limited comments about pseudodiclidophorids. Only 5 pseudodiclido- phorids were studied, and none has completely lost the clamps; one has transverse striations [43], and one, Allopseudodiclidophora opelu Yamaguti, 1965 (Figure 4F) has a ‘‘long anchor-bearing appendage’’ [57]; several have outstandingly wide posterior bodies that evoke the possibility of this part working as a sucker, as suggested for the microcotylid Aspinatrium gallieni Euzet & Ktari, 1971 [58]. These observations suggest that reduction in clamps, coincidental with development of secondary organs of attachment is a rare event, but has occurred in multiple lineages of Species in Lethacotyle fijiensis’’ Holotype Adults Juveniles Adult Adult Juvenile Reference This paper This paper This paper Manter & Price, 1953 [15] Ramalingam, 1968 [18] Ramalingam, 1968 [18] n 1 8 2 2 1 1 Body Length 5130 4340 (2300–5720, n = 8) 1300, 1700 3156–3759 1540 950 Body Width 750 973 (500–1270, n = 9) 320, 350 663–770 380 130 Pharynx Length 53 66 (53–75, n = 9) 38, 45 64 50 37 Pharynx Width 45 59 (45–70, n = 9) 38,43 50 33 25 Buccal Sucker Length 70 71 (50–83, n = 18) 60, 60 49–52 (diameter) 37 27 Buccal Sucker Width 42–57 59 (42–75, n = 18) 38, 50 25 25 Anterior-Genital Pore Distance 800 648 (360–803, n = 9) Number of Genital Corona Spines 24 24 (21–27, n = 9) 24–25 24 Length of Genital Corona Spines 52 (43–66, n = 10) 5065.7 (35–66, n = 138) 24 15 Number of Testes 34 29 (21–34, n = 7) 30 31 Testis Length 4367.9 (25–55, n = 34) 52 (25–82, n = 57) Testis Width 4267.9 (27–52, n = 34) 145 (63–262, n = 57) Testicular Mass Length 975 913 (588–1163, n = 6) 370 Testicular Mass Width 125 352 (125–489, n = 6) Sclerotized Vagina Length 150 157 (125–175, n = 9) Sclerotized Vagina Width 85 81 (38–100, n = 9) Unsclerotised Vagina Length 175 170 (60–250, n = 9) Anterior-Vagina Pore Distance 850 739 (407–938, n = 9) Ovary Length 542 591 (114–935, n = 8) Ovary Width 192 340 (192–550, n = 8) Haptor Total Length 282 325 (245–400, n = 7) 207, 275 130 90 Haptor Total Width 850 764 (588–850, n = 6) 452, 525 260 280 Hamulus Length 24, 30 28 (24–33, n = 15) 27, 32 24 33 30 Posterior Hook Length 16, 16 18 (10–24, n = 13) 15, 16 16 18 22 Small Hook Length 14 (11–16, n = 8) 14 12 12 All measurements are in mm, in the form: mean (minimum–maximum), except for a few measurements with sample .30, for which measurements are in the form: mean 6 standard deviation (minimum–maximum). doi:10.1371/journal.pone.0079155.t005 All measurements are in mm, in the form: mean (minimum–maximum), except for a few measurements with sample .30, for which measurements are in the form: mean 6 standard deviation (minimum–maximum). References Marine Parasitology: CSIRO, Collingwood, Australia & CABI, Oxon, UK. pp. 63–72. 8. Yamaguti S (1963) Systema Helminthum Volume IV Monogenea and Aspidocotylea: John Wiley & Sons. 27. Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PD (2005) DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society of London B Biological Sciences 360: 1847–1857. 9. Kearn GC (2004) Leeches, Lice and Lampreys. A natural history of skin and gill parasites of fishes. Dordrecht: Springer. 432 p. 28. Ratnasingham S, Hebert PDN (2007) BOLD: The Barcode of Life Data System (www. barcodinglife. org). Molecular Ecology Notes 7: 355–364. 10. Boeger WA, Kritsky DC (1993) Phylogeny and a revised classification of the Monogenoidea Bychowsky, 1937 (Platyhelminthes). Systematic Parasitology 26: 1–32. 29. Hassouna N, Michot B, Bachellerie J-P (1984) The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes. Nucleic Acids Research 12: 3563–3583. 11. Bychowsky BE (1961) Monogenetic Trematodes their systematics and phylogeny. English translation edited by W. J. Hargis Jr. Washington: American Institute of Biological Sciences. 627 p. 30. Olson PD, Littlewood DTJ (2002) Phylogenetics of the Monogenea - evidence from a medley of molecules. International Journal for Parasitology 32: 233–244. 12. Llewellyn J (1956) The adhesive mechanisms of monogenetic trematodes - the attachment of Plectanocotyle gurnardi (V. Ben. & Hesse) to the gills of Trigla. Journal of the Marine Biological Association of the United Kingdom 35: 507–514. 31. Jovelin R, Justine J-L (2001) Phylogenetic relationships within the polyopistho- cotylean monogeneans (Platyhelminthes) inferred from partial 28S rDNA sequences. International Journal for Parasitology 31: 393–401. 13. Llewellyn J (1958) The adhesive mechanisms of monogenetic trematodes - the attachment of species of the Diclidophoridae to the gills of gadoid fishes. Journal of the Marine Biological Association of the United Kingdom 37: 67–79. 32. Olson PD, Tkach VV (2005) Advances and trends in the molecular systematics of the parasitic Platyhelminthes. Advances in Parasitology: Academic Press. pp. 165–243. 14. Wong W-L, Gorb SN (2013) Attachment ability of a clamp-bearing fish parasite, Diplozoon paradoxum (Monogenea), on gills of the common bream, Abramis brama. Journal of Experimental Biology 216: 3008–3014. 33. Posada D, Crandall KA (2001) Evaluation of methods for detecting recombination from DNA sequences: Computer simulations. Proceedings of the National Academy of Sciences of the United States of America 98: 13757– 13762. 15. References Caledonia emphasizes parasite biodiversity in coral reef fish. Folia Parasitologica 57: 237–262. Caledonia emphasizes parasite biodiversity in coral reef fish. Folia Parasitologica 57: 237–262. 1. Justine J-L (1998) Non-monophyly of the monogeneans? International Journal for Parasitology 28: 1653–1657. 22. Justine J-L, Beveridge I, Boxshall GA, Bray RA, Moravec F, et al. (2010) An annotated list of fish parasites (Copepoda, Monogenea, Digenea, Cestoda and Nematoda) collected from Emperors and Emperor Bream (Lethrinidae) in New Caledonia further highlights parasite biodiversity estimates on coral reef fish. Zootaxa 2691: 1–40. 2. Perkins EM, Donnellan SC, Bertozzi T, Whittington ID (2010) Closing the mitochondrial circle on paraphyly of the Monogenea (Platyhelminthes) infers evolution in the diet of parasitic flatworms. International Journal for Parasitology 40: 1237–1245. 3. Mollaret I, Jamieson BGM, Adlard RD, Hugall A, Lecointre G, et al. (1997) Phylogenetic analysis of the Monogenea and their relationships with Digenea and Eucestoda inferred from 28S rDNA sequences. Molecular and Biochemical Parasitology 90: 433–438. 23. Marie AD, Justine J-L (2005) Monocotylids (Monogenea: Monopisthocotylea) from Aetobatus cf. narinari off New Caledonia, with a description of Decacotyle elpora n. sp. Systematic Parasitology 60: 175–185. 24. Fyler CA, Caira JN, Jensen K (2009) Five new species of Acanthobothrium (Cestoda: Tetraphyllidea) from an unusual species of Himantura (Rajiformes: Dasyatidae) from northern Australia. Folia Parasitologica 56: 107–128. 4. Mollaret I, Jamieson BGM, Justine J-L (2000) Phylogeny of the Monopistho- cotylea and Polyopisthocotylea (Platyhelminthes) inferred from 28S rDNA sequences. International Journal for Parasitology 30: 171–185. 5. Littlewood DTJ, Rohde K, Clough KA (1999) The interrelationships of all major groups of Platyhelminthes: phylogenetic evidence from morphology and molecules. Biological Journal of the Linnean Society 66: 75–114. 25. Justine J-L (2005) Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of measurements of specimens prepared using different methods, and a description of P. caledonicus n. sp. Systematic Parasitology 62: 1–37. 6. Hayward C (2005) Monogenea Polyopisthocotylea (ectoparasitic flukes). In: Rohde K, editor. Marine Parasitology: CSIRO, Collingwood, Australia & CABI, Oxon, UK. pp. 55–63. 26. Kritsky DC, McAleese WJ, Bakenhaster MD (2011) Heteronchoineans (Monogenoidea) from the gills of crevalle jack, Caranx hippos (Perciformes, Carangidae), from Everglades National Park, Florida, with a redescription of Protomicrocotyle mirabilis (Gastrocotylinea, Protomicrocotylidae). Comparative Parasitology 78: 265–274. 7. Whittington ID (2005) Monogenea Monopisthocotylea (ectoparasitic flukes). In: Rohde K, editor. Author Contributions Conceived and designed the experiments: Conceived and designed the experiments: JLJ CR. Performed parasite collection and preparation of specimens: JLJ. Performed microscopical observations on new specimens: CR JLJ. Performed microscopical observations on Museum specimens: CR JLJ EPH. Drawn specimens: CR JLJ. Performed analysis of clamp: body ratios, including statistical tests: CR. Performed molecular analyses: CS DG. Performed molecular phylogenetic analysis: CS. Discussed manu- script: JLJ CR DG CS EPH. Performed the experiments: JLJ CR DG CS EPH. Analyzed the data: JLJ CR DG CS EPH. Wrote the paper: JLJ CR DG CS EPH. Acknowledgments Various fish specimens were identified (from photographs) by Ronald Fricke, Jack Randall, Michel Kulbicki, Samuel Igle´sias and Bernard Se´ret. Eric Bureau, student in training, collected some specimens. David Gibson (BMNH) helped with linguistic discussions, and in the acquisition of rare literature and comments about species in WoRMS. Patricia Pilitt (USNPC) provided photomicrographs of the holotype of L. fijiensis, and Takashi Iwaki (MPM) kindly took photographs of additional museum specimens. Patricia Clamps of protomicrocotylids as vestigial organs Vestigial organs are structures that have apparently lost their ancestral function in a species, and for which homologous and functional organs are known in related species. Typical examples are the loss or reduction of flight organs in some island-dwelling November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 15 The Monogenean Which Lost Its Clamps Figure 9. Tree of gastrocotylinean monogeneans. Tree of gastrocotylinean monogeneans, based on a phylogenetic analysis of 28S sequences. doi:10.1371/journal.pone.0079155.g009 Figure 9. Tree of gastrocotylinean monogeneans. Tree of gastrocotylinean monogeneans, based on a phylogenetic analysis of 28S sequences. doi:10.1371/journal.pone.0079155.g009 various sensory attributes in larvae, used to seek hosts [56,63]. In Lethacotyle and protomicrocotylids, the loss and modification of organs concerns the haptor and clamps, body parts of the monogeneans which are clearly an adaptation to ectoparasitism. The occurrence of vestigial clamps or the complete absence of clamps, however, does not demonstrate that these parasites are ‘‘simplified’’. In contrast, reduction has occurred in the evolutionary context for development of novel structures for attachment (flaps and striations) which are unique among any of the lineages of the monogeneans. species (in insects or birds, independently), limbs bones in cetaceans, or the loss of eyes and pigmentation in cavern-dwelling species which have occurred under changing regimes for selection [59–62]. Parasites, in old anthropogenic interpretations, were considered ‘‘simpler’’ than free-living animals because they had lost certain organs (such as the intestine in cestodes)(e.g. [56]). More nuanced observations have demonstrated the considerable specialization and structural and biochemical complexity of helminths which often have complexes of novel organ systems in relation to parasitism, such as November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 16 The Monogenean Which Lost Its Clamps Pilitt (USNPC) and Eileen Harris (BMNH) are thanked for arranging specimen loans. Pilitt (USNPC) and Eileen Harris (BMNH) are thanked for arranging specimen loans. Supporting Information File S1 PDF of all figures and measurements of clamp and body surfaces. Total number of figures: 120. (PDF) The Monogenean Which Lost Its Clamps 41. Rasband WS (1997–2013) ImageJ U. S. 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Rohde K, Hayward CJ (1999) Scomberomorocotyle munroi n. g., n. sp. (Scomberomorocotylinae n. subf.), a thoracocotylid monogenean from Scomber- omorus munroi (Scombridae) off Australia and Papua New Guinea. Systematic Parasitology 43: 1–6. y p y pp 62. Helfman G, Collette B, Facey D, Bowen BW (2009) The diversity of fishes: Biology, evolution and ecology. New York: Wiley-Blackwell. 720 p. 63. Rohde K (1989) At least eight types of sense receptors in an endoparasitic flatworm: a counter-trend to sacculinization. Naturwissenschaften 76: 383–385. 85. Bravo Hollis M (1979) Monoge´neos de peces de la coleccio´n Winter II. Sobre un protomicrocotylido nuevo de Caranx hippos caninus Gu¨nther del Pacı´fico Mexicano. Anales del Centro de Ciencias del Mar y Limnologı´a 6: 189–196. 64. References Manter HW, Prince DF (1953) Some Monogenetic Trematodes of marine fishes from Fiji. Proceedings of the Helminthological Society of Washington 20: 105– 112. 34. Swofford DL (2002) PAUP*. 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Available from http://beast.bio. ed.ac.uk/Tracer. 19. Llewellyn J (1970) Taxonomy, genetics and evolution of parasites. Monogenea. Journal of Parasitology 56: 493–504. 38. Lebedev BI (1972) [The taxonomy of monogeneans of suborder Gastrocotyli- nea]. In Investigations on the Fauna, Systematics, and Biochemistry of Helminths in the Far East. (In Russian). Proceedings of the Far-East Science Centre, USSR Academy of Sciences 11: 121–145. 20. Justine J-L, Beveridge I, Boxshall GA, Bray RA, Miller TL, et al. (2012) An annotated list of fish parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda, Nematoda) collected from Snappers and Bream (Lutjanidae, Nemipteridae, Caesionidae) in New Caledonia confirms high parasite biodiver- sity on coral reef fish. Aquatic Biosystems 8: 22. 39. Lebedev BI (1986) [Monogenea: suborder Gastrocotylinea]. Leningrad: Nauka. 200 p. 40. Euzet L, Trilles J-P (1960) Sur deux Monoge`nes nouveaux de Sphyraena sphyraena (L.) (Teleostei Sphyraenidae). Bulletin de la Socie´te´ zoologique de France 85: 189–198. 21. Justine J-L, Beveridge I, Boxshall GA, Bray RA, Moravec F, et al. (2010) An annotated list of parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda and Nematoda) collected in groupers (Serranidae, Epinephelinae) in New November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 17 The Monogenean Which Lost Its Clamps MacCallum GA (1918) Notes on the genus Telorchis and other trematodes. Zoopathologica 1: 81–97. 65. Yamaguti S (1953) Parasitic worms mainly from Celebes. Part 2. Monogenetic trematodes of fishes. Acta Medicinae Okayama 8: 203–256+209 Pl. 86. Hoberg EP (2002) Foundations for an integrative parasitology: collections, archives, and biodiversity informatics. Comparative Parasitology 69: 124–131. 66. Chauhan BS (1945) Trematodes from Indian marine fishes. Part I. On some new monogenetic trematodes of the sub-orders Monopisthocotylea Odhner, 1912 and Polyopisthocotylea Odhner, 1912. Proceedings of the Indian Academy of Sciences - Section B 21: 129–159. 87. Hoberg EP, Pilitt PA, Galbreath KE (2009) Why museums matter: a tale of pinworms (Oxyuroidea: Heteroxynematidae) among pikas (Ochotona princeps and O. collaris) in the American West. Journal of Parasitology 95: 490–501. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 18
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Author Correction: Challenging the sustainability of urban beekeeping using evidence from Swiss cities
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www.nature.com/npjUrbanSustain AUTHOR CORRECTION OPEN Author Correction: Challenging the sustainability of urban beekeeping using evidence from Swiss cities Joan Casanelles-Abella and Marco Moretti npj Urban Sustainability (2022)2:14 ; https://doi.org/10.1038/s42949-022-00059-9 Correction to: npj Urban Sustainability https://doi.org/10.1038/ s42949-021-00046-6, published online 12 January 2022 1234567890():,; The original version of this Article contained a typographic error in the Abstract: “an average 10.14 hives per km2 (9370 in total)” should have read “an average 8.1 hives per km2 (6370 in total)”. The authors also became aware of an inaccurate assumption in the original version of the paper, which incorrectly read “we additionally considered different carrying capacity scenarios, ranging from 0.5 to an unrealistic value of 75 hives per km2” in the second last sentence of the first paragraph in the section “Assessing the sustainability of urban beekeeping” under Results. In the corrected version, “ranging from 0.5 to” is replaced by “ranging from 1 to” to avoid confusion. Published online: 09 June 2022 Published in partnership with RMIT University This has now been corrected in both the PDF and HTML versions of the Article. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. © The Author(s) 2022
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Investigation of reactions between trace gases and functional CuO nanospheres and octahedrons using NEXAFS-TXM imaging
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Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1, Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 In order to take full advantage of novel functional materials in the next generation of sensorial devices scalable processes for their fabrication and utilization are of great importance. Also understanding the processes lending the properties to those materials is essential. Among the most sought-after sensor applications are low-cost, highly sensitive and selective metal oxide based gas sensors. Yet, the surface reactions responsible for provoking a change in the electrical behavior of gas sensitive layers are insufficiently comprehended. Here, we have used near-edge x-ray absorption fine structure spectroscopy in combination with x-ray microscopy (NEXAFS-TXM) for ex-situ measurements, in order to reveal the hydrogen sulfide induced processes at the surface of copper oxide nanoparticles, which are ultimately responsible for triggering a percolation phase transition. For the first time these measurements allow the imaging of trace gas induced reactions and the effect they have on the chemical composition of the metal oxide surface and bulk. This makes the new technique suitable for elucidating adsorption processes in-situ and under real operating conditions. In recent years micro- and nano-scaled particles of various morphologies and sizes have been investi- gated in order to utilize their unique properties for a multitude of technological applications, including energy conversion and storage1, plasmonics2, drug delivery3, catalysis4,5 and gas sensing6. Among the impediments preventing a quick commercial adaptation of groundbreaking research results is the lack of scalable interfaces between the nano-regime and macroscopic equipment. One appealing technology in that regard is the inkjet printing process, which provides a precise and scalable method to deposit functional nanoparticles onto arbitrary structures. Using this technique colloidal suspensions of metal oxide inks may be deposited and used as a gas sensitive layer on low-power consuming, micro-machined silicon-based structures. This enables the investigation of the gas sensitive behavior of metal oxide par- ticles as a function of size, shape and temperature. In light of the need of highly sensitive and selective gas sensor solutions for advanced analytical applications the understanding of the underlying processes as well as the possibility to scale the production processes are of primary concern. p y p p p y Until now, most works are focusing on using n-type metal oxide semiconductors, such as SnO2, WO3 or ZnO for gas sensors. Only limited work has been reported on p-type semiconducting gas sensors 1Institute for Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Hahn-Meitner-Platz 1, 14109 Berlin, Germany. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Investigation of reactions between trace gases and functional CuO nanospheres and octahedrons using NEXAFS-TXM imaging received: 28 June 2015 accepted: 05 November 2015 Published: 03 December 2015 Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1, Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1, Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles. The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: + + + → + + . ( ) + − + N H H O Cu OH N H O Cu O 2 4 4 4 2 1 2 4 2 2 2 3 2 + + + → + + . + − + N H H O Cu OH N H O Cu O 2 4 4 4 2 2 4 2 2 2 3 2 ( ) 1 In order to increase the yield and to control the shape and the size of the particles, NaOH has been added to the reaction12,13. Besides the positive effect on the yield, NaOH may be used to control the morphology and the size of the resulting Cu2O particles. To this end, the NaOH concentration has been varied from 0.002–0.5 M NaOH. Up to a NaOH concentration of 0.04 M, the particle size shows a slight decrease of the hydrodynamic radius from about 240 nm to about 160 nm observed by dynamic light scattering14–16 shown in Fig. 1a. For these samples a spherical shape and a reasonably narrow size dis- tribution can be inferred from scanning electron microscopy (SEM) pictures as exemplarily depicted in Fig. 1b. A sharp transition occurs for NaOH concentrations exceeding 0.04 M (Fig. 1a), when the shape of Cu2O particles changes from spherical to octahedral. The addition of NaOH into the reaction solution has been proved to provide the necessary alkalinity for the use of N2H4 as reducing agent17,18. Two fac- tors are mainly influencing the morphology evolution of Cu2O nanoparticles: One is the capping agent, Polyvinylpyrrolidone (PVP). As a surfactant, PVP is preferentially adsorbed onto the {111} planes of the Cu2O crystals, which can decrease the surface energy of the {111} surface and reduce its growth rate. This favors the Cu2O crystal growth into octahedral structures19,20. Another factor is the amount of NaOH in the system. During the reaction, Cu(OH)2 is produced by NaOH reacting with Cu2+ ions, which is the key intermediate. The production of Cu(OH)2 can decrease the reduction rate of Cu2O, which slows down the growth rate of Cu2O crystals indirectly. Without addition of NaOH, the crystal growth rate is too fast to be controlled by PVP. Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1, Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 2Institut für Makromolekulare Chemie, University of Freiburg, 79104 Freiburg, Germany. 3Institut für Physikalische Chemie, University of Freiburg, 79104 Freiburg, Germany. 4Laboratory for Gas Sensors, Department of Microsystems Engineering, University of Freiburg, Georges-Köhler Allee 102, 79110 Freiburg. †Present address: Laboratory for Synchrotron Radiation – Catalysis and Sustainable Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland. Correspondence and requests for materials should be addressed to S.P. (email: Stefan.palzer@imtek.uni-freiburg.de) Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 1 www.nature.com/scientificreports/ based on materials such as Cu2O and CuO. However, p-type gas sensitive materials show significant surface reactivity with reducing and oxidizing gases albeit at lower operating temperatures as compared to n-type materials and consequently they might play a pivotal role in future, low-energy consuming gas sensing devices. In the past Cu2O and CuO nanostructures have been fabricated using numerous approaches, few of which are compatible with large-scale production. And while gas-surface interactions are proposed to explain the gas sensitive behavior of metal oxide layers, analytical tools for directly observing adsorption processes are scarce7. In this regard, techniques of x-ray absorption spectroscopy offer unique possibilities, but have so far not been used to directly monitor the chemical structure of the gas sensitive material itself. In previous works the oxidation state of platinum upon gas exposure has been investigated8,9 in order to study its effect on the gas sensitive material. To monitor the gas sensitive material directly Cu2O as well as CuO offer ideal conditions to demonstrate a novel approach com- bining near-edge x-ray absorption fine structure spectroscopy with x-ray microscopy NEXAFS-TXM10 and gas sensitive characterizations. This technique is able to probe both, the O-K-edge as well as the Cu-L2,3-edge11 to reveal specific surface processes upon exposure to a trace gas.i , g pi p p p g Here, we focus on the reactions between the highly toxic trace gas hydrogen sulfide (H2S) and CuO to highlight the capabilities of the technique. We employ the novel approach to track the fundamental changes in CuO caused by the interaction with H2S using the NEXAFS-TXM method to shed light onto fundamental surface processes. To address both applied as well as fundamental issues of next generation metal oxide based sensors we present a new route for synthesizing Cu2O nanoparticles with high yield, and demonstrate how to interface and to use them as sensor material. Using on-chip annealing the Cu2O nanoparticles may be converted to CuO. Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1, Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 Interestingly, the shape of the nanoparticles remains the same, even though the crystal structure changes from cubic to monoclinic and the oxygen content increases to about 50%. Hence, the here presented route allows for producing either CuO or Cu2O nanoparticles, con- trolling their shape and size and using their specific gas sensitive characteristics. The Cu2O nanoparticles are dispersed in a solution that is subsequently used as ink for depositing the nanoparticles employing an inkjet printing system. The solvent of the ink is then evaporated without leaving contaminations in the sensing layer, i.e. without affecting the gas sensing properties after deposition. The presented approach is able to pave the way towards a new generation of gas sensitive materials with tailor-made properties and reproducible, stable base-line resistances. The gas sensing properties of the CuO particles are inves- tigated and in addition the ex-situ NEXAFS-TXM measurements reveal the changes of the chemical composition due to the annealing process as well as those caused by exposure of Cu2O and CuO to H2S. The induced chemical changes of the Cu2O/CuO particles by H2S are irreversible at room temperature. Therefore, it was possible to map all stages of the surface reactions between H2S and Cu2O/CuO ex-situ in the high vacuum setup. This demonstrates the suitability of the NEXAFS-TXM method to provide a spatially resolved analysis of the chemical bonds formed during gas-surface interaction, which are at the heart of gas detection using semiconducting metal-oxides as functional material. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles. The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: Adding NaOH to the reaction, the crystal growth rate will be slowed down. In this case, the previously mentioned formation of Cu2O octahedrons due to the presence of PVP occurs. Moreover, continuously increasing the concentration of NaOH, [Cu(OH)4]2− can be formed via the complexation of Cu2+ with OH−, which will increase the edge lengths of the octahedrons as shown in Fig. 1c,d, a similar result also has been found by W. Zhu and his co-workers13. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 2 www.nature.com/scientificreports/ Figure 1. Both the shape as well as the particle size of the Cu2O particles crucially depend on the NaOH concentration. (a) The particle radius has been determined using dynamic light scattering. (b) Concentrations up to 0.03 M NaOH result in spherical particles with a diameter around 200 nm and a reasonably narrow particle size distribution. (c) Above 0.04 M NaOH the shape changes to octahedrons and (d) further increasing the NaOH concentration results in larger particle sizes. (b–d) show scanning electron microscopy (SEM) images for particles produced using 0.03, 0.04 and 0.3 M NaOH, respectively. Figure 1. Both the shape as well as the particle size of the Cu2O particles crucially depend on the NaOH concentration. (a) The particle radius has been determined using dynamic light scattering. (b) Concentrations up to 0.03 M NaOH result in spherical particles with a diameter around 200 nm and a reasonably narrow particle size distribution. (c) Above 0.04 M NaOH the shape changes to octahedrons and (d) further increasing the NaOH concentration results in larger particle sizes. (b–d) show scanning electron microscopy (SEM) images for particles produced using 0.03, 0.04 and 0.3 M NaOH, respectively. Characterization of the as-synthesized particles and solid-state transformation to CuO nanoparticles. Using the scalable wet chemistry process it is possible to control shape and size of the Cu2O particles. The deposition of the particles is achieved by employing a DIMATIX DMP-283121 system. Tuning the rheological properties by adding polyethylene glycol (PEG) 400 is necessary to match the requirements imposed by the inkjet printing system. Due to a solid state phase transformation from Cu2O to CuO at temperatures above 250 °C and ambient pressure it is furthermore possible to convert the so-produced particles using an on-chip annealing technique. Here we used elevated temperatures for one hour, which results in pure CuO particles of the same shape as the Cu2O particles. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: In order to examine the long-range as well as the short-range structure of the nanoparticles NEXAFS spectra and x-ray diffraction (XRD) pattern have been recorded for both spherical and octahedral samples before and after annealing. The results are presented in Fig. 2.h t gh p g Figure 2a–d shows the TXM-micrographs of the respective samples. The O-K-edge of the particles directly after synthesis cannot be investigated due to the remaining PEG, because they have been inkjet printed in order to ensure complete correspondence between NEXAFS analysis and gas sensitive char- acterization. However, the Cu-L2,3 edge shown in Fig. 2e reveals the differences in the near range order when comparing spherical and octahedron particles after synthesis. The Cu-L2,3-edge reveals that the spherical particles consist of a mixture of Cu+ and Cu2+ in an environment with a highly electronega- tive partner, like oxygen. The signals at ~931 and 950.9 eV are typical for Cu2+ and the features at 933.8 and 953.6 eV can be assigned to Cu+, respectively11,22–29. The XRD pattern of the spherical particles after synthesis shown in Fig. 2g does not show any evidence on the coexistence of a copper-(I)- and copper-(II)-oxide lattice structure within this sample. Without heat treatment, five obvious diffraction peaks can be found in the pattern, which are indexed to the {110}, {111}, {200}, {220} and {311} planes of cubic Cu2O (Fig. 2g,h blue lines, JCPDS card no. 65–3288). This means that the detected Cu2+ exists only in amorphous CuO phases or at defect sites within the spherical particles. On the contrary, as shown in Fig. 2e, the as-synthesized octahedral particles show features in the NEXAFS spectrum which can be fully assigned to the chemical nature of copper-(I)-oxide. These differences between the different particle morphologies can only be detected by NEXAFS spectroscopy. Beam damage as a reason for Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 3 www.nature.com/scientificreports/ Figure 2. (a) TXM-micrograph of the Cu2O spheres after synthesis: overlay of 930.8 eV (yellow) and 933.8 eV (blue) represents the chemical inhomogeneity of the sample. (b) TXM-micrograph of the Cu2O octahedron after synthesis (933.8 eV). (c) TXM-micrograph of the CuO spheres after oxidation (532.7 eV). (d) TXM-micrograph of the CuO octahedron after oxidation (532.7 eV). (e) NEXAFS spectra at the Cu- L2,3-edge of the Cu2O nanoparticles after synthesis. (f) NEXAFS spectra at the O-K-edge and Cu-L2,3-edge of the CuO nanoparticles after oxidation. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: In the XRD pattern, the measured intensity is plotted versus the scattering angle 2Θ . (g) XRD pattern of Cu2O nanoparticles after synthesis. (h) XRD pattern of CuO nanoparticles after oxidation. The reference values for the expected Bragg peaks for Cu2O and CuO are depicted via the δ –function at the corresponding angles. Figure 2. (a) TXM-micrograph of the Cu2O spheres after synthesis: overlay of 930.8 eV (yellow) and 933.8 eV (blue) represents the chemical inhomogeneity of the sample. (b) TXM-micrograph of the Cu2O octahedron after synthesis (933.8 eV). (c) TXM-micrograph of the CuO spheres after oxidation (532.7 eV). (d) TXM-micrograph of the CuO octahedron after oxidation (532.7 eV). (e) NEXAFS spectra at the Cu- L2,3-edge of the Cu2O nanoparticles after synthesis. (f) NEXAFS spectra at the O-K-edge and Cu-L2,3-edge of the CuO nanoparticles after oxidation. In the XRD pattern, the measured intensity is plotted versus the scattering angle 2Θ . (g) XRD pattern of Cu2O nanoparticles after synthesis. (h) XRD pattern of CuO nanoparticles after oxidation. The reference values for the expected Bragg peaks for Cu2O and CuO are depicted via the δ –function at the corresponding angles. this can be excluded because possible beam induced reactions play a minor role in the investigation of hard-condensed matter like inorganic colloidal particles. Additionally, the vacuum environment during investigation prevents the sample from oxidation and the soft x-rays (< 1 keV) do not have the energy needed for crystal rearrangement. Therefore, the found differences between both morphologies can be related to the nature of the immediate neighbors and the chemical state of the investigated element. Furthermore, this may be a factor contributing to the different behavior of the spherical particles in comparison to the octahedrons, which is further investigated in the ESI.t p g After oxidation both types of particles reveal a complete solid-state transformation from Cu2O to CuO, which is demonstrated by the NEXAFS spectra shown in Fig. 2f and with the XRD patterns in Fig. 2h. During the oxidation, the oxygen containing PEG in the samples is removed, which can be confirmed by the TXM images in Fig. 2c,d. Additionally, the spectrum of the O-K-edge in Fig. 2f shows no evidence of any other oxygen containing substance. All the recorded features in the O-K- and Cu-L2,3-spectra for the spherical as well as the octahedral particles can be assigned to copper-(II)-oxide. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: Analyzing the peak widths using the Scherrer equation yields a crystallite size of 21.8 ±  0.8 nm for the octahedral particles (for 0.3 M NaOH), and 9.5 ±  1.6 nm for the spherical Cu2O particles (0.02 M NaOH), whereas the respective particle sizes are ~1000 nm and ~305 nm (analyzed by TXM consistent with the results from dynamic light scattering investigation). Spatially resolved surface reactions. The currently used NEXAFS-TXM setup at the electron stor- age ring BESSY II does not allow in-situ measurements because of the vacuum setup. Therefore, we have opted for an exemplary gas-surface reaction that is non-reversible at room temperature in normal atmosphere to demonstrate the potential of our approach. Namely, we have used the known, highly spe- cific reaction of CuO towards H2S exposure. Even in oxygen depleted atmospheres30,31 the exothermic reaction32: ( ) 2 + → + , ( ) CuO H S CuS H O 2 2 2 causes fundamental changes in the electrical behavior of gas sensitive layers. At temperatures below 200 °C this conversion is irreversible which means that the reaction product is frozen and samples may be transferred to the NEXAFS-TXM setup without altering the composition of the surface or bulk. Especially since the CuO crystal is transformed to CuS the presence of surface adsorbed species on either CuO or CuS surfaces do not limit the validity of the NEXAFS-TXM results in the low tempera- ture regime. In this operational mode the CuO layer may be used to determine the H2S concentration by using a H2S induced percolation phase transition and measuring the time tPercol necessary to establish a conducting path via emerging CuS clusters. A second, competing reaction governs the CuO layer’s behavior for high temperatures33,34, + → + + , ( ) ( ) − − H S O SO H O e 2 3 2 2 3 3 ads 2 2 2 2 ( ) 3 i.e. the layer shows a typical reaction towards reducing gases. In order to confirm the proposed reac- tions at the employed gas-sensitive materials using the Cu-L2,3 edge and the O-K edge we have taken the respective spectra of the reference materials Cu, CuS, CuO, and Cu2O. This way we can demonstrate the formation of CuS even without accessing the S-K edge, which is unavailable at the currently used beam line. The reference spectra are depicted in Fig. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: The XRD patterns of the spherical (purple) and octahedral (dark cyan) nanoparticles after annealing are shown in Fig. 2h. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 4 www.nature.com/scientificreports/ Figure 3. NEXAFS-spectra at the O-K- and Cu-L2,3 edge of reference materials. Black: copper-(II)- sulfide; red: copper; blue: copper-(II)-oxide; green: copper-(I)-oxide. The signal at 932.2 eV in the CuS sample is attributed to the transition of Cu 2p3/2 to 3d states and shows a distinctly different behavior when compared to CuO. The O-K edge demonstrates the fundamental differences even more clearly since it does not show a clear absorption edge, which means that there is no oxygen present in the bulk of the reference material. Figure 3. NEXAFS-spectra at the O-K- and Cu-L2,3 edge of reference materials. Black: copper-(II)- sulfide; red: copper; blue: copper-(II)-oxide; green: copper-(I)-oxide. The signal at 932.2 eV in the CuS sample is attributed to the transition of Cu 2p3/2 to 3d states and shows a distinctly different behavior when compared to CuO. The O-K edge demonstrates the fundamental differences even more clearly since it does not show a clear absorption edge, which means that there is no oxygen present in the bulk of the reference material. After the annealing step, the original Cu2O signals disappear completely, while new peaks appear, which correspond to the {110}, {002}, and {111} planes of CuO (Fig. 2g and h beige-colored lines, JCPDS card no. 45–0937). This is further evidence that the Cu2O nanoparticles have completely transformed to CuO nanoparticles via heating. Analyzing the peak widths using the Scherrer equation yields a crystallite size of 21.8 ±  0.8 nm for the octahedral particles (for 0.3 M NaOH), and 9.5 ±  1.6 nm for the spherical Cu2O particles (0.02 M NaOH), whereas the respective particle sizes are ~1000 nm and ~305 nm (analyzed by TXM consistent with the results from dynamic light scattering investigation). After the annealing step, the original Cu2O signals disappear completely, while new peaks appear, which correspond to the {110}, {002}, and {111} planes of CuO (Fig. 2g and h beige-colored lines, JCPDS card no. 45–0937). This is further evidence that the Cu2O nanoparticles have completely transformed to CuO nanoparticles via heating. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: 4a,b shows the electrical response of the gas sensitive layer for both shapes and at tempera- tures of 450 °C and 250 °C for 1 ppm of H2S, respectively. At the high temperature of 450 °C, both par- ticle morphologies react in accordance with equation (3), i.e. the resistivity of the layer increases upon exposure to H2S. In the low temperature regime both shapes exhibit a steep decline in resistivity because H2S exposure causes the emergence of a continuous path of highly conducting CuS. Since the reaction towards H2S is unique and stable CuS structures are formed for temperatures at or below 250 °C the system used in this work offers an ideal test case to demonstrate the feasibility of directly monitoring chemical reactions of gas sensitive materials. The corresponding NEXAFS-TXM analysis is depicted in Fig. 4e,h. In the high temperature regime the morphology of the nanoparticles remains stable which is verified by NEXAFS-TXM analysis of the layer after exposure to H2S at 450 °C shown in Fig. 4e. The NEXAFS spectra in Fig. 4d reveal that the chemical composition of the bulk phase is not changed due to the H2S atmosphere in this temperature regime. All detected signals of the NEXAFS spectra at the O-K- and Cu-L2,3-edge can be assigned to the signals of copper-(II)-oxide and are identical to the spectra prior to gas exposure (compare Figs 2 and 3). This means that only the surface of the nanoparticles reacts during the gas sensing in the high temperature regime. g g g g p g In stark contrast to this, the morphology and the chemical composition of the particles change if they are exposed to H2S in the low temperature regime which is demonstrated by the NEXAFS-TXM in Fig. 4f,g. The detected signals at the O-K-edge of both kinds of particles are shown in Fig. 4h, which can be no longer assigned to CuO bonds. This means that the oxygen lattice of the particles is disturbed and differences have to be assigned to the different reaction kinetics of spherical and octahedral particle morphology. The reaction described in equation (2) is not limited to the surface but rather takes place in the bulk phase as well. Thus, the CuO is completely converted into copper sulfide in the low temperature regime. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: 3 and a detailed discussion thereof can be found in the ESI. In our experiments both CuO shapes have been exposed to low levels of hydrogen sulfide in the same temperature interval. To evaluate the gas sensitive behavior of the layers the electrical conductivity is determined using interdigitated electrode structures, which form part of a micro-machined hotplate device35. The combination of inkjet printing technology and the hotplate device allows for quickly testing Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 5 www.nature.com/scientificreports/ Figure 4. Sensing responses towards hydrogen sulfide in two temperature regimes. (a) At 450 °C the sensors show a chemisorption induced increase in electrical resistance. (b) At 250 °C stable CuS structures form resulting in a sharp resistance decline without recovery of the sensing signal. Using NEXAFS in combination with x-ray microscopy all surface processes during gas sensing have been investigated spatially resolved. For high temperature operation upon H2S exposure the CuO short-range order remains unaltered (c) and (d) and the NEXAFS spectra for both kind of particles does not change (e), while in the low temperature regime the structure of the nanoparticles changes dramatically (f,g). (h) In this temperature regime the CuS structures appear which is confirmed via spectroscopy of the Cu-L2,3 and O-K edge, respectively. Figure 4. Sensing responses towards hydrogen sulfide in two temperature regimes. (a) At 450 °C the sensors show a chemisorption induced increase in electrical resistance. (b) At 250 °C stable CuS structures form resulting in a sharp resistance decline without recovery of the sensing signal. Using NEXAFS in combination with x-ray microscopy all surface processes during gas sensing have been investigated spatially resolved. For high temperature operation upon H2S exposure the CuO short-range order remains unaltered (c) and (d) and the NEXAFS spectra for both kind of particles does not change (e), while in the low temperature regime the structure of the nanoparticles changes dramatically (f,g). (h) In this temperature regime the CuS structures appear which is confirmed via spectroscopy of the Cu-L2,3 and O-K edge, respectively. novel nano-sized gas sensitive materials. Using NEXAFS-TXM compatible mounts, the experimental conditions have been duplicated in order to monitor all steps of the gas-surface reaction. Figure 4 shows both, the gas sensitive response of the CuO layer and the corresponding NEXAFS-TXM analysis, relating the electrical response of the CuO nanoparticles to the chemical processes at the surface and the bulk phase. Fig. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For higher concentrations the formation of CuS structures becomes dominant, yet unstable, as the sensing resistance recovers after exposure. At 250 °C the CuS structures are stable on the surface and no recovery of the sensing signal is observed. (b) However, applying a heating protocol the sensor characteristics can be reproducibly recovered. The temperature is indicated in the middle panel by depicting the corresponding heater resistivity RH. (c) Measuring tPercol enables selectively determining the H2S concentration using the trace gas induced phase transition from semiconducting to conducting overall behavior. g ( ) g p y gif p At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For higher concentrations the formation of CuS structures becomes dominant, yet unstable, as the sensing resistance recovers after exposure. At 250 °C the CuS structures are stable on the surface and no recovery of the sensing signal is observed. (b) However, applying a heating protocol the sensor characteristics can be reproducibly recovered. The temperature is indicated in the middle panel by depicting the corresponding heater resistivity RH. (c) Measuring tPercol enables selectively determining the H2S concentration using the trace gas induced phase transition from semiconducting to conducting overall behavior. Because temperature apparently plays a pivotal role in the behavior of the CuO layers we have expanded the gas sensitive measurements to intermediate temperatures and further concentrations. The experimental results are depicted in Fig. 5. Lowering the temperature to 350 °C both reaction strengths are on the same order of magnitude which is highlighted by the shape dependent response to 1 ppm H2S. In order to further investigate the shape and size dependent gas reaction of CuO further experiments have been performed. The results are presented in the ESI. While sphere-based layers still react according to equation (2), i.e. by increasing the electrical resistivity upon gas exposure, octahedron-based layers undergo the percolation phase transition. Increasing the applied H2S concentration above 1 ppm causes the process described by equation (2) to become dominant for both shapes, i.e. the percolation phase transition always takes place. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: This is underlined by the detected signals at the Cu-L2,3-edge at 932.3 eV and 952.1 eV which can be assigned to CuS (compare Ref. 29 and Fig. 3). However, the detected signals in the region between 934 and 941 eV of the Cu-L3-edge are not in complete accordance with the copper-(II)-sulfide structure. It cannot be ruled out that a distinct amount of Cu2S is also formed under these experimental conditions. By comparing the spectra in Fig. 4h to the reference spectra of CuS in Fig. 3 and to the literature29 this is further emphasized. Therefore, measurements at the S-K-edge would have to be carried out which is not possible at the used TXM at the moment. Additionally, measurements at other x-ray microscopes at the sulfur-K-edge with similar spatial resolution allowing single particle analysis are currently not available. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 6 www.nature.com/scientificreports/ Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For higher concentrations the formation of CuS structures becomes dominant, yet unstable, as the sensing resistance recovers after exposure. At 250 °C the CuS structures are stable on the surface and no recovery of the sensing signal is observed. (b) However, applying a heating protocol the sensor characteristics can be reproducibly recovered. The temperature is indicated in the middle panel by depicting the corresponding heater resistivity RH. (c) Measuring tPercol enables selectively determining the H2S concentration using the trace gas induced phase transition from semiconducting to conducting overall behavior. Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. At 450°C the sensors show a chemisorption induced increase in electrical resistance as a function of the Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: The arrows in (b) indicate the location of peaks and photon energies used to characterize the different chemical species in (a). Figure 6. (a) TXM-micrographs of the spherical particles after printing and direct exposure to H2S at 200 °C (overlay 930.8 eV (red), 931.4 eV (green), 932.3 eV (blue)). The false color representation is used as a tool here in order to visualize the chemical inhomogeneity of the sample. Each color corresponds to a particular type of chemical bond. This information is extracted from the spectrum of each pixel of the image. (b) NEXAFS spectra of the O-K-edge and the Cu-L2,3-edge of the marked particles in (a) are represented in green, red and blue and are labeled 1, 2, and 3, respectively. For comparison, the spectrum of an exemplary octahedron particle under similar experimental conditions is depicted as well. The arrows in (b) indicate the location of peaks and photon energies used to characterize the different chemical species in (a). Imaging local surface reactions. Because the gas surface reactions crucially depend on the metal oxide material we also tested the as-synthesized Cu2O nanoparticles with respect to their gas sensitivity. Unfortunately, due to remaining PEG the sensing layer resistances composed of Cu2O are in excess of 108 Ω making the particles unsuitable for use as gas sensing material. In order to assess the influence of H2S exposure on Cu2O particles a NEXAFS sample was produced without using the inkjet printing system, i.e. without using PEG. Future work will entail tuning the rheological properties without using PEG thus allowing for an electrical read-out of inkjet printed Cu2O layers. To investigate the gas-surface reactions now, the as-synthesized particles are exposed to 5 ppm H2S in dry synthetic air for 2 hours at 200 °C. Afterwards, the chemical composition of the particles is examined by NEXAFS-TXM. Fig. 6a shows a resulting TXM-micrograph of the spherical particles, which is given in false color representa- tion to directly map the chemical inhomogeneity of this sample after the H2S exposure experiment. The spatially resolved spectroscopy approach allows for extracting information about the chemical bonds and oxidation state prevailing in each pixel of the image. The false colors are assigned subject to the marked peak position in Fig. 6b at the Cu-L2,3-edge. The red color in Fig. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: Nevertheless, when removing H2S at 350 °C the CuS structures recede and both the spherical as well as the octahedral layer return to p-type semiconductivity. Lowering the tem- perature to 250 °C, the conversion to conductive behavior becomes permanent, i.e. even after removing H2S from the test chamber the CuS structures remain stable as indicated by the stable, low resistivity which can be seen in Fig. 4b. Consequently, a temperature treatment might be used to reset the func- tional layers. This approach is demonstrated in Fig. 5b where the temperature of the gas sensitive layer is increased to 450 °C after the end of the gas exposure. This is indicated by the temperature dependent resistivity of the microheater RH in the center panel of the graph. By applying 450 °C to the layer for 30 min it returns to p-type semiconductivity thus resetting the original gas sensing conditions. Fig. 5c shows the behavior of the layers after the onset of the exposure to a concentration of 1 ppm, 5 ppm, and 10 ppm H2S, respectively, at 250 °C in greater detail. It demonstrates how the percolation time tPercol may be used to infer the H2S concentration. To characterize the layer’s behavior the time tPercol is defined as the time from the onset of gas exposure until the resistivity of the layer drops below the threshold value of 300 Ω. This novel approach may be used to determine the H2S concentration in a fundamentally new, highly specific way because only H2S causes the percolation phase transition. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 7 7 www.nature.com/scientificreports/ Figure 6. (a) TXM-micrographs of the spherical particles after printing and direct exposure to H2S at 200 °C (overlay 930.8 eV (red), 931.4 eV (green), 932.3 eV (blue)). The false color representation is used as a tool here in order to visualize the chemical inhomogeneity of the sample. Each color corresponds to a particular type of chemical bond. This information is extracted from the spectrum of each pixel of the image. (b) NEXAFS spectra of the O-K-edge and the Cu-L2,3-edge of the marked particles in (a) are represented in green, red and blue and are labeled 1, 2, and 3, respectively. For comparison, the spectrum of an exemplary octahedron particle under similar experimental conditions is depicted as well. Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: 6a is assigned to photon energy 930.8 eV, which means that at this energy a chemical bond of Cu2+ in an oxygen environment will show a maximum of contrast in x-ray absorption whereas the other copper components show a much smaller absorption. The blue color in Fig. 6a is assigned to photon energy 932.3 eV, which is the characteristic absorption energy of Cu2+ in a sulfide environment. The green color presents a transition state between the oxygen and sulfide environment of Cu2+. This cannot be detected in a conventional x-ray absorp- tion spectroscopy experiment with a spot size of a few microns. The NEXAFS-TXM method employed here features a spatial resolution on the order of 20 nm and demonstrates how NEXAFS-TXM helps uncovering the underlying surface processes. The respective NEXAFS spectra of the marked particles in Fig. 6a are shown in Fig. 6b. Additionally, a representative NEXAFS spectrum of an octahedron particle prepared under similar experimental conditions as the spherical particles is shown in Fig. 6b. The cor- responding TXM-micrograph is presented in ESI Figure S1.h p g g p p g The analysis of the NEXAFS spectra has utilized the O-K-edge in the energy range 525 eV–555 eV as well as at the Cu-L2,3-edge in the energy range 925 eV–970 eV. In this way information about the chemical state of both atomic species constituting the gas sensitive material can be obtained.h p g g The comparison of the spectra of the octahedral particles with literature data and the measured ref- erences are in good accordance for copper-(I)-oxide22,23,24,27. In detail: The recorded NEXAFS spectra at the O-K-edge show the typical pronounced absorption peak at 532.7 eV and minor features at higher photon energy for copper-(I)-oxide. The main absorption feature can be explained by the transition of an electron from the 1 s core level to the final states of p symmetry at the oxygen site. The minor features at higher photon energy are caused by the hybridization of the O 2p states with the Cu d and s states23. The recorded main signals of the octahedron particles at the Cu-L3-edge (2p3/2; around 933.8 eV) and the Cu-L2-edge (2p1/2; around 953.6 eV) are ascribed to the electron transition from the Cu p states to the unoccupied 3d level due to the unusual crystal structure of Cu2O22. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Results and Discussion Large-scale synthesis route for well-defined Cu2O nanoparticles.  The synthesis of Cu2O nano- particles relies on a precipitation reaction in alkaline media: p y 2 In contrast, the NEXAFS spectra at the O-K-edge of all marked spherical particles in Fig. 6a sho significant difference in comparison to the spectrum of the octahedral particles and to the Cu2O refere Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 8 www.nature.com/scientificreports/ For the marked particle 3, no distinct absorption signal is recorded, which leads to the assumption that the oxygen lattice structure of the blue area in Fig. 6a is more or less disturbed. The Cu-L2,3-edge spec- trum of particle 3 clearly shows that a copper sulfide structure is formed. The peak at 932.3 eV indicates the existence for Cu2+ in a sulfide environment, whereas the region between 934 to 940 eV does not precisely show the minima and maxima of the pure CuS structure (compare Fig. 3 and the Ref. 29). This means that there is a coexistence of CuS and Cu2S. Additionally, the shoulder around 930.8 eV marks the existence of Cu2+ in an environment with an electronegative anion, like oxygen27. Similar spectra can be obtained for all the blue areas of Fig. 6a for both absorption edges. The recorded spectra of particle 1 and 2 presents in our view transitions states in this solid-state transformation process, because the recorded signals for both particles cannot be fully assigned to the Cu2O, CuO or to the CuS structure.h g p y g These results clearly indicate that the as-synthesized spherical particles undergo a solid-state trans- formation into copper sulfide upon H2S exposure while octahedral particles do show a fundamentally different behavior. This is a direct observation of the profound influence of particle shape on gas-surface reactions. Possibly, the exposed crystallographic plane and the associated surface energies play a central role in this process36 and consequently influence on the gas sensitive behavior is expected. Additionally, this example illustrates the high potential for combining the spatial resolution of x-ray microscopy with the chemical information obtained from NEXAFS spectroscopy for functional materials development. Conclusion Because of the highly complex nature of gas-solid interactions and the current lack of possibilities to directly monitor surface reactions the behavior of solid-state gas sensors based on functional metal oxides is inadequately understood. Within this work the use of NEXAFS-TXM as a means to shed light onto chemisorption processes at metal oxide surface has been demonstrated using nanoparticles syn- thesized using a wet chemistry approach. Here, the combination of a bottom-up approach to produce nanoparticles with well-defined shape and form with the inkjet technology proved to be a promising method to directly apply basic research results in sensorial devices. The NEXAFS-TXM samples have been prepared copying all steps of the gas detection, i.e. after synthesis of Cu2O, after transformation to CuO, after exposure to H2S at low temperatures and after resetting the functional layer, i.e. after convert- ing CuS back to CuO. Using narrow bandwidth x-ray absorption spectroscopy it was possible to reveal the underlying changes to the material system, morphology and chemical composition. The reaction between CuO/Cu2O surfaces and H2S has provided a suitable sample system since the surface states of the different reactions may be retained for examination in the HZB NEXAFS-TXM. Consequently, the existing explanations for changes in electrical conductivity observed during the gas characterization could be substantiated using spectral analysis of the Cu-L2,3 and O-K edges. In particular, the NEXAFS-TXM analysis demonstrated that for temperatures below 250 °C CuO is converted to CuS whenever H2S is present. The emergence of highly conducting CuS structures as well as the chemical structure of CuO have been imaged spatially resolved revealing the sensing mechanism responsible for the selective detec- tion of H2S. This process is not limited to the particle surface but encompasses the whole particle thus completely destroying the CuO crystalline structure. Furthermore, the transformation is reversible in the absence of H2S and applying temperatures of 450 °C to the layer. This makes the reaction mechanism a suitable starting point for a new type of gas sensor based on the percolation effect induced by H2S. Future setups based on this approach should offer the possibility for in-situ measurements under real-world conditions by combining a gas measurement apparatus with a NEXAFS setup, thus enabling a spatially resolved imaging of the chemisorption processes at metal oxide surfaces under gas exposure. Methods D i l The glass ampule was opened directly before the measurement and some crystals of the reference materials have been spread over the prepared grids and directly transferred into the oxidation protective vacuum of the TXM measurement.i y p A chemical vapor deposition (CVD) process on a TEM grid with holey carbon support film was used to prepare the copper metal reference (film thickness ~30 nm). The sample was stored and placed on the sample holder under argon atmosphere. Only during the time for the transfer from one apparatus to another the sample to the TXM microscope was not protected by inert gas. Near edge X-ray absorption fine structure-transmission X-ray microscopy (NEXAFS-TXM). The NEXAFS-TXM spectra were recorded on the O-K-edge and the Cu-L2,3-edge with the HZB-TXM which is installed at the undulator beamline U41-FSGM at the electron storage ring BESSY II, Berlin, Germany. It provides a high spatial resolution close to 10 nm (half-pitch) and a spectral resolution up to E/Δ E ≈  104. Typical spectra are presented for each set of measurements. The TXM allows measure- ments to be taken at room or liquid nitrogen temperature in a vacuum of 1.3 ×  10−9 bar. The spectra were recorded at room temperature in transmission mode by taking a sequence of images over a range of photon energies covering the investigated absorption edges with a calculated E/Δ E >  5800 for the Cu-L2,3-edge and E/Δ E >  12000 for the O-K-edge. Note that the exit slit of the monochromator was set to 9 μ m for the Cu-L2,3-edge and 7 μ m for the O-K-edge resulting in the given calculated monochroma- ticity values. The exposure time for one image with 1340 ×  1300 pixels was 24 s for the Cu-L2,3-edge and 4 s for the O-K-edge to achieve a sufficient signal to noise ratio in the images. Taking an image stack with up to 226 images at different energies needs inherently about 45 to 120 min because of all necessary movements, exposure time, and camera read out time and image storage. The NEXAFS spectra were normalized since the photon flux varies as a function of photon energy (hν ) and time in the object field (x, y). Methods D i l The remaining protocol was the same as in the standard synthesis without NaOH. Inkjet printing. After washing the Cu2O particles are dispersed in pure ethanol and 60 s of ultrasound are applied prior to printing in order to obtain a homogeneous dispersion. 2 ml of the dispersion are transferred into the printer cartridges. The dispersions containing spherical particles were modified with 0.5 ml PEG 400 to improve printability.h The inkjet deposition is performed using a DIMATIX DMP 2831 printer system with 10 pl print heads. The nozzle is kept at room temperature and the substrate at 30 °C. The nozzle droplet firing voltage was set to 27 ±  3 V. The drop-to-drop distance (pitch) was set to 40 μ m. For gas sensor devices 6 stacked lay- ers were deposited. For NEXAFS measurement particles were deposited on a silicon nitride membrane. Sample preparation printer parameters have been identical for gas sensor devices and NEXAFS-TXM samples. Conversion of Cu2O to CuO. For the gas sensing experiments Cu2O particles were converted on-chip by the integrated heaters at a temperature of 350 °C for several hours prior to exposing the sensors to trace gases. This is well above the limit for stable Cu2O. gh 2 For the NEXAFS-TXM characterization the Cu2O particles were oxidized in dry ambient air in a quartz tube furnace (ATV PEO 603) by linearly ramping the temperature for 1 h to the destination tem- perature of 400 °C and holding it for 1 h before cooling down. XRD. X-ray diffraction measurements were operated in the 2θ  range from 20 to 80° on a Bruker D8-Advance X-ray diffractometer with Cu Kα 1 radiation (λ = 1.5406 Å). The radiation source was oper- ated at a voltage of 40 kV and a current of 40 mA. The step size of the measurements is 0.02° with an integration time of 3.5 seconds per step. Sample preparation of reference-samples for NEXAFS-TXM. The copper grids with carbon support film (200 meshes, Science Services, Munich, Germany) have been pretreated by 10 s of glow discharge. Reference materials for Cu2O (Sigma-Aldrich; product no. 566284; purity > 99.99%), CuO (Sigma-Aldrich, product no.203130; purity > 99.999%) and CuS (Sigma Aldrich, product no. 450820, purity > 99.99%) are commercially ordered. These high purity materials are delivered in a glass ampule, which is completely sealed under inert gas atmosphere. Methods D i l Dynamic light scattering. Particle sizes were determined by dynamic light scattering (DLS), using a He:Ne gas laser (λ  =  632.8 nm, 22.5 mW) and a goniometer (ALV/SP-86; from ALV Company, Langen, Germany) equipped with an index match bath. As detector two Perkin Elmer photomultipli- ers (SPCM-CD2696 Rev.G) were used and cross correlated by an ALV 5000/E correlator. The intensity auto-correlation function was transformed into the intermediate scattering function by using the Siegert relation. Diffusion coefficients were calculated by cumulant analysis and used to determine the hydro- dynamic radii via the Stokes–Einstein equation. The measurements were performed at an angle of 100°. Synthesis of Cu2O nanoparticles. Cu2O nanospheres with radii ranging from 150 nm to 1600 nm were prepared with high yield needed for inkjet printing. In the synthesis protocol Cu(NO3) 2•3H2O was dissolved in a 1000 ml three-necked flask in 1/3 of the total water (500 ml) and then kept stirring for about 30 min at 250 rpm. Meanwhile 5 g Polyvinylpyrrolidone (PVP, average MW= 55000) were dissolved in the remaining water. After complete dissolution the PVP solution was added dropwise to the Cu(NO3) solution and the reaction mixture was then stirred for another 2 hours. Then 453 μ l of hydrazine solution (35% N2H4 in water) was added to the reaction mixture drop-wisely. The color of the solution changed into orange immediately after the introduction of N2H4, which indicates the production of Cu2O nano- particles. The resulting solution was kept stirring for 30 min at room temperature and was then washed with H2O and EtOH several times by centrifugation and redispersion (Multifuge 3 SR, Heraeus: 900 g, 45 min, 21 °C). In a final step the Cu2O nanoparticles were redispersed in a small amount of EtOH and stowed away in the dark until further use. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 9 www.nature.com/scientificreports/ To further optimize the synthesis for higher yield this standard protocol was modified by the addition of NaOH. To achieve the wanted NaOH concentration in the reaction mixture water in the Cu(NO3) solution was reduced by a certain amount to be later on replaced by the same volume of a 2 mol/l NaOH stock solution. After drop-wise addition of the PVP solution the reaction mixture was stirred for 2 hours and then the NaOH stock solution volume added under stirring (250 rpm). The N2H4 was then added after 5 min further stirring. References Walden, P. et al. Micromachined hotplate platform for the investigation of ink-jet printed, functionalized metal oxid nanoparticles, J. Microelectromech. S. 24, 1384–1390 (2015). 35. Walden, P. et al. Micromachined hotplate platform for the investigation of ink-jet printed, functionalized metal oxide nanoparticles, J. Microelectromech. S. 24, 1384–1390 (2015). p 6. Jiang, H. B. et al. Anatase TiO2 crystals with exposed high-index facets. Angew. Chem. Int. Edit. 50, 3764 –3768 (2011). 36. Jiang, H. B. et al. Anatase TiO2 crystals with exposed high-in J g 2 y p g g ( ) 37. Hitchcock, A. P. (2015). aXis2000: Analysis of X-ray Images and Spectra, McMaster University, Hamilton , Canada. URL http:// unicorn.mcmaster.ca/aXis2000.html. g 2 y p g g 37. Hitchcock, A. P. (2015). aXis2000: Analysis of X-ray Images and Spectra, McMaster University, Hamilton , Canada. URL http:// unicorn.mcmaster.ca/aXis2000.html. Methods D i l The normalization was performed by dividing the intensity I(x, y, hν ) recorded on a single nanostructure by the intensity I0 (x +  Δ x, y +  Δ y, hν ) recorded in its sample free proximity at position (x +  Δ x, y +  Δ y). Both I(x, y, hν ) and I0(x, y, hν ) were recorded within the same image stack since bare regions in the vicinity of the nanostructures permit the measurement of I0.ht g y p 0 The image stacks were aligned and analyzed with the aXis2000 software37. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 10 www.nature.com/scientificreports/ Gas sensitive characterization. The apparatus used to control the temperature of the functional MOX layers as well as the composition of the gas matrix is described in detail in38. 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Surf. Interface Anal. 23, 171–179 (1995). 32. Galtayries, A. & Bonnelle, J.-P. XPS and ISS studies on the interaction of H2S with polycrystalline Cu, Cu2O, and CuO surfaces. Surf. Interface Anal. 23, 171–179 (1995). f f 33. Zhang, F. et al. CuO nanosheets for sensitive and selective determination of H2S with high recovery ability. J. Phys. Chem. C 19214–19219 (2010). f f 33. Zhang, F. et al. CuO nanosheets for sensitive and selective determination of H2S with high recovery ability. J. Phys. Chem. C 19214–19219 (2010). ( ) 34. Steinhauer, S. et al. Gas sensing properties of novel CuO nanowire devices. Sensor. Actuat. B–Chem. 187, 50–57 (2013). g p p ( ) 35. Author Contributions K.H. performed the NEXAFS-TXM measurements and the related analysis. P.G. supported K.H. during the NEXAFS-TXM measurement. J.H. and A.H. performed the particle synthesis and characterization. A.H. performed the dynamic light scattering experiments. J.K. produced the gas sensitive MEMS devices and performed the gas sensitive characterisation. E.B. and Y.L. supervised the particle synthesis and characterization. K.H., J.K. and S.P. designed the experiments and analysed the data. P.G., E.B., Y.L. edited the manuscript. K.H. and S.P. wrote the manuscript. Acknowledgementst A.H. thanks “Stiftung Industrieforschung” for financial support. This work has been partially funded by the German Federal ministry for Education and Science (BMBF) under grant number 16SV5943 (SensOdor). We thank HZB for the allocation of neutron/synchrotron radiation beamtime. The article Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 11 www.nature.com/scientificreports/ processing charge was funded by the German Research Foundation (DFG) and the Albert Ludwigs University Freiburg in the funding programme Open Access Publishing. processing charge was funded by the German Research Foundation (DFG) and the Albert Ludwigs University Freiburg in the funding programme Open Access Publishing. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Henzler, K. et al. Investigation of reactions between trace gases and functional CuO nanospheres and octahedrons using NEXAFS - TXM imaging. Sci. Rep. 5, 17729; doi: 10.1038/ srep17729 (2015). How to cite this article: Henzler, K. et al. Investigation of reactions between trace gases and functional CuO nanospheres and octahedrons using NEXAFS - TXM imaging. Sci. Rep. 5, 17729; doi: 10.1038/ srep17729 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com- mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 12
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Measuring ice- and liquid-water properties in mixed-phase cloud layers at the Leipzig Cloudnet station
Atmospheric chemistry and physics
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Measuring ice- and liquid-water properties in mixed-phase clou layers at the Leipzig Cloudnet station Johannes Bühl, Patric Seifert, Alexander Myagkov, and Albert Ansmann Leibniz Institute for Tropospheric Research (TROPOS), Permoserstr. 15, 04318 Leipzig, Germany Correspondence to: Johannes Bühl (buehl@tropos.de) Received: 12 January 2016 – Published in Atmos. Chem. Phys. Discuss.: 27 January 2016 Revised: 23 June 2016 – Accepted: 18 July 2016 – Published: 26 August 2016 Received: 12 January 2016 – Published in Atmos. Chem. Phys. Discuss.: 27 January 2016 Revised: 23 June 2016 – Accepted: 18 July 2016 – Published: 26 August 2016 Abstract. An analysis of the Cloudnet data set collected at Leipzig, Germany, with special focus on mixed-phase lay- ered clouds is presented. We derive liquid- and ice-water content together with vertical motions of ice particles falling through cloud base. The ice mass flux is calculated by combining measurements of ice-water content and particle Doppler velocity. The efficiency of heterogeneous ice forma- tion and its impact on cloud lifetime is estimated for different cloud-top temperatures by relating the ice mass flux and the liquid-water content at cloud top. Cloud radar measurements of polarization and Doppler velocity indicate that ice crys- tals formed in mixed-phase cloud layers with a geometrical thickness of less than 350 m are mostly pristine when they fall out of the cloud. spectrum of vertical air motions in detail. Laboratory mea- surements have already delivered a lot of useful information, e.g., about the ice nucleation efficiency of aerosol particles with temperature (Murray et al., 2012; DeMott et al., 2015). Observations of the process of ice nucleation in nature, how- ever, are limited. By means of active remote sensing, quan- tities that are directly connected with ice nucleation events, e.g., the ice-water content (IWC) of ice crystals from cloud layers, can be measured (D. Zhang et al., 2010; Bühl et al., 2013). In the European Union research project BACCHUS (Impact of Biogenic vs. Anthropogenic emissions on Clouds and Climate: towards a Holistic UnderStanding) the ice nu- cleating properties of aerosols are investigated. It is one ma- jor task of this project to study the life cycle of aerosols from its source through the clouds by means of aircraft, in situ, and remote-sensing observations. Combined remote- sensing observations in the framework of Cloudnet (Illing- worth et al., 2007) constitute one main pillar of the BAC- CHUS project. Beyond other things, Cloudnet provides a tar- get classification scheme for identifying the physical phase of hydrometeors. Measuring ice- and liquid-water properties in mixed-phase clou layers at the Leipzig Cloudnet station Johannes Bühl, Patric Seifert, Alexander Myagkov, and Albert Ansmann Leibniz Institute for Tropospheric Research (TROPOS), Permoserstr. 15, 04318 Leipzig, Germany A similar multi-sensor approach is used by the ARM (Atmospheric Radiation Measurement) program (Shupe, 2007), which recently performed several measure- ment campaigns in the Arctic in order to study the interaction between aerosols and clouds (Zhang et al., 2014). 1 Introduction Understanding the process of heterogeneous ice formation is currently one of the major topics in weather and climate re- search (Cantrell and Heymsfield, 2005; Hoose et al., 2008). Heterogeneous ice formation drives the generation of rain (Mülmenstädt et al., 2015) and impacts cloud stability (Mor- rison et al., 2005) and atmospheric radiative transfer (Sun and Shine, 1994). It is therefore a crucial component in the hydrological cycle in the Earth’s atmosphere. The interaction between aerosol and clouds in general involves very complex processes. Vertical motions keep mixed-phase clouds alive by activating aerosol particles to cloud droplets, while at the same time ice crystals nucleate and remove water from the cloud. To understand these complex interactions it is neces- sary to know all influences, process aspects, involved aerosol particles, cloud droplets, ice crystal ensembles, as well as the Since 2011, the Leipzig Aerosol and Cloud Remote Obser- vations System (LACROS) (Wandinger, 2012) has belonged to the Cloudnet consortium. In this article, remote measure- ments of LACROS analyzed with Cloudnet algorithms are used to describe ice formation processes under ambient con- ditions. Such remote-sensing measurements fill a critical gap in the study of mixed-phase processes because they deliver the information about the entire cloud column from the base Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ doi:10.5194/acp-16-10609-2016 © Author(s) 2016. CC Attribution 3.0 License. Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ doi:10.5194/acp-16-10609-2016 © Author(s) 2016. CC Attribution 3.0 License. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds For a residence time of 20 min within a mixed-phase cloud layer, particles could still be considered pristine. Additionally, Yano and Phillips (2010) found that within this time, secondary processes like riming do not influence heterogeneous ice formation significantly. According to Fukuta and Takahashi (1999), a residence time of 20 min corresponds to a geometrical thickness of a mixed- phase cloud-top layer of 350 m. Hence, for the present study only clouds with a geometrical thickness of below 350 m are selected to avoid altering of the ice crystals by riming, splin- tering, or aggregation processes. g g y y Shallow mixed-phase cloud layers like altocumulus, al- tostratus, or stratocumulus have been used before by dif- ferent groups as atmospheric laboratories in order to study aerosol–cloud-dynamics interaction under ambient condi- tions (Fleishauer et al., 2002; Y. Zhang et al., 2010; D. Zhang et al., 2010; Bühl et al., 2013; Schmidt et al., 2015; Seifert et al., 2015). These cloud types are especially well suited for process studies purposes because they show narrow con- straints on basic environmental variables like temperature, pressure, humidity, and the number of potentially involved microphysical processes (Tao and Moncrieff, 2009). The well-defined base and top of shallow cloud layers are opti- mal to study aerosol effects on ice nucleation as well as the impact of up- and downdraft on cloud ice production. As an additional benefit, these shallow cloud layers can easily be penetrated by lidar and cloud radar systems, which is not pos- sible for deep convective clouds due to massive signal atten- uation and strong turbulence within their cores. For climate research these shallow cloud layers are important due to their hard-to-predict impact on Earth’s radiative budget. From the meteorological point of view, the understanding of ice forma- tion processes in deep convective mixed-phase clouds may be more important. However, such clouds are difficult to ob- serve and may not allow to resolve the basic ice processes and aerosol- and dynamics-related aspects of ice formation. Both questions can be answered only by studying the process of ice formation itself in the atmosphere. p All of the statistical analysis of ice formation in our for- mer studies (Kanitz et al., 2011; Bühl et al., 2013; Schmidt et al., 2015; Seifert et al., 2015) has been done manually. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10610 (2013), in which properties of ice and cloud water have been analyzed separately and independently. Secondly, statistics about Doppler velocity (terminal fall velocity of the ice crys- tals plus vertical velocity of air) and radar depolarization of the ice crystals are compiled in order to directly assess ice crystal sedimentation rates and to derive basic information about the shape of particles at the same time (not only quan- titative knowledge about the particles themselves is gathered, but also the usability of cloud layers as atmospheric “lab- oratories” is characterized). Only if ice crystals are pristine (i.e., do not show signs of riming growth, aggregation, or secondary ice formation) is there a direct link between the properties of the ice (e.g., size, shape, and mass) and their formation process within the mixed-phase cloud-top layer. These measurements of ice particle properties are compared with laboratory studies of Fukuta and Takahashi (1999) in order to assess the quality of the Cloudnet measurements. Based on our data set, the IWC produced by particles falling from cloud layers is derived and compared with the avail- able liquid water within the cloud-top layer. Together with the quality-assured measurements of fall velocity (Doppler velocity averaged over a complete cloud case), a direct con- nection between the liquid water in the cloud-top layer and the resulting ice mass flux is established, which can be re- garded as a quantitative measure of heterogeneous ice forma- tion in the atmosphere. With this approach, the impact of ice formation on cloud lifetime is also estimated for the temper- ature regime between −35 and 0 ◦C. Fukuta and Takahashi (1999) also provide comprehensive laboratory measurements of the growth of ice crystals. They found different distinct features in the resulting shape of ice crystals for different growth times and calculated corresponding residence times within a cloud layer, taking into account increasing fall speed with increasing particle size. For a residence time of 20 min within a mixed-phase cloud layer, particles could still be considered pristine. Additionally, Yano and Phillips (2010) found that within this time, secondary processes like riming do not influence heterogeneous ice formation significantly. According to Fukuta and Takahashi (1999), a residence time of 20 min corresponds to a geometrical thickness of a mixed- phase cloud-top layer of 350 m. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Hence, for the present study only clouds with a geometrical thickness of below 350 m are selected to avoid altering of the ice crystals by riming, splin- tering, or aggregation processes. h i d f ll i i h to the top, which is not possible with aircraft measurements alone. In this way, the temperature level at which ice nucle- ation takes place can be derived and at the same time the resulting ice water falling from the layer can be analyzed. (2013), in which properties of ice and cloud water have been analyzed separately and independently. Secondly, statistics about Doppler velocity (terminal fall velocity of the ice crys- tals plus vertical velocity of air) and radar depolarization of the ice crystals are compiled in order to directly assess ice crystal sedimentation rates and to derive basic information about the shape of particles at the same time (not only quan- titative knowledge about the particles themselves is gathered, but also the usability of cloud layers as atmospheric “lab- oratories” is characterized). Only if ice crystals are pristine (i.e., do not show signs of riming growth, aggregation, or secondary ice formation) is there a direct link between the properties of the ice (e.g., size, shape, and mass) and their formation process within the mixed-phase cloud-top layer. These measurements of ice particle properties are compared with laboratory studies of Fukuta and Takahashi (1999) in order to assess the quality of the Cloudnet measurements. Based on our data set, the IWC produced by particles falling from cloud layers is derived and compared with the avail- able liquid water within the cloud-top layer. Together with the quality-assured measurements of fall velocity (Doppler velocity averaged over a complete cloud case), a direct con- nection between the liquid water in the cloud-top layer and the resulting ice mass flux is established, which can be re- garded as a quantitative measure of heterogeneous ice forma- tion in the atmosphere. With this approach, the impact of ice formation on cloud lifetime is also estimated for the temper- ature regime between −35 and 0 ◦C. Fukuta and Takahashi (1999) also provide comprehensive laboratory measurements of the growth of ice crystals. They found different distinct features in the resulting shape of ice crystals for different growth times and calculated corresponding residence times within a cloud layer, taking into account increasing fall speed with increasing particle size. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 3 Automated selection and classification of cloud layers in a Cloudnet data set The goal of this study is to obtain a data set of mixed-phase cloud layers that fulfill certain quality criteria such as tem- poral and spatial homogeneity. As stated above, the contin- uous, homogenized Cloudnet-processed data set is used as a basis for the approach. The automated Cloudnet algorithm reduces data from a set of remote-sensing instruments on a common grid that has a temporal resolution of 30 s and a height resolution of 30.2 m (similar to the one of the cloud radar). In a further step, the physical state of the atmosphere in all height bins is classified into different categories, e.g., containing cloud droplets, ice particles, or both. Other defi- nitions concerning aerosol are also present but do not play a role in the context of this work. A detailed description of the target categorization scheme of Cloudnet is given in Illing- worth et al. (2007). Basically, liquid-water droplets are de- tected by a threshold in lidar signal followed by a characteris- tic decrease of the latter above liquid cloud base. Ice particles are in general defined to be present if the radar-observed ver- tical velocity of the targets indicates falling particles and the dew-point temperature within a range gate is below 0 ◦C. If, in addition, the analysis of the lidar signal of the considered pixel meets the criteria for the presence of liquid droplets, the pixel is categorized as mixed phase. The height of the melt- ing layer is derived either from the meteorological data (dew- For the statistical analysis, a cloud must pass certain qual- ity criteria: a coherent cloud structure must be found for more than 15 min, no seeding of particles from higher-level clouds must be present, and for at least 85 % of the cloud’s occur- rence time a liquid- or mixed-phase cloud top must be de- tected (height range where water vapor saturation over liquid water is close to 1; see Fig. 1). The properties of the detected clouds (e.g., CTH, geometrical cloud thickness δh, standard deviation of cloud-top height σCTH, cloud-top temperature (CTT), radar reflectivity factor (Z), IWC, liquid-water con- tent (LWC), LDR, lidar attenuated backscatter coefficient (β), and lidar volume linear depolarization ratio) are stored for further analysis. See Fig. 1 for an overview where the different properties are derived for one cloud case. 2 Data set point temperature is 0 ◦C) or from measurements of radar lin- ear depolarization ratio (LDR) larger than −15 dB. Thus, the decision between liquid-only, mixed-phase, or ice-only cloud layers is made primarily based on the modeled temperature and changes in the vertical-velocity profile. However, on the basis of temperature only, there is no way to unambiguously decide between drizzle and/or falling ice crystals below 0 ◦C. The data analyzed within the frame of this work have been collected with LACROS (Wandinger, 2012) at TRO- POS Leipzig, Germany (51.3◦N, 12.4◦E), between 2011 and 2015. The time coverage of Cloudnet observations at Leipzig is about 85 %. Instruments relevant for the present work are the PollyXT Raman/depolarization lidar (Althausen et al., 2009; Engelmann et al., 2016), the Jenoptik ceilometer CHM15kx, the MIRA-35 cloud radar (Görsdorf et al., 2015), and the HATPRO (Humidity and Temperature Profiler) mi- crowave radiometer (Rose et al., 2005). The measurements of these instruments are analyzed by the Cloudnet algorithms (Illingworth et al., 2007) to derive microphysical properties of hydrometeors on a continuous basis. Additionally, model input of environmental variables like temperature and hu- midity is used. For the Cloudnet data set of Leipzig, fore- cast data of COSMO-EU (Consortium for Small-scale Mod- eling – Europe) were used from 2011 to May 2014. Since June 2014, forecast data of the integrated forecast system of the ECMWF (European Centre for Medium-Range Weather Forecasts) have been used. In the rare cases when these data are not available, COSMO-EU is used as a fall-back option. The resulting Cloudnet data set is the basis for the following analysis of cloud layers over Leipzig presented in the follow- ing. g y The target classification of Cloudnet only takes into ac- count single range gates. Taking into account measurements of a complete cloud case facilitates the disambiguation be- tween a mixed-phase and a liquid-only case. Hence, for this work, an automated algorithm has been developed that runs on this basic target classification product of Cloudnet. Sin- gle 30 s profiles are analyzed to search for liquid water at T < 0 ◦C. If liquid water is found, the base and top height of the liquid layer is stored and the height range below this liquid-water bin is searched for ice. If ice is found below, the height of transition between liquid and ice is also stored. 2 Data set This procedure is done for all profiles of the data set. Af- terwards neighboring cloud profiles are merged to coherent cloud layers if they lie within 300 s of temporal and 350 m of vertical distance. The 300 s horizontal separation is derived from experience. Increasing the value increases the homo- geneity of the cloud cases but reduces the total number of cases at the same time. The 350 m cloud thickness is mo- tivated by Fukuta and Takahashi (1999), as it probably ex- cludes secondary ice formation processes and particle rim- ing. Cloud-top height (CTH) of the cloud layers is specified to be larger than 1500 m in order to exclude clouds influenced by the boundary layer. D. Zhang et al. (2010) went with a similar approach. A set of connected profiles constitutes a cloud layer for which we assume that the cloud properties are similar. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10611 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Such an approach is time consuming and cloud selection criteria can not be applied on a fully objective basis. Until now, some Cloudnet stations have been running continuously for more than 10 years (e.g., Chilbolton and Lindenberg), providing a wealth of measurement values each day. There- fore, the analysis of clouds within such a data set can only be effective with an automated algorithm. For the present work, a method has been developed to automatically evaluate measurements from the Cloudnet data set collected between 2011 and 2015 at TROPOS. A modified cloud-classification scheme from Bühl et al. (2013) is used to automatically dis- criminate liquid- and mixed-phase cloud layers. The method is generally applicable to any Cloudnet data set of arbitrary size. Hence, the method can be used to quickly analyze any data set with the same objective criteria, thus harmonizing Cloudnet measurements from all over the world. The paper is structured as follows. Section 2 gives a short overview about the data set used in the context of this work. In Sect. 3 the methodology to analyze the data set is pre- sented. At the beginning of Sect. 4 the ice-detection capabil- ity of different cloud radar systems is analyzed. After that, quantitative statistics of ice and liquid water within mixed- phase cloud layers are derived. The focus of the present work is twofold. Firstly, quanti- tative statistics about ice and water mass in shallow mixed- phase cloud layers are derived from the Cloudnet data set, taking into account values of each Cloudnet profile individu- ally. This constitutes a step forward compared to Bühl et al. www.atmos-chem-phys.net/16/10609/2016/ Atmos. Chem. Phys., 16, 10609–10620, 2016 3 Automated selection and classification of cloud layers in a Cloudnet data set The pic- ture also shows that some measurement values are taken only from a height level 60 m below the mixed-phase cloud base. At this point, cloud droplets should be absent and ice parti- cles should still be largely unaltered by evaporation or aggre- gation processes. Hence their size and shape should only be related to processes that take place within the mixed-phase cloud-top layer. In the context of this work, all measurement values derived in this way are marked with the index CB Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ (2014), state of water saturation is indicated for the different parts of the clouds. Figure 1. Schematic representation of the different measurement Figure 1. Schematic representation of the different measurement and averaging schemes in a mixed-phase cloud layer. Water droplets within the mixed-phase top layer are detected by lidar. The ice pre- cipitation below is mainly detected by the cloud radar. IWC and LWC are provided by Cloudnet and are a function of height (h) and time (t). IWP and LWP are the column integrated values of LWC and IWC over the liquid cloud top and the ice precipitation respectively. IWCCB represents the mean of all IWC values mea- sured about 60 m below current cloud-base height (CBH). Follow- ing Zhang et al. (2014), state of water saturation is indicated for the different parts of the clouds. mixed Figure 2. Flowchart of the mixed-phase cloud discrimination method from Bühl et al. (2013) as it is applied in the current work. Most clouds are successfully analyzed with combined lidar/radar. (for “cloud base”). In addition, by this definition of coher- ent cloud layers the average vertical velocities can be used as an estimate of the particle fall velocity. In cloud layers, the size of turbulent eddies is restricted to the layer depth. The maximum scale length of free turbulence within the layer is hence approximately 2 times its geometrical thick- ness (Moin, 2009). For cloud layers of 350 m of vertical geo- metrical extent, such small-scale fluctuations cancel out over the course of 15 min or longer. The average Doppler velocity of falling particles measured over the timespan of cloud layer occurrence is therefore free of influences of small-scale tur- bulence. However, large-scale vertical air motions equal to or longer than 15 min still influence the measurements. used (Engelmann et al., 2016), if available. In Fig. 3 three example cases with different CTTs from different dates are shown together. Cloud radar measurements of Z, LDR, and v are shown together with the attenuated backscatter coeffi- cient from the lidar. The CTTs of the three cases are chosen in such a way that distinct differences in LDR measurements are visible between the cases. As an example for cloud detec- tion/selection, all clouds with δh < 350 m and σCTH < 150 m detected on 2 October 2012 at Leipzig are marked in Fig. 4. www.atmos-chem-phys.net/16/10609/2016/ The CTT statistics of all selected and classified cloud layers with these selection criteria (δh < 350 m and σCTH < 150 m) are shown in Fig. 5a and b. It is visible that no mixed-phase clouds are detected below −40 ◦C. The result of this auto- mated analysis is within the statistical accuracy of 15 % of the results of the study of Bühl et al. (2013), which was done on the basis on manual cloud selection. After cloud identification, the cloud-classification scheme from Bühl et al. (2013) is used to discriminate between liquid- and mixed-phase cloud layers (see Fig. 2). This clas- sification method reduces the dependence on model temper- ature by taking into account information from all cloud pro- files to make a decision between the microphysical states “liquid” or “mixed phase”. Depolarization measurements from lidar and radar are used to directly identify ice crys- tals falling from a cloud layer. Mixed-phase clouds close to 0 ◦C also often show a melting layer, which is the most un- ambiguous sign of the presence of ice particles (Di Giro- lamo et al., 2012). High LDR values are also produced by the needle-like ice crystals prevailing for clouds with a CTT between −8 and −2 ◦C (Fukuta and Takahashi, 1999). Such clear LDR signals make the decision between ice and liq- uid water fortunately very easy close to the 0 ◦C level, where model temperature in most cases is not accurate enough and the increase in particle fall speed due to melting is not signif- icant. For low values of Z (typically below −30 dBZ) and no detection of a melting layer, the depolarized signal is usually too weak to be detected by the cross-polarized channel of the MIRA-35 cloud radar. In this case, measurements of volume linear depolarization ratio from a collocated PollyXT lidar is www.atmos-chem-phys.net/16/10609/2016/ Atmos. Chem. Phys., 16, 10609–10620, 2016 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10612 ing ice- and liquid-water properties in mixed-phase clouds MIRA-35 Cloud Radar PollyXT Lidar Cloud layer detection Melting layer present? LDR > -30 dB at T < 0 °C Terminal velocity < 0.8 m s ¹ ⁻ Lidar volume depol. > 0.2 liquid mixed NO YES YES YES YES YES NO NO NO / N.A. Virga present? NO Cloudnet Data Synthesis Figure 2. Flowchart of the mixed-phase cloud discrimination method from Bühl et al. (2013) as it is applied in the current work. Most clouds are successfully analyzed with combined lidar/radar. 10612 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Figure 1. Schematic representation of the different measurement and averaging schemes in a mixed-phase cloud layer. Water droplets within the mixed-phase top layer are detected by lidar. The ice pre- cipitation below is mainly detected by the cloud radar. IWC and LWC are provided by Cloudnet and are a function of height (h) and time (t). IWP and LWP are the column integrated values of LWC and IWC over the liquid cloud top and the ice precipitation respectively. IWCCB represents the mean of all IWC values mea- sured about 60 m below current cloud-base height (CBH). Follow- ing Zhang et al. (2014), state of water saturation is indicated for the different parts of the clouds. MIRA-35 Cloud Radar PollyXT Lidar Cloud layer detection Melting layer present? LDR > -30 dB at T < 0 °C Terminal velocity < 0.8 m s ¹ ⁻ Lidar volume depol. > 0.2 liquid mixed NO YES YES YES YES YES NO NO NO / N.A. Virga present? NO Cloudnet Data Synthesis Figure 2. Flowchart of the mixed-phase cloud discrimination Figure 1. Schematic representation of the different measurement and averaging schemes in a mixed-phase cloud layer. Water droplets within the mixed-phase top layer are detected by lidar. The ice pre- cipitation below is mainly detected by the cloud radar. IWC and LWC are provided by Cloudnet and are a function of height (h) and time (t). IWP and LWP are the column integrated values of LWC and IWC over the liquid cloud top and the ice precipitation respectively. IWCCB represents the mean of all IWC values mea- sured about 60 m below current cloud-base height (CBH). Follow- ing Zhang et al. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10613 Figure 3. Three example case studies of mixed-phase clouds identified with the automated algorithm described in Sect. 3. Figure 3. Three example case studies of mixed-phase clouds identified with the automated algorithm ree example case studies of mixed-phase clouds identified with the automated algorithm described in Sect. 3. Figure 4. Example of automated detection of mixed-phase cloud layers on the basis of the Cloudnet target classification scheme for 2 October 2012. Clouds are marked due to the selection criteria ex- plained in the text. Blue squares mark liquid-only layers and red squares mark mixed-phase layers. The colors are only for a very basic visualization of the layer detection. The decision between mixed-phase and liquid clouds in the following analysis is more complex and described in the text. restrictions to certain scenarios or they do not take into ac- count any temperature dependence of the ice properties. Uncertainties in the measurements of Z add to these er- rors. Amongst these, for the quantitative understanding of ice formation in the atmosphere, knowledge about the ac- curacy and – especially – about the signal detection thresh- old of the cloud radar is critical. In the case of ground-based radar, different factors can affect the measured values of Z, e.g., unknown attenuation in rain and uncertainties in radar calibration. Attenuation induced by water vapor and liquid cloud layers is corrected in Cloudnet. Additionally, attenu- ation is avoided by excluding clouds from the analysis that are measured above other clouds or rain. The LACROS cloud radar is calibrated by the manufacturer with the method de- scribed in Görsdorf et al. (2015). The calibration is estimated to be accurate to 3 dB, resulting in an additional bias in the IWC retrieval of about 35 % (for the range between −60 and 0 dBZ and −40 to 0 ◦C), making them an estimation within the order of magnitude. Figure 4. Example of automated detection of mixed-phase cloud layers on the basis of the Cloudnet target classification scheme for 2 October 2012. Clouds are marked due to the selection criteria ex- plained in the text. Blue squares mark liquid-only layers and red squares mark mixed-phase layers. The colors are only for a very basic visualization of the layer detection. The decision between mixed-phase and liquid clouds in the following analysis is more complex and described in the text. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds The starting point for the characterization of the IWC data set is Fig. 6. In this figure, the signal-to-noise ratio (SNR) detected within cloud virgae (streams of ice particles falling from cloud top in which water is close to saturation over ice; see Fig. 1) is depicted together with the detected aver- age LDR (color scale). The LACROS cloud radar can detect between the methods is about a factor of 2 for the temper- ature interval −30 to −15 ◦C and about a factor of 5 in the interval −15 to 0 ◦C. Other methods to derive the IWC from continuous remote-sensing observations, as they are for in- stance summarized in Shupe et al. (2008), suffer from their Atmos. Chem. Phys., 16, 10609–10620, 2016 4.1 Ice mass retrieval and detection thresholds A quantitative retrieval of ice mass is done by Cloudnet via the method of Hogan et al. (2006). IWC values are ob- tained for each range bin with a simple empirical function depending on Z and the ambient temperature. The uncer- tainty of the method is estimated by Hogan et al. (2006) to be (+50/−30) % below a temperature of −10 ◦C and (+100/−50) % above. A possible bias of (+15/−10) % is estimated by Hogan et al. (2006). Comparison between the retrieval of Hogan et al. (2006) and three other Z−T param- eterizations from Protat et al. (2007) shows that the spread Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ Measuring ice- and liquid-water properties in mixed-phase clouds 4.2 Particle Doppler velocity and radar depolarization of pristine ice crystals In contrast to the extensive properties ZCB and IWCCB, the measurements of the cloud radar can also be used to de- rive the intensive properties of the ice crystals (e.g., vCB and LDR). The latter are connected to size, shape, and orienta- tion of the ice particles. Values of LDR and vCB averaged for each cloud case are shown in Fig. 8c and d. Note that LDR is dependent on both particle shape and particle orientation, so this information is not unambiguous (Reinking et al., 1997). However, if particles are oriented, high LDR values indicate prolate (column-shaped) particles and low values point to- wards more oblate particles like dendrites. For randomly ori- ented aspherical particles, LDR is always elevated. In this way, LDR gives only basic information about particle shape, but LDR has the advantage that it can be derived easily to- gether with vCB values with a vertical-pointing radar. Zthr = −10 ×  2log(50002/r2)  −45dBZ (1) (1) due to the quadratic decrease of received radiation with range. The corresponding thresholds of IWC (IWCthr) for different radar systems are drawn within the plots. Please note that the ice detection threshold is dependent on not only the radar signal threshold but also temperature, according to the retrieval of Hogan et al. (2006). For spaceborne sys- tems Zthr is nearly constant for the complete troposphere. The measurement distance of about 400–800 km leads to a range-induced signal variation of maximum 5 % between 0 and 12 km height. For ground-based systems, however, the detection threshold varies significantly for different heights. This phenomenon is depicted in Fig. 7c, where mean ZCB is plotted against CTH instead of CTT. The height-dependent detection threshold of the LACROS cloud radar is shown. The single values (30 s integration time and 30 m height resolution) of LDR and vCB from all cases are shown in Fig. 8a and b. The values are taken from the virgae where the target classification of Cloudnet states “ice only” (red- zone in Fig. 1). These representations already show inter- esting features. In Fig. 7a, for example, it is shown that at temperatures above −10 ◦C the average value of ZCB is of- ten below −30 dBZ. The depolarization measurements show a clear feature of elevated LDR values in this temperature range, pointing towards the presence of highly prolate and oriented ice particles. The vertical-velocity measurements in Fig. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10614 Figure 6. The 90 % percentile of cloud radar SNR is shown for each cloud case together with mean detected LDR. For ±2.5 ◦C intervals mean values (white squares) and standard deviation (black bars) are given. Figure 5. Distribution of cloud-top temperature for all pure liq- uid (a) and mixed-phase (b) cloud layers detected between 2011 and 2015 over Leipzig. Figure 5. Distribution of cloud-top temperature for all pure liq- uid (a) and mixed-phase (b) cloud layers detected between 2011 and 2015 over Leipzig. Figure 6. The 90 % percentile of cloud radar SNR is shown for each cloud case together with mean detected LDR. For ±2.5 ◦C intervals mean values (white squares) and standard deviation (black bars) are given. a signal down to a SNR of −23 dB. From Fig. 6 it becomes obvious that particle detection at higher temperatures above −10 ◦C are often close to the detection limit. In this temper- ature regime, the detection of some ice below cloud bases might be missed and clouds could be erroneously be classi- fied as liquid clouds. In contrast, ice detection seems to be quite reliable below −10 ◦C, where all cases have a mean SNR well above the detection threshold. It is also visible from the figure that LDR values can only be detected if a certain SNR threshold is reached. olds within the troposphere of −27 dBZ and −33 dBZ re- spectively. Hence, the CloudSat and EarthCare satellites are both able to detect most of the ice formation in clouds with CTT < −10 ◦C. At temperatures warmer than this, probably 90 % of the ice signals below the cloud layers will be missed (see Fig. 7a). Figure 7a depicts all measurements of ZCB sorted by CTT. In Fig. 7b the values of ZCB are shown averaged for individ- ual cloud cases. The equivalent values of IWCCB are shown in Fig. 7c. The LACROS MIRA-35 cloud radar has a de- tection threshold of Zthr = −45 dBZ at a range of 5000.0 m (Görsdorf et al., 2015). For other ranges r we hence find a threshold of J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10615 Figure 7. (a) All values of column-normalized ZCB. Maximum values in each column are marked with white bars. (b) ZCB averaged for each cloud case together with averaged LDR values. (c) IWCCB averaged for each cloud case. (d) Values of ZCB depicted depending on CTH instead of CTT; the cutoff at lower heights appears due to the selection criterion CTH > 1500 m. Thresholds for Z and IWC are illustrated within the graphs as solid lines with labels. Figure 7. (a) All values of column-normalized ZCB. Maximum values in each column are marked with white bars. (b) ZCB averaged for each cloud case together with averaged LDR values. (c) IWCCB averaged for each cloud case. (d) Values of ZCB depicted depending on CTH instead of CTT; the cutoff at lower heights appears due to the selection criterion CTH > 1500 m. Thresholds for Z and IWC are illustrated within the graphs as solid lines with labels. val is too large for a definite identification of this phe- nomenon. indicating the different prevailing particle habits over the temperature range of heterogeneous ice formation. Fukuta and Takahashi (1999) also found several distinct features in the distribution of ice particle size, shape, and mass with temperature. Some of these features can be seen within the measurements of LDR and vCB. We note that by our definition of “pristine particles” we follow the laboratory experiments of Fukuta and Takahashi (1999). We consider all particles pristine that have not un- dergone riming growth, aggregation, or splintering. As ex- plained above, these processes should be excluded by the cloud selection criteria. Non-pristine crystals that would re- sult, for example, from ice particle break-up, aggregation, or graupel formation would be asymmetric and would there- fore increase the LDR values. However, the LDR values we find are very close to the literature values of Reinking et al. (1997), which yield −28 dB for plate-like crystals and about −20 dB for columnar-shaped particles. These calculations, however, depend strongly on the orientation of the ice crys- tals (i.e., at what angle they “wobble”). Larger angles of ori- entation would increase the measured LDR values. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Finding these very low values of down to −30 dB is therefore an in- dication that pristine particles dominate and secondary ice formation only plays a minor role within the selected cloud layer type. – An enhanced growth of ice crystal mass around −14 ◦C was found by Fukuta and Takahashi (1999). The effect can also be seen in Fig. 7a and b as a strong increase of ZCB at this temperature. – The high values of LDR measured at a CTT of −5 ◦C correspond to a needle- or column-like particle shape (see Fig. 8a and c). In the temperature range around −14 ◦C LDR values can be found to be around −28 dB, corresponding to plate-like crystal shapes. Please note that these features are also displayed in Fig. 3. In Reink- ing et al. (1997) the LDR values of −15 to −20 dB are computed for these ice crystals shapes. g p g −14 ◦C LDR values can be found to be around −28 dB, corresponding to plate-like crystal shapes. Please note that these features are also displayed in Fig. 3. In Reink- ing et al. (1997) the LDR values of −15 to −20 dB are computed for these ice crystals shapes. – Hints about the presence of these isometric ice crystals are found in the increase of Doppler velocity in Fig. 8d. Measured Doppler velocities peak at around −10 and −22 ◦C, while minima of LDR can be found at −12 and −22 ◦C. This connection also points towards more isometric, compact ice crystals around these tempera- tures. Actually, the increase of Doppler velocities in the temperature interval between −5 and −0 ◦C bin is also found in Fukuta and Takahashi (1999). However, the un- certainty of the measurements in this temperature inter- 4.2 Particle Doppler velocity and radar depolarization of pristine ice crystals 8b also show features of enhanced Doppler velocities, The LACROS cloud radar has a depolarization decoupling of −33 dB, which stands out from all radars currently op- erated within the framework of Cloudnet. Only this techni- cal prerequisite makes high-quality measurements of LDR possible. Also, the detection threshold of −47 dBZ at a range of 5000 m is outstanding. Satellite missions equipped with cloud radars like CloudSat (Stephens et al., 2002) and EarthCare (Illingworth et al., 2014) have detection thresh- Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ 4.3 IWCCB and LWC at cloud top In the previous sections the properties of the ice particles pro- duced within mixed-phase clouds were investigated. For the estimation of cloud stability by approaches like the one pre- sented by Korolev and Field (2008), however, the ratio of IWCCB/LWC = ILCR (ice- to liquid-water content mass ra- www.atmos-chem-phys.net/16/10609/2016/ www.atmos-chem-phys.net/16/10609/2016/ Atmos. Chem. Phys., 16, 10609–10620, 2016 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10616 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Figure 8. All values of (a) LDR and (b) vCB measured with cloud radar MIRA-35 in the virgae below cloud layers over Leipzig. The visible spread in vCB is due to vertical air motion (see velocity plots in Fig. 3). Averaged values for the individual cloud cases are depicted in (c, d) respectively. Maximum values in each column are marked with white bars. MIRA 35 i h i b l l d l L i i Th i ibl Figure 8. All values of (a) LDR and (b) vCB measured with cloud radar MIRA-35 in the virgae below cloud layers over Leipzig. The visible spread in vCB is due to vertical air motion (see velocity plots in Fig. 3). Averaged values for the individual cloud cases are depicted in (c, d) respectively. Maximum values in each column are marked with white bars. tio) at cloud top is important. For that estimation, the LWC has to be retrieved in addition to the IWC. phase layer have the same properties as within the layer, this estimate of ILCR is representative for the average ratio be- tween ice- and liquid-water content within the mixed-phase cloud layer. The uncertainty of ILCR is still quite large. As mentioned above, the absolute accuracy of the measurements of LWC and IWCCB is 1 order of magnitude due to unknown biases of the retrieval itself and the radar calibration. Nev- ertheless, the standard deviation within a temperature inter- val of about −5 ◦C is only a factor of 2. That comparably low value might be partially due to the reason that both the IWCCB and the LWC retrieval method rely on the same tem- perature field, reducing this part of the variability. Systematic uncertainties of both the IWCCB and LWC, however, remain. In this work, the LWC of a cloud layer is calculated for each cloud profile adiabatically between cloud bases and cloud tops, assuming an adiabaticity of 1. Cloudnet also pro- vides operationally adiabatic profiles scaled with the liquid- water path (LWP) measured with the microwave radiome- ter (Merk et al., 2016). However, the LWP measurements of the microwave radiometer have an uncertainty of about ±20 g m−2. www.atmos-chem-phys.net/16/10609/2016/ Since the average LWP of the cloud under study is actually around 20 g m−2, the adiabatically calculated pro- files are used in the context of this work. An overview about the LWP of all cloud layers under study is given in Fig. 9a. Zhang et al. (2014) found a similar relationship between LWP and T for Arctic supercooled midlevel clouds. For the current work, the retrieved adiabatic LWP can be considered as a maximum guess. The actual LWP may be lower, which is described by the adiabaticity factor f . Merk et al. (2016) report f to be within 0.6 to 1.0. Airborne studies of mixed- phase clouds found rather good agreement between observed and adiabatic LWC profiles for shallow cloud layers (Larson et al., 2006; Noh et al., 2013). Hence, the adiabatic LWC profiles serve as an estimation until better calibration meth- ods for the microwave radiometers are available. Such meth- ods are currently under investigation by different groups, e.g., Maschwitz et al. (2013). An alternative approach may be LWP measurements with depolarization lidar (Hu et al., 2010; Donovan et al., 2015). temperature range of heterogeneous ice formation (−40 to 0 ◦C). temperature range of heterogeneous ice formation (−40 to 0 ◦C). temperature range of heterogeneous ice formation (−40 to 0 ◦C). at temperatures below −20 ◦C it can be seen that the flux of ice mass is only weakly dependent on temperature. In this temperature range IWCCB (Fig. 7c) is decreasing with tem- perature while vCB (Fig. 8d) is increasing. Also, the peak at −15 ◦C is less pronounced compared to Fig. 7b and c as it coincides with a minimum in particle fall velocity. LWP (2) Defined in this way, Tl is time measured in seconds. Assum- ing static conditions, Tl is the time the liquid cloud-top layer would have depleted all its liquid water by ice sedimentation alone. It is a theoretical quantity, but it gives an impression of the relative impact of ice formation on different cloud layers. An overview of Tl for all cloud cases under study is shown in Fig. 10a, indicating that Tl varies over 4 orders over the Defined in this way, Tl is time measured in seconds. Assum- ing static conditions, Tl is the time the liquid cloud-top layer would have depleted all its liquid water by ice sedimentation alone. It is a theoretical quantity, but it gives an impression of the relative impact of ice formation on different cloud layers. An overview of Tl for all cloud cases under study is shown in Fig. 10a, indicating that Tl varies over 4 orders over the It is demonstrated in this work that a detailed insight into the microphysics of mixed-phase cloud layers is possible with a combination of the LACROS instrumentation and Cloudnet. Vertical-velocity measurements show the dynami- cal state of the turbulent layer and cloud radar measurements 4.4 Estimating the ice mass flux from a cloud layer The ILCR connects measurements of ice- and liquid-water mass. However, ice crystals formed inside the mixed- phase cloud-top layer are falling with vCB > 0.2 m s−1 (see Fig. 8d), while the majority of cloud droplets have negligible fall velocities. The same number of particles creates a differ- ent IWC when falling at different terminal velocities, because the stream of particles is “stretched” differently. Hence, the ice flux F = IWCCB × vCB at cloud base gives the most ac- curate description of ice formation per time interval inside the cloud-top layer. In this very simple picture, F describes the flux quite coarsely. However, since both vCB and IWCCB are calculated from the same radar signal, a direct multipli- cation can be applied. The resulting parameter is an estima- tion within the order of magnitude, but it can safely be com- pared to the other flux values presented here. Figure 10a dis- plays averaged F for all cloud cases under study. Especially In Fig. 9b, IWCCB is divided by the mean LWC in the mixed-phase cloud top in order to derive an estimate of ILCR. Assuming that particles directly below the mixed- Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ www.atmos-chem-phys.net/16/10609/2016/ J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10617 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Measuring ice- and liquid-water properties in mixed-phase clouds 10617 Figure 9. (a) LWP of all clouds under study is shown in dependence of temperature and mean cloud-top thickness. (b) The ratio between IWCCB and mean LWC is calculated for each cloud profile and averaged for each cloud case. Figure 9. (a) LWP of all clouds under study is shown in dependence of temperature and mean cloud-top thickness. (b) The ratio between IWCCB and mean LWC is calculated for each cloud profile and averaged for each cloud case. Figure 10. (a) The ice mass flux at cloud base. (b) The estimated static lifetime index Tl = LWP/F of each cloud. Figure 10. (a) The ice mass flux at cloud base. (b) The estimated static lifetime index Tl = LWP/F of each cloud. ce mass flux at cloud base. (b) The estimated static lifetime index Tl = LWP/F of each cloud. 5 Summary and conclusions The concept of ice mass flux also opens the possibility to derive basic information about the impact of ice forma- tion on static cloud lifetime. Water particles most probably glaciate at cloud top and fall through the mixed-phase layer. Having connected vCB with IWCCB to the ice flux, it is also possible to relate this quantity to the available LWP within the ice-generating liquid cloud layer. Since ice particles grow through the Wegener–Bergeron–Findeisen process (Korolev and Field, 2008), there is an indirect connection between the amount of available water vapor and ice crystal growth. Hence, a dynamic view of ice formation in the cloud layers can be established by dividing F and LWP profile-wise. Quantitative retrievals of ice crystal properties like basic in- formation about particle shape and Doppler velocity have been found to be quantitatively in line with theoretical com- putations of Reinking et al. (1997) and laboratory studies of Fukuta and Takahashi (1999). The dominating part of the ice particles falling from mixed-phase cloud layers with a geo- metrical thickness of the mixed-phase top layer < 350 m are apparently mostly pristine. Hence, these particles are prob- ably the result of primary ice formation, and secondary ice formation is only a minor process in these cloud layers. Ad- ditionally, a profile-based connection between the measured LWP and the retrieved IWCCB has been established. The flux of ice mass at cloud-base height is found to increase within 2 orders of magnitude within the CTT range from −40 to 0 ◦C. The relative influence of the loss of ice on static lifetime in- dex is found to increase even by 4 orders of magnitude within the same range of CTT. LWP F = LWP IWCCB × vCB != Tl (2) J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds show the ice flux from that layer. Together with the retrieval of ice nuclei properties with Raman lidar (Mamouri and Ans- mann, 2015) the life cycle of an ice nucleus in mixed-phase clouds from entrainment over activation to ice nucleation and sedimentation can be closed. quests, please contact Johannes Bühl (buehl@tropos.de) at TROPOS. We will gladly provide the requested data used in this study. Please note that the full data set contains about 600 GB of data. quests, please contact Johannes Bühl (buehl@tropos.de) at TROPOS. We will gladly provide the requested data used in this study. Please note that the full data set contains about 600 GB of data. It is an important finding that the dominating number of ice crystals in mixed-phase cloud layers with δh < 350 m is pristine. This means that the flux of ice crystals measured at cloud base is directly connected to the rate of ice nucleation within the mixed-phase layer. The direct measurement of the complete process of ice nucleation seems therefore feasible with remote sensing. However, in future, more advanced par- ticle typing methods such as presented in Myagkov et al. (2016a, b) should be applied to further characterize shape and size of the particles on an operational basis. Acknowledgements. The research leading to these results has re- ceived funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 654109 (ACTRIS-2) and previously from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 262254 (ACTRIS) and 603445 (BACCHUS) as well as from the Cloudnet project (European Union contract EVK2-2000- 00611). Funding was also received from the HD(CP)2 project (FKZ 01LK1209C and 01LK1212C) of the German Ministry for Education and Research. The relative impact of the loss of ice water on a mixed- phase cloud layer has been measured. However, it has to be noted again that the cloud static lifetime index presented here might not directly be connected to the absolute lifetime of a cloud. Even the definition of a cloud lifetime is difficult because particles are mixed between cloud parcels and the apparent motion of clouds can be independent from horizon- tal wind speed. However, the static lifetime value presented here can be used to study the impact of ice on predominantly liquid cloud layers occurring at different temperature levels. References Althausen, D., Engelmann, R., Baars, H., Heese, B., Ansmann, A., Müller, D., and Komppula, M.: Portable Raman lidar PollyXT for automated profiling of aerosol backscatter,extinction, and de- polarization, J. Atmos. Ocean. Tech., 26, 2366–2378, 2009. Bühl, J., Ansmann, A., Seifert, P., Baars, H., and Engelmann, R.: Towards a quantitative characterization of heterogeneous ice formation with lidar/radar: Comparison of CALIPSO/CloudSat with ground-based observations, Geophys. Res. Lett., 40, 4404– 4408, doi:10.1002/grl.50792, 2013. Cantrell, W. and Heymsfield, A.: Production of Ice in Tropo- spheric Clouds: A Review, B. Am. Meteor. Soc., 86, 795–807, doi:10.1175/BAMS-86-6-795, 2005. DeMott, P. J., Prenni, A. J., McMeeking, G. R., Sullivan, R. C., Petters, M. D., Tobo, Y., Niemand, M., Möhler, O., Snider, J. R., Wang, Z., and Kreidenweis, S. M.: Integrating laboratory and field data to quantify the immersion freezing ice nucleation activ- ity of mineral dust particles, Atmos. Chem. Phys., 15, 393–409, doi:10.5194/acp-15-393-2015, 2015. Di Girolamo, P., Summa, D., Cacciani, M., Norton, E. G., Peters, G., and Dufournet, Y.: Lidar and radar measurements of the melting layer: observations of dark and bright band phenomena, Atmos. Chem. Phys., 12, 4143–4157, doi:10.5194/acp-12-4143- 2012, 2012. The presented algorithm to classify mixed-phase clouds in Cloudnet data sets is universal. It is applicable not only on Cloudnet data sets but also in general on all data sets that separate an atmospheric column into liquid, ice, and mixed phase. The evaluation of mixed-phase clouds predicted by weather models seems therefore possible if suitable data out- put is given. Donovan, D. P., Klein Baltink, H., Henzing, J. S., de Roode, S. R., and Siebesma, A. P.: A depolarisation lidar-based method for the determination of liquid-cloud microphysical properties, Atmos. Meas. Tech., 8, 237–266, doi:10.5194/amt-8-237-2015, 2015. Meas. Tech., 8, 237–266, doi:10.5194/amt-8-237-2015, 2015. Engelmann, R., Kanitz, T., Baars, H., Heese, B., Althausen, D., g Skupin, A., Wandinger, U., Komppula, M., Stachlewska, I. 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(2013), in which the mass ratio of ice and liquid water in mixed-phase layered clouds was estimated with a ratio of IWP (ice water path) and LWP on manually selected clouds. The ratio of IWCCB and LWP, combined with the particle Doppler velocity, gives a much more direct measure of the actual impact of the ice on the liquid water within a mixed-phase layer. Edited by: J. Huang Reviewed by: three anonymous referees Edited by: J. Huang Reviewed by: three anonymous referees Edited by: J. Huang Reviewed by: three anonymous referees www.atmos-chem-phys.net/16/10609/2016/ www.atmos-chem-phys.net/16/10609/2016/ Atmos. Chem. Phys., 16, 10609–10620, 2016 10618 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10619 clouds, J. Atmos. Sci., 59, 1779–1804, doi:10.1175/1520- 0469(2002)059<1779:OMSOMM>2.0.CO;2, 2002. clouds, J. Atmos. 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https://openalex.org/W4387707365
https://jklst.org/index.php/home/article/download/43/31
English
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Launching a new English language program
Journal of knowledge learning and science technology
2,023
cc-by
7,045
Abstract In the rapidly globalizing world, English language proficiency is becoming increasingly pivotal for academic and professional success. This research article presents a comprehensive study surrounding the launch of a novel English language program, termed the IELTS 4.0, by the Mentor Language School. The article delves into the objectives, development, and promotion processes of the program, aiming to elucidate its distinctive features and potential benefits. Drawing from both qualitative and quantitative data, the research evaluates the program's curriculum design, pedagogical strategies, and its early impact on students' language proficiency outcomes. Initial feedback reveals a positive reception, with particular emphasis on its modern teaching methodologies, expert-led instruction, and cultural immersion components. Additionally, the program's structured approach, segmented into Beginner, Intermediate, Advanced, and IELTS Preparation levels, facilitates tailored learning experiences, catering to diverse student needs. The article concludes by underscoring the potential of the IELTS 4.0 program in shaping the future landscape of English language education, with recommendations for further enhancements based on early observations and feedback. Keywords: Pedagogical Strategies, Language Proficiency, Cultural Immersion Article Information: Article history: Received: 15/07/2023 Accepted: 05/08/2023 Online: 06/10/2023 Publish DOI: https://doi.org/10.60087/jklst.vol2.n3.p ̣̣̣̣45 i Correspondence author: Thuan, Phan Kim Email: thuanphanvus84@gmail.com Article Information: Article history: Received: 15/07/2023 Accepted: 05/08/2023 Online: 06/10/2023 Published: 20/12/2023 DOI: https://doi.org/10.60087/jklst.vol2.n3.p ̣̣̣̣45 i Correspondence author: Thuan, Phan Kim Email: thuanphanvus84@gmail.com Launching a new English language program Thuan, Phan Kim MAE, Hoa Sen University 1. Introduction Mentor Language School is a reputable language teaching organization that has been providing high-quality language programs to students from various backgrounds for many years. Our organization has a strong commitment to meeting the needs of our students, and we are continually striving to provide innovative and effective language education programs. This report introduces our new English language program, the IELTS 4.0 program, and outlines the project's development and promotion process. It includes an overview of the curriculum, objectives, and marketing strategies used to promote the program, as well as feedback received since its launch. The report aims to showcase the program's unique features and benefits while providing insight into its development and marketing process. ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 Journal of Knowledge Learning and Science Technology journal homepage: https://jklst.org/index.php/home ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 Journal of Knowledge Learning and Science Technology journal homepage: https://jklst.org/index.php/home ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 Journal of Knowledge Learning and Science Technology journal homepage: https://jklst.org/index.php/home ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 Journal of Knowledge Learning and Science Technology journal homepage: https://jklst.org/index.php/home 2.1. Explanation of the program's goals The IELTS 4.0 program is designed to help high school students graduate from high school and assist other students in improving their English language proficiency to increase their chances of securing better job opportunities. The program aims to achieve the following goals: (Nick Kenny and Bruce Rogers, 2015). 35 Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 Enhance students' language skills: The IELTS 4.0 program is designed to help students develop their English language skills, including reading, writing, listening, and speaking. By improving their language proficiency, students can communicate more effectively and confidently, which can increase their chances of academic and career success. Prepare students for academic success: The program aims to help high school students improve their English language proficiency to meet the requirements for graduation. It provides a structured curriculum and expert instructors to ensure that students have the necessary language skills to excel in their academic studies. Increase employability: The IELTS 4.0 program also targets students who are looking to improve their English language proficiency to increase their chances of finding better job opportunities. By enhancing their language skills, students can communicate effectively with colleagues, clients, and customers in the workplace, making them more desirable to potential employers. Provide a high-quality English language program: The program is designed to provide a high-quality English language learning experience for students. It includes a comprehensive curriculum, expert instructors, and modern teaching methods to ensure that students receive the best possible education. Overall, the IELTS 4.0 program aims to provide students with the language skills and knowledge necessary to achieve academic success and improve their employability, ultimately leading to better opportunities for their future. Provide a high-quality English language program: The program is designed to provide a high-quality English language learning experience for students. It includes a comprehensive curriculum, expert instructors, and modern teaching methods to ensure that students receive the best possible education. Overall, the IELTS 4.0 program aims to provide students with the language skills and knowledge necessary to achieve academic success and improve their employability, ultimately leading to better opportunities for their future. Level 2: Intermediate The intermediate level is designed for students who have some experience with the English language but still need to improve their language proficiency. In this level, students will learn more advanced vocabulary, sentence structure, and grammar rules. They will also learn how to read and write more complex texts and hold more advanced conversations. By the end of this level, students should be able to communicate more effectively in English and understand more complex information. Level 1: Beginner The beginner level is designed for students who have little or no experience with the English language. In this level, students will learn basic vocabulary, sentence structure, and grammar rules. They will also learn how to read and write simple texts and hold basic conversations. By the end of this level, students should be able to understand and communicate basic information in English. 2.2. Detailed program description and curriculum 2.2.1. Detailed program description The IELTS 4.0 program is a comprehensive English language program designed to help high school students graduate from high school and assist other students in improving their English language proficiency to increase their chances of securing better job opportunities. The program is divided into four levels, each designed to help students develop their language skills in reading, writing, listening, and speaking. (Nick Kenny and Bruce Rogers, 2015) Level 3: Advanced The advanced level is designed for students who have a good understanding of the English language but still need to improve their language proficiency. At this level, students will learn advanced vocabulary, sentence structure, and grammar rules. They will also learn how to read and write academic texts and hold Thuan Phan Kim 36 advanced conversations. By the end of this level, students should be able to communicate fluently in English and understand complex academic information. The IELTS preparation level is designed for students who need to prepare for the IELTS exam. In this level, students will learn advanced vocabulary, sentence structure, and grammar rules specifically for the IELTS exam. They will also learn test-taking strategies and practice taking mock exams to prepare for the actual exam. By the end of this level, students should be prepared to take the IELTS exam and achieve their desired score. 2.3. Our strengths and features that distinguish our program from others. These include: Potential Weakness Descriptions Cost Our program may be more expensive than other language programs, due to the personalized nature of the instruction and the use of specialized materials and technology. The IELTS 4.0 program offered by our language teaching organization is designed to provide students with a high-quality English language learning experience that sets it apart from other programs. Here are some strengths and features that distinguish our program: Strengths and Features Description Tailored curriculum The program's curriculum is designed to be flexible and customizable, depending on each student's specific needs and goals. This approach ensures that students receive personalized instruction that meets their individual learning needs. Experienced instructors The program has experienced and highly qualified instructors who are trained to provide expert guidance and support to students. Instructors are native English speakers who have extensive experience teaching English to students from diverse backgrounds. Advanced technology The program uses advanced technology to enhance the learning experience. Multimedia resources, online tools, and interactive activities are incorporated to make learning engaging and effective. Students can access the online learning platform, which includes video lectures, quizzes, and practice exercises. Small class sizes The program emphasizes small class sizes to ensure that students receive personalized attention and guidance from their instructors. This approach allows instructors to provide individualized feedback and support to each student, which can lead to better learning outcomes. IELTS test preparation The program includes a dedicated IELTS preparation level that is designed to help students prepare for the IELTS exam. This level provides students with the skills and knowledge necessary to succeed on the exam, including test-taking strategies and mock exams. Academic and career support The program provides academic and career support to students, including academic counseling, college application assistance, and career counseling. The organization is committed to helping students achieve their academic and career goals. Overall, the IELTS 4.0 program offered by our language teaching organization stands out from other programs due to its tailored curriculum, experienced instructors, advanced technology, small class sizes, dedicated IELTS test preparation level, and academic and career support. These strengths and features ensure that students receive a high-quality English language learning experience that can help them achieve their goals. 2.3. Our strengths and features that distinguish our program from others. 2.3. Our strengths and features that distinguish our program from others. The IELTS 4.0 program offered by our language teaching organization is designed to provide students with a high-quality English language learning experience that sets it apart from other programs. Here are some strengths and features that distinguish our program: Strengths and Features Description Tailored curriculum The program's curriculum is designed to be flexible and customizable, depending on each student's specific needs and goals. This approach ensures that students receive personalized instruction that meets their individual learning needs. Experienced instructors The program has experienced and highly qualified instructors who are trained to provide expert guidance and support to students. Instructors are native English speakers who have extensive experience teaching English to students from diverse backgrounds. Advanced technology The program uses advanced technology to enhance the learning experience. Multimedia resources, online tools, and interactive activities are incorporated to make learning engaging and effective. Students can access the online learning platform, which includes video lectures, quizzes, and practice exercises. Small class sizes The program emphasizes small class sizes to ensure that students receive personalized attention and guidance from their instructors. This approach allows instructors to provide individualized feedback and support to each student, which can lead to better learning outcomes. IELTS test preparation The program includes a dedicated IELTS preparation level that is designed to help students prepare for the IELTS exam. This level provides students with the skills and knowledge necessary to succeed on the exam, including test-taking strategies and mock exams. Academic and career support The program provides academic and career support to students, including academic counseling, college application assistance, and career counseling. The organization is committed to helping students achieve their academic and career goals. Overall, the IELTS 4.0 program offered by our language teaching organization stands out from other programs due to its tailored curriculum, experienced instructors, advanced technology, small class sizes, dedicated IELTS test preparation level, and academic and career support. These strengths and features ensure that students receive a high-quality English language learning experience that can help them achieve their goals. 2.4. Some potential weaknesses While we believe our new English language program has many strengths, it is important to acknowledge that there are also some potential weaknesses. 2.2.2. Curriculum The curriculum for each level includes the following components: Fig. 1 Detailed program description and curriculum Reading • Developing reading comprehension skills • Building vocabulary through reading • Identifying and analyzing the main ideas and supporting details of texts Writing • Developing writing skills for different purposes (e.g., essays, emails, reports) • Building vocabulary for writing • Developing organization and structure in writing Listening • Developing listening comprehension skills • Building vocabulary through listening • Identifying and analyzing the main ideas and supporting details of spoken language Speaking • Developing speaking skills for different purposes (e.g., interviews, presentations, group discussions) • Building vocabulary for speaking • Developing fluency and accuracy in speaking CURRICULUM Speaking Writing Reading Listening The curriculum for each level includes the following components: Reading Speaking Writing CURRICULUM Speaking Listening Reading g • Developing reading comprehension skills • Building vocabulary through reading • Identifying and analyzing the main ideas and supporting details of texts Writing • Developing writing skills for different purposes (e.g., essays, emails, reports) • Building vocabulary for writing • Developing organization and structure in writing Listening • Developing listening comprehension skills • Building vocabulary through listening • Identifying and analyzing the main ideas and supporting details of spoken language Speaking • Developing speaking skills for different purposes (e.g., interviews, presentations, group discussions) • Building vocabulary for speaking • Developing fluency and accuracy in speaking Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 37 Overall, the IELTS 4.0 program provides a comprehensive and structured curriculum that is designed to help students develop their language skills and achieve their goals in academic and professional contexts. 2.5. Some threats While we see many opportunities in the current situation for the English program, we should also pay attention to some threats. While we see many opportunities in the current situation for the English program, we should also pay attention to some threats. 1. Competition from other language teaching organizations offering similar English language programs, potentially limiting enrollment and revenue. 2. Economic downturns or recessions affect the ability of individuals to afford language education, decreasing the program's revenue. 3. Changes in government policies or regulations impacting the availability of student visas or funding for language education, potentially limiting the program's reach and appeal. 4. Political instability or social unrest affecting the safety and security of students, potentially impacting enrollment and the program's reputation. 3. Project development process 3.1. Market research to identify student preferences and needs. 2.4. Some potential weaknesses reased online education platform use enables the program to reach a wider audience. Increased online education platform use enables the program to reach a wider audience. Access to a diverse pool of experienced and qualified English language teachers, allowing for the development of a high-quality program. Access to a diverse pool of experienced and qualified English language teachers, allowing for the development of a high-quality program. The ability to incorporate new technologies and teaching methods to improve the program's effectiveness. The ability to incorporate new technologies and teaching methods to improve the program' The ability to incorporate new technologies and teaching methods to improve the program's effectiveness. The ability to incorporate new technologies and teaching methods to improve the program's effectiveness. 2 5 Some threats 2.4. Some potential weaknesses While we believe our new English language program has many strengths, it is important to acknowledge that there are also some potential weaknesses. These include: Potential Weakness Descriptions Cost Our program may be more expensive than other language programs, due to the personalized nature of the instruction and the use of specialized materials and technology. Potential Weakness Descriptions Cost Our program may be more expensive than other language programs, due to the personalized nature of the instruction and the use of specialized materials and technology. 38 Thuan Phan Kim Availability Our program may not be available in all locations, as we may not have the resources to expand to all areas. Time commitment Our program requires a significant time commitment from students, as it is designed to be comprehensive and thorough. Students will need to dedicate themselves to regular study and practice in order to see the full benefits of the program. Teaching style Our program may not be suitable for students who prefer a more traditional teaching style, as it is designed to be interactive and engaging, using a range of multimedia materials and real-life scenarios. rogram may not be available in all locations, as we may not have the resources to expand areas Overall, while our program has many strengths, it is important to acknowledge that there may be some limitations or weaknesses that could impact its suitability for certain students or situations. Overall, while our program has many strengths, it is important to acknowledge that there may be some limitations or weaknesses that could impact its suitability for certain students or situations. In addition to the strengths and weaknesses outlined above, the IELTS 4.0 program offers students a range of opportunities to enhance their language skills and gain valuable experiences. These are some of the opportunities of the current situation of the program: Growing demand for English language proficiency globally, creating opportunities for the program to attract new students. In addition to the strengths and weaknesses outlined above, the IELTS 4.0 program offers students a range of opportunities to enhance their language skills and gain valuable experiences. These are some of the opportunities of the current situation of the program: Growing demand for English language proficiency globally, creating opportunities for the program to attract new students. Increased online education platform use enables the program to reach a wider audience. 3.1. Market research to identify student preferences and needs. To ensure that our IELTS 4.0 program meets the preferences and needs of our students, we conducted extensive market research. Our research involved surveys, focus groups, and interviews with current and potential students, as well as analysis of industry trends and best practices. The findings from our research helped us to tailor the program's curriculum and features to meet the specific needs and preferences of our target audience. We are confident that our program will provide a high-quality English language learning experience that meets the needs and expectations of our students. Based on the findings of the market research, the IELTS 4.0 program should be designed to meet the preferences and needs of potential students. A blended learning approach that includes both online and in- person classes would be ideal, with a curriculum that is relevant, up-to-date, and includes multimedia nal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 202 39 content. The class size should be kept small, with no more than 15 students per class, and teachers should be highly qualified and experienced. By meeting these preferences and needs, the new English program will be well-positioned to attract and retain students. 3.2. Development of program objectives and curriculum To develop an effective program, several steps must be taken to ensure that it meets the needs of the target audience and achieves its intended learning outcomes: Needs Assessment: Identify the target audience's English proficiency level, learning needs, and preferences. Program Objectives: Establish specific, measurable, achievable, relevant, and time-bound objectives. Needs Assessment: Identify the target audience's English proficiency level, learning needs, and preferences. Program Objectives: Establish specific, measurable, achievable, relevant, and time-bound objectives. Curriculum Design: Develop a curriculum that aligns with the program objectives and includes clear learning outcomes, lesson plans, and assessments. Needs Assessment: Identify the target audience s English proficiency level, learning needs, and preferences. Program Objectives: Establish specific, measurable, achievable, relevant, and time-bound objectives. Curriculum Design: Develop a curriculum that aligns with the program objectives and includes clear learning outcomes, lesson plans, and assessments. Curriculum Design: Develop a curriculum that aligns with the program objectives and includes clear learning outcomes, lesson plans, and assessments. Teaching Methodology: Design an interactive, learner-centred methodology that caters to different learning styles. Teaching Methodology: Design an interactive, learner-centred methodology that caters to different learning styles. Course Materials and Resources: Select and design relevant, up-to-date materials and resources that support the curriculum and teaching methodology. Course Materials and Resources: Select and design relevant, up-to-date materials and resources that support the curriculum and teaching methodology. Program Evaluation: Conduct regular evaluation and feedback to measure program effectiveness and make necessary improvements. Continuous Improvement: Continuously update and improve the program based on feedback received. Collaboration among program designers, language experts, and potential participants is crucial to ensure program success. 3.3. Timeline and milestones Developing a timeline and milestones for the new English program is an essential step in ensuring that the program is implemented effectively and efficiently. The following is an example of a timeline and milestones for a 12-week program (Peter Sommerville, 2023). Week Activities Weeks 1-2 Conduct a needs assessment, establish program objectives, design the curriculum, teaching methodology, and course materials. Weeks 3-4 Recruit and train English language teachers who meet the program's requirements and qualifications. Train teachers on the program's objectives, curriculum, teaching methodology, and assessment methods. Weeks 5-6 Finalize the program schedule, lesson plans, and course materials. Establish communication channels with participants and provide orientation on the program's objectives, schedule, and expectations. Weeks 7-11 Implement the program. Participants attend classes and engage in learning activities. Conduct regular assessments to measure progress and adjust teaching strategies and materials if necessary. Week 12 Conduct program evaluations to measure the effectiveness of the program in achieving its objectives. Participants who have successfully completed the program will receive a certificate of completion. Overall, developing a timeline and milestones for the new English program will help ensure that the program is delivered effectively and efficiently and that its objectives are achieved within the desired timeframe. 3.4. Budget and resources 40 Thuan Phan Kim Developing a budget and identifying the necessary resources for the new English program is an important step in ensuring the program's success. The following are some of the main expenses and resources that may be needed for the program: (Mike McKeever, 2011) Developing a budget and identifying the necessary resources for the new English program is an important step in ensuring the program's success. The following are some of the main expenses and resources that may be needed for the program: (Mike McKeever, 2011) Personnel Costs: The largest expense for the program is likely to be personnel costs, including salaries and benefits for the English language teachers and program staff. Course Materials and Resources: Course materials and resources such as textbooks, workbooks, audio- visual aids, and technology equipment will be needed to support the curriculum and teaching methodology. Facility Costs: The program may require a dedicated facility or classrooms equipped with appropriate technology and learning resources. Marketing and Promotion: A budget for marketing and promotion may be required to promote the program and attract potential participants. This may include website development, online advertising, and brochures. 3.3. Timeline and milestones Administrative Costs: Administrative costs such as licensing fees, insurance, and legal fees should also be included in the budget. Program Evaluation: A budget for program evaluation should be included to measure the effectiveness of the program and identify areas for improvement. Program Evaluation: A budget for program evaluation should be included to measure the effectiveness of the program and identify areas for improvement. Contingency: It is also important to include a contingency budget to account for unexpected costs or emergencies. Contingency: It is also important to include a contingency budget to account for unexpected costs or emergencies. In addition to budgeting for expenses, resources such as expert advisors, consultants, and partners may also be needed to support the program's development and implementation. Other resources, such as research materials, training materials, and software may also be required. Overall, developing a budget and identifying the necessary resources for the new English program is crucial in ensuring that the program is adequately funded and that the resources needed to deliver high-quality instruction are available. The following table displays the break-even analysis of the new product. The following table displays the break-even analysis of the new product. Table 1. The break-even analysis of the new product. Table 1. The break-even analysis of the new product. Table 1. The break-even analysis of the new product. Account Rate in thousand VND Revenue Tuition (on each course) 20,00 Total per student (on each course) 20,000 Cost Variable costs per student Material 500 Copying 500 Total variable 1,000 Gross Profit 19,000 Fixed cost Account Rate in thousand VND Revenue Tuition (on each course) 20,00 Total per student (on each course) 20,000 Cost Variable costs per student Material 500 Copying 500 Total variable 1,000 Account Rate in thousand VND Revenue Tuition (on each course) 20,00 Total per student (on each course) 20,000 500 500 1,000 19,000 500 500 1,000 19,000 Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 3.5. Recruitment and training of teachers for the new program Recruiting and training teachers for the new English program is an essential step in ensuring that the program is delivered effectively and efficiently. The following are some considerations for teacher recruitment and training: (Peter Sommerville, 2023) Requirements and qualifications: The program development team should identify the requirements and qualifications for English language teachers. These may include minimum educational requirements, teaching experience, language proficiency, and teaching certifications. Recruitment: The program development team should develop a recruitment plan that identifies potential candidates and outlines the recruitment process. Recruitment channels may include job boards, social media, personal networks, and professional associations. Selection: Once applications are received, a selection process should be established to identify the most suitable candidates. This may include an initial screening, interviews, and reference checks. Selection: Once applications are received, a selection process should be established to identify the most suitable candidates. This may include an initial screening, interviews, and reference checks. Orientation: Newly hired teachers should receive an orientation that includes an overview of the program's objectives, curriculum, teaching methodology, and assessment methods. They should also be provided with relevant teaching resources and technology equipment. Training: Teachers should receive ongoing training and professional development to support their growth and development. This may include workshops, mentoring, coaching, and online training. Training: Teachers should receive ongoing training and professional development to support their growth and development. This may include workshops, mentoring, coaching, and online training. Assessment and Feedback: Teachers should receive regular assessment and feedback on their performance, including observations, evaluations, and feedback from participants. This can help identify areas for improvement and support ongoing development. Assessment and Feedback: Teachers should receive regular assessment and feedback on their performance, including observations, evaluations, and feedback from participants. This can help identify areas for improvement and support ongoing development. Overall, recruiting and training teachers for the new English program is a critical step in ensuring the program's success. By identifying the requirements and qualifications for teachers, developing a recruitment plan, providing orientation and training, and assessing performance, the program can attract and retain qualified and effective teachers to deliver high-quality instruction to participants. 3.6. Establishment of program pricing When establishing program pricing, it's crucial to consider multiple factors to ensure accessibility, affordability, and profitability. The team should evaluate program costs, conduct market research, assess program value, consider the target market, and develop a pricing strategy that aligns with program objectives. Promotions and discounts can also attract and retain participants. Ultimately, the pricing strategy should generate enough revenue to cover costs and generate profit to support the program's growth and sustainability. 3.3. Timeline and milestones 2, Issue 3, December 2023 41 And each class with maximum number of students is 15, the profit will be: Account Rate in thousand VND Revenue Tuition (on each course) (with 15 students) 300,000 Total of each class (15 students) 300,000 Cost Variable costs per student Material 7,500 Copying 7,500 Total variable 15,000 Gross Profit of 15 students class 285,000 Fixed cost Personnel Costs (Teaching salaries/ benefits) 57,600 Facility Costs 30,000 Marketing and Promotion 35,000 Administrative Costs 10,000 Program Evaluation 5,000 Contingency Budget 19,000 Total fixed cost 156,600 FC Fixed costs 156,600 CR Class revenue 300,000 Personnel Costs (Teaching salaries/ benefits) 57,600 Facility Costs 30,000 Marketing and Promotion 35,000 Administrative Costs 10,000 Program Evaluation 5,000 Contingency Budget 19,000 Total fixed cost 156,600 FC Fixed costs 156,600 Unit Revenue Unit revenue or price/unit 20,000 Unit VC Unit variable cost 1,000 GP Gross Profit 19,000 BE Break-even point in units 8.2 And each class with maximum number of students is 15, the profit will be: Account Rate in thousand VND Revenue Tuition (on each course) (with 15 students) 300,000 Total of each class (15 students) 300,000 Cost Variable costs per student Material 7,500 Copying 7,500 Total variable 15,000 Gross Profit of 15 students class 285,000 Fixed cost Personnel Costs (Teaching salaries/ benefits) 57,600 Facility Costs 30,000 Marketing and Promotion 35,000 Administrative Costs 10,000 Program Evaluation 5,000 Contingency Budget 19,000 Total fixed cost 156,600 FC Fixed costs 156,600 CR Class revenue 300,000 Personnel Costs (Teaching salaries/ benefits) 57,600 Facility Costs 30,000 Marketing and Promotion 35,000 Administrative Costs 10,000 Program Evaluation 5,000 Contingency Budget 19,000 Total fixed cost 156,600 FC Fixed costs 156,600 Unit Revenue Unit revenue or price/unit 20,000 Unit VC Unit variable cost 1,000 GP Gross Profit 19,000 BE Break-even point in units 8.2 42 Thuan Phan Kim CVC Class variable cost 15,000 GP Gross Profit 285,000 NP Net profit 128,400 Class variable cost Gross Profit Net profit 4.3. Measuring the effectiveness of marketing strategies To measure the marketing strategies' effectiveness for the new English program, the program development team can track website traffic, monitor social media engagement, analyze SEM results, track referral marketing, and monitor enrollment numbers. By analyzing data regularly, the team can evaluate the strategies and make adjustments as needed to improve the program's visibility and enrollment numbers. The target audience for the new English program: IELTS 4.0 course would be: 4.1. The target audience for the new English program: IELTS 4.0 course would be 4.1. The target audience for the new English program: IELTS 4.0 course would be: 1. High school students who are seeking to graduate from high school without attending the final English exam. This may include students who struggle with the English language or who have failed previous English exams. 1. High school students who are seeking to graduate from high school without attending the final English exam. This may include students who struggle with the English language or who have failed previous English exams. Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 43 2. Individuals who want to improve their English language proficiency to get a better job or advance their careers. This may include individuals who work in industries that require strong English language skills, such as international business, hospitality, and customer service. 2. Individuals who want to improve their English language proficiency to get a better job or advance their careers. This may include individuals who work in industries that require strong English language skills, such as international business, hospitality, and customer service. 4.2. Marketing channels used to reach the target audience. B i bi ti f k ti h l th d l t t i th ' Events and workshops: The team may host events or workshops to showcase the program's curriculum, teaching methodology, and outcomes, and to engage with potential participants directly. Events and workshops: The team may host events or workshops to showcase the program's curriculum, teaching methodology, and outcomes, and to engage with potential participants directly. By using a combination of marketing channels, the program development team can increase the program's visibility and attract potential participants who are most likely to benefit from the program. By using a combination of marketing channels, the program development team can increase the program's visibility and attract potential participants who are most likely to benefit from the program. 4.2. Marketing channels used to reach the target audience. To reach the target audience for the new English program: IELTS 4.0 course, the program development team may consider using a variety of marketing channels to promote the program and attract potential participants. Here are some examples of marketing channels that may be effective for reaching the target audience: (Simon Kingsnorth, 2019) Social media advertising: The program development team may use social media advertising on platforms such as Facebook, TikTok, and YouTube to target potential participants based on their demographics, interests, and behavior. Search engine marketing (SEM): The team may use search engine marketing to target potential participants who are actively searching for English language courses or IELTS exam preparation. Referral marketing: The team may encourage current and former program participants to refer friends and family members to the program in exchange for discounts or other incentives. Public relations (PR): The team may use PR strategies to generate media coverage and build awareness of the program, such as issuing press releases or pitching stories to local media outlets. Public relations (PR): The team may use PR strategies to generate media coverage and build awareness of the program, such as issuing press releases or pitching stories to local media outlets. the program, such as issuing press releases or pitching stories to local media outlets. Community partnerships: The team may establish partnerships with local schools, community organizations, or businesses to promote the program to their networks and members. Events and workshops: The team may host events or workshops to showcase the program's curriculum, teaching methodology, and outcomes, and to engage with potential participants directly. By using a combination of marketing channels, the program development team can increase the program's visibility and attract potential participants who are most likely to benefit from the program. Community partnerships: The team may establish partnerships with local schools, community organizations, or businesses to promote the program to their networks and members. Community partnerships: The team may establish partnerships with local schools, community organizations, or businesses to promote the program to their networks and members. Events and workshops: The team may host events or workshops to showcase the program's curriculum, teaching methodology, and outcomes, and to engage with potential participants directly. 5. Accountability Here are some of the important positions at a language center and their duties: (Peter Sommerville, 2023) Director of the Language Center: The Director of the Language Center oversees the overall management of the center. He is responsible for developing and implementing the center's policies and procedures, overseeing budget management, and ensuring that the center is meeting its objectives and goals. Academic Director: The Academic Director is responsible for developing and overseeing the center's academic programs, including curriculum development, teacher training, and evaluation. They ensure that the center's academic programs meet industry standards and that the teaching methodology is effective. Operations Manager: The Operations Manager oversees the daily operations of the center, including managing administrative staff, scheduling classes, and managing the facility. They ensure that the center runs smoothly and efficiently. Here are some of the important positions at a language center and their duties: (Peter Sommerville, 2023) Director of the Language Center: The Director of the Language Center oversees the overall management of the center. He is responsible for developing and implementing the center's policies and procedures, overseeing budget management, and ensuring that the center is meeting its objectives and goals. Academic Director: The Academic Director is responsible for developing and overseeing the center's academic programs, including curriculum development, teacher training, and evaluation. They ensure that the center's academic programs meet industry standards and that the teaching methodology is effective. Academic Director: The Academic Director is responsible for developing and overseeing the center's academic programs, including curriculum development, teacher training, and evaluation. They ensure that the center's academic programs meet industry standards and that the teaching methodology is effective. Operations Manager: The Operations Manager oversees the daily operations of the center, including managing administrative staff, scheduling classes, and managing the facility. They ensure that the center runs smoothly and efficiently. Thuan Phan Kim 44 Course Coordinator: The Course Coordinator is responsible for coordinating the center's courses and ensuring that they are running effectively. They work closely with teachers to ensure that course content and delivery meet the center's objectives and standards. Language Teachers: Language Teachers are responsible for delivering high-quality language instruction to students. They develop lesson plans, deliver lectures, and provide feedback to students. They also evaluate students' progress and adjust lesson plans as needed. 6. Results and Feedback Enrollment numbers and revenue generated are two key indicators of the success of a language program. However, success should not be measured solely by these numbers, as they can be influenced by various factors such as program pricing, competition, and marketing efforts. Enrollment numbers and revenue generated are two key indicators of the success of a language program. However, success should not be measured solely by these numbers, as they can be influenced by various factors such as program pricing, competition, and marketing efforts. To determine the success of the program, it is important to consider other factors such as student satisfaction, teacher satisfaction, student outcomes, and program reputation. To determine the success of the program, it is important to consider other factors such as student satisfaction, teacher satisfaction, student outcomes, and program reputation. Student satisfaction can be measured through surveys and feedback forms, which can provide information about how students perceive the program, their overall experience, and areas for improvement. Student satisfaction can be measured through surveys and feedback forms, which can provide information about how students perceive the program, their overall experience, and areas for improvement. Teacher satisfaction can be measured through teacher evaluations, which can provide information about how teachers perceive the program, their level of engagement, and their satisfaction with their roles and responsibilities. Teacher satisfaction can be measured through teacher evaluations, which can provide information about how teachers perceive the program, their level of engagement, and their satisfaction with their roles and responsibilities. Student outcomes can be measured through assessments, evaluations, and graduation rates. These can provide information about how well students are learning and achieving their goals. Student outcomes can be measured through assessments, evaluations, and graduation rates. These can provide information about how well students are learning and achieving their goals. Program reputation can be measured through word-of-mouth recommendations, online reviews, and other forms of feedback from students and stakeholders. This can provide insight into how the program is perceived in the community and its overall reputation. By considering these factors in addition to enrollment numbers and revenue generated, the language centre can determine the overall success of the program and make informed decisions about how to improve and grow the program over time. 5. Accountability Administrative Staff: Administrative staff members provide support to the center, including managing registrations and payments, answering phone calls, messages, comments and emails, and managing the center's database. Each of these positions plays an important role in the success of a language center, and they work together to ensure that the center is providing high-quality language instruction and meeting the needs of its students. [1] Mike McKeever (2011). How to Write a Business Plan 10th Edition. Delta Printing Solutions, Inc. [2] Nick Kenny and Bruce Rogers (2015). The Complete Guide To IELTS Student's Book. Cengage Learning. [3] Peter Sommerville (2023). Managing Teacher Quality and Training: A Guide for Language School Managers (e-book). Oxford, UK. [4] Simon Kingsnorth (2019). Digital Marketing Strategy. Kogan Page Limited. 8. Conclusion The report on the new English program, IELTS 4.0, for students who want to graduate from high school without attending the final English exam and other students who want to improve their English to get a better job, found that there is a significant demand for such a program in the market. Through market research, the target audience's preferences and needs were identified, which informed the program's objectives and curriculum. The report outlined the timeline and milestones for the program's development, as well as the budget and resources required for its implementation. The recruitment and training of teachers for the program were also discussed, as were the program's pricing strategies. In terms of marketing, the report recommended various channels for reaching the target audience, including social media, email campaigns, and advertising. The importance of monitoring and evaluating the program's effectiveness was emphasized, and several plans for future improvements and adjustments were proposed, including curriculum revisions, teacher training and development, and enhanced student support. The report also highlighted the importance of establishing monitoring and evaluation systems to assess the program's effectiveness and identify areas for improvement. Overall, the report found that the IELTS 4.0 In terms of marketing, the report recommended various channels for reaching the target audience, including social media, email campaigns, and advertising. The importance of monitoring and evaluating the program's effectiveness was emphasized, and several plans for future improvements and adjustments were proposed, including curriculum revisions, teacher training and development, and enhanced student support. The report also highlighted the importance of establishing monitoring and evaluation systems to assess the program's effectiveness and identify areas for improvement. Overall, the report found that the IELTS 4.0 program has the potential to be a successful and profitable venture for the language center, given the high demand for such a program in the market. 7. Monitoring & evaluation systems Monitoring and evaluation systems are essential for assessing the effectiveness of the English program and identifying areas for improvement. Some key elements of a monitoring and evaluation system for the program above include: Student and teacher feedback: Collecting feedback from students and teachers through surveys, evaluations, and other feedback mechanisms can provide valuable insights into the program's effectiveness, areas for improvement, and overall satisfaction. Assessment data: Monitoring and analyzing student assessment data, such as test scores and graduation rates, can provide insight into student outcomes and identify areas where the program may need improvement. Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 45 Program metrics: Tracking program metrics, such as enrollment numbers, revenue generated, and marketing effectiveness, can provide data-driven insights into the program's performance and areas for improvement. Program metrics: Tracking program metrics, such as enrollment numbers, revenue generated, and marketing effectiveness, can provide data-driven insights into the program's performance and areas for improvement. Performance indicators: Establishing performance indicators, such as student satisfaction rates, teacher engagement levels, and student outcomes, can help to measure the program's success over time and provide a basis for ongoing improvement efforts. Regular program reviews: Conducting regular program reviews, including external evaluations and internal assessments, can provide a comprehensive overview of the program's performance and help to identify areas for improvement. By establishing a robust monitoring and evaluation system that incorporates feedback from students, teachers, and other stakeholders, the language center can ensure the program remains relevant and effective over time and make data-driven decisions about program improvements and adjustments.
https://openalex.org/W4297147185
https://rgu-repository.worktribe.com/file/1764089/1/SEDDIKI%202022%20Development%20and%20pilot%20%28VOR%29
English
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Development and Pilot Evaluation of an Online Retrofit Decision-Making Tool for Homeowners
Buildings
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cc-by
11,764
SEDDIKI, M., BENNADJI, A., ALABID, J., GRAY, D. and DEVECI, G. 2022. Development and pilot evaluation of an online retrofit decision-making tool for homeowners. Buildings [online], 12(10), article 1513. Available from: https://doi.org/10.3390/buildings12101513 SEDDIKI, M., BENNADJI, A., ALABID, J., GRAY, D. and DEVECI, G. 2022. Development and pilot evaluation of an online retrofit decision-making tool for homeowners. Buildings [online], 12(10), article 1513. Available from: https://doi.org/10.3390/buildings12101513 SEDDIKI, M., BENNADJI, A., ALABID, J., GRAY, D. and DEVECI, G. 2022. Development and pilot evaluation of an online retrofit decision-making tool for homeowners. Buildings [online], 12(10), article 1513. Available from: https://doi.org/10.3390/buildings12101513 Citation: Seddiki, M.; Bennadji, A.; Alabid, J.; Gray, D.; Deveci, G. Development and Pilot Evaluation of an Online Retrofit Decision-Making Tool for Homeowners. Buildings 2022, 12, 1513. https://doi.org/10.3390/ buildings12101513 Keywords: decision-making; online tool; retrofit; pilot evaluation; homeowners; moisture-safe Academic Editor: Antonio Caggiano Article Mohammed Seddiki 1,*, Amar Bennadji 1, Jamal Alabid 2, David Gray 3 and Gokay Deveci 1 1 Scott Sutherland School of Architecture and Built Environment, Robert Gordon University, Garthdee House, Garthdee Road, Aberdeen AB10 7QB, UK 1 Scott Sutherland School of Architecture and Built Environment, Robert Gordon University, Garthdee House Garthdee Road, Aberdeen AB10 7QB, UK 2 Faculty of Engineering & Informatics, University of Bradford, Bradfor BD7 1DP, UK 3 C lt l d C ti B i S h l R b t G d U i it Ab d AB10 7QB UK 2 Faculty of Engineering & Informatics, University of Bradford, Bradfor BD7 1DP, UK 3 Cultural and Creative Business School, Robert Gordon University, Aberdeen AB10 7QB, UK * Correspondence: m.seddiki1@rgu.ac.uk Abstract: Many retrofit projects went wrong in the UK principally because of the application of inappropriate retrofit solutions, which resulted in damp issues, with some leaving houses in worse conditions than pre-retrofit. Various online tools were developed to inform homeowners about the benefits of retrofitting. Prior to this study, little was known about users’ evaluation of these tools and the effects of calculator use. Furthermore, no retrofit tool aims to raise the awareness of homeowners about moisture risks in a retrofit project. The originality of this study is to develop and evaluate an online moisture-safe retrofit decision-making tool for homeowners. The adopted methodology consisted of two phases. Phase one aimed to develop the tool. In phase two, semi- structured interviews were conducted to evaluate the tool. The results indicate that the tool has been well received by homeowners. The tool significantly increased participants’ awareness of moisture risks related to a retrofit project. Most participants considered the tool an eye-opener, while few of them found it scary. However, the tool did not result in an increased willingness to invest in energy efficiency measures. The discouragement was related to high investment costs and long payback periods of some retrofit measures. Based on our findings, we formulate a set of design recommendations to improve the proposed tool and help retrofit calculators, in general, overcome challenges. Development and pilot evaluation of an online retrofit decision-making tool for homeowners. 2022 © 2022 by the authors. Licensee MDPI, Basel, Switzerland. © 2022 by the authors. Licensee MDPI, Basel, Switzerland. © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This document was downloaded from https://openair.rgu.ac.uk buildings buildings buildings 1. Introduction Several retrofit tools are available in the UK to help homeowners understand how their houses and flats use energy and how their home’s energy is used. The identified tools in the UK are “Home Energy Saving” tool developed by Halifax (part of Lloyds Banking Group) [9], “Energy Efficiency calculator” developed by the UK government [10], “Home Energy Check tool” developed by home energy Scotland [11], “AECB Home Energy Check” developed by AECB—the sustainable building association [12], “Quick Scan tool” devel- oped by the city of Aberdeen (Scotland) [13], and “Reducing home heat loss” developed by energy saving trust [14]. Another tool available for England that is worth mentioning even though it is not destinated for homeowners and targets professionals is the TABULA tool which was developed as part of the European project EPISCOPE [15]. Most available tools for homeowners evaluate retrofit options by taking into consid- eration energetic, financial, and environmental criteria, while other key characteristics that need to be considered when selecting an appropriate package of improvements are neglected, such as the disruptions caused by the renovation work or more importantly the risks related to moisture after a retrofitting work [6]. In fact, the perceived hassle of installation has been found to be one of the factors influ- encing house owners’ preferences on energy retrofits [16]. As indicated in Glew et al. [17], disruption is an important indicator because it can be a serious barrier to the implementa- tion of retrofit measures, especially if the property cannot be used while the retrofit work is carried out. Another crucial aspect is the moisture risk in retrofitting projects. Many homeowners are reluctant to undertake renovation works, particularly in traditional houses, because they are afraid that this may cause damp and mould growth issues in the building [18] which would negatively affect the value of the building, the energy performance, and the health of occupants. These worries are sustained by the fact that during the last decade, a large number of retrofit projects went wrong in the UK mainly because of the application of inappropriate retrofit solutions which resulted in damp and mould growth issues leaving homeowners with houses in a worse condition than pre-retrofit [19]. 1. Introduction In 2020, the UK residential sector emitted 67.7 MtCO2, accounting for 20.8% of all carbon dioxide emissions [1]. UK homes’ greenhouse gas emissions need to fall by at least 24% by 2030 from 1990 levels to meet the UK’s ambitious goal, which is reaching zero emissions from existing buildings in 2050 [2]. The current rate of retrofitting progress is around 9000 measures installed per week across the whole UK housing stock and needs to rise by around seven times to meet its climate targets. The UK has the ambition to retrofit almost all homes to a minimum of Energy Performance Certificate (EPC) band C standard by 2035 [3]. Published: 23 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. y Researchers have found that insufficient support measures hinder homeowners from investing in energy renovations and recommended states to identify a set of support measures that is appropriate to homeowners by involving them in the preparation and implementation of their strategies [4]. It has been found that information on the capital cost of energy efficiency measures and the amount of energy savings is one of the strongest incentives to encourage homeowners to invest in energy renovation [5]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). g gy There have been several projects that aimed to develop simple energy tools to assist homeowners in their renovation projects by providing them with information on energy improvement solutions suitable for their homes. Seddiki et al. [6] provided a comprehensive https://www.mdpi.com/journal/buildings Buildings 2022, 12, 1513. https://doi.org/10.3390/buildings12101513 Buildings 2022, 12, 1513 2 of 18 literature review on existing energy retrofit web decision tools available for homeowners. For instance, Request2Action, which is a European Union Intelligent Energy programme funded project, developed a web-based tool named Home Energy Check (HEC) that aims to familiarise the user with the different available energy improvement solutions and to highlight the implementation of EPCs recommendations and energy savings achieved [7]. Elsewhere, the city of Ghent in Belgium developed a user-friendly web application, “Check Je Huis”, that guides homeowners/tenants in their renovation projects. The appli- cation helps users to identify the energy efficiency of their home and the possibilities of improving them [8]. 1. Introduction Not only does this undermine the UK target to meet net zero by 2050, but it also costs a fortune to remove the applied measures and redo the work for already vulnerable householders having generally to rectify problems themselves, not to mention the disturbance, stress, and often health issues caused to homeowners. In fact, damp and mould development due to poorly designed and installed retrofit measures are likely to cause respiratory problems, respiratory infections, allergies, or asthma [20]. A report produced by the UK Office of Gas and Electricity Markets (Ofgem’s) indicated that 6.9% of the almost 1.5 million measures installed in the UK between January 2013 and March 2015 were inspected, and 9.9% of the examined measures haven’t been installed correctly in the first instance and required additional work to be undertaken [21]. After the observation that many retrofit projects failed, the Secretaries of State of the Department for Energy and Climate Change (DECC-now BEIS) and the Department for Communities and Local Government (DCLG-now MHCLG) commissioned an industry-led report “Each Home Counts review” [21]. The aims of the review were to restore the confi- dence of consumers and of Government in the retrofit industry in order to facilitate new central and local government policies to promote and support a national programme of domestic retrofit. The recommendation made in the report “Each Home Counts review” led Buildings 2022, 12, 1513 3 of 18 3 of 18 the UK government to introduce the PAS2035 standard, which provides a comprehensive methodology on how to perform a retrofit project. The standard introduced a new actor, namely the retrofit coordinator, which is responsible for overseeing the assessment of dwellings, elaborating a risk assessment and management plan, as well as the subsequent specification, monitoring, and evaluation of energy efficiency measures, in accordance with PAS 2035. The retrofit coordinator should have an adequate training to assess moisture risks related to the implementation of retrofit measures which is anticipated to prevent catas- trophic situations. However, PAS 2035 is currently compulsory only for public buildings, while for private buildings, landlords and homeowners could install retrofitting measures simply on the basis of an assessment of installers or domestic energy assessors which are not qualified to assess moisture risks and related issues in retrofit measures [22]. q Hence, including moisture risks assessment in an energy retrofit online tool for home- owners presents various advantages. 1. Introduction On one hand, this could reassure reluctant homeown- ers that are afraid of moisture issues on the feasibility of managing the risks and would likely encourage them to invest in retrofit measures. On the other hand, for homeowners that are not aware of moisture risks, the online tool would prevent them from implementing risky solutions recommended by a retrofit assessor or an installer and would raise their awareness on moisture risks. This paper extends the current knowledge by including moisture risks in an online energy retrofit tool for homeowners. In contrast, existing tools evaluate retrofit options by considering only energy, financial, and environmental criteria. Furthermore, little is known about the (positive and negative, intended and unintended) effects retrofit calculators may have on the awareness and the willingness to pursue renovations of their users. The proposed paper extends existing findings by shedding light on the experiences of retrofit calculator users. The paper focuses on the (1) users’ acceptance of and satisfaction with the developed retrofit calculator, (2) effects of the tool on moisture awareness and behaviour, and (3) effects of the tool on the willingness to invest in energy efficiency. 2.2. Creation and Validation of Energy Models of Building Typologies Energy models were developed using the Standard Assessment Procedure (SAP), which is the methodology used by the UK government to assess and compare the energy and environmental performance of dwellings. SAP quantifies a dwelling’s performance in terms of energy use per unit floor area, a fuel-cost-based energy efficiency rating (the SAP Rating) and emissions of CO2 (the Environmental Impact Rating). Other SAP outputs include space heating demand, total delivered energy, cooling load, and so on. PAS 2035 recommends using Full SAP for retrofit assessments [22]. The assumptions regarding building typologies can be found in Bennadji et al. [23]. g yp g j [ ] To validate the energy models, the calculated energy efficiency ratings (the SAP Rating) of the building typologies were compared with the median energy efficiency rating from statistics as it is the only indicator available for building typologies in the four UK countries. Moreover, it was not possible to compare the SAP rating of the 20 building types separately as the data available in the statistics indicates median SAP ratings for different UK countries either per dwelling type or per dwelling age, and there are no data that combines these two factors in a matrix of typologies. Median SAP ratings were retrieved from the office of national statistics for England and Wales [24], the Scottish house condition survey 2019 [25], and the Northern Ireland house condition survey 2016 [26]. g yp g j To validate the energy models, the calculated energy efficiency ratings (the SAP Rating) of the building typologies were compared with the median energy efficiency rating from statistics as it is the only indicator available for building typologies in the four UK countries. Moreover, it was not possible to compare the SAP rating of the 20 building types l h d l bl h d d f d ff To validate the energy models, the calculated energy efficiency ratings (the SAP Rating) of the building typologies were compared with the median energy efficiency rating from statistics as it is the only indicator available for building typologies in the four UK countries. Moreover, it was not possible to compare the SAP rating of the 20 building types separately as the data available in the statistics indicates median SAP ratings for different UK countries either per dwelling type or per dwelling age, and there are no data that combines these two factors in a matrix of typologies. 2. Materials and Methods As indicated in Seddiki et al. [6], four major methodologies have been adopted in developing energy retrofit tools for homeowners; empirical data-driven methods, pre- simulated databases, simplified normative calculation methods, and advanced calculation methods. In this paper, the tool development was based on a database of multicriteria assessments carried out for 20 building typologies representative of the UK housing stock. In a pre-simulated database, the energy performance of several combinations of building envelope, HVAC systems, and renewable energy integration is predicted using detailed numerical models of representative buildings. The choice to base the developed moisture- safe tool on a pre-simulated database is motivated by the fact that this approach allows the creation of extremely easy to use tools as they require few inputs and are highly accurate as simulations are performed in advanced simulation engines. The methodology used to create the tool consists of different steps (Figure 1): First, building typologies representative of the UK housing stock were defined, then the energy model of each building typology was created and validated against statistical data. Then, individual retrofit measures as well as packages of solutions were generated. Afterward, the retrofitting measures were evaluated according to four criteria: energy saving, capital cost, disruption level, and moisture risks. Then, the results of the multicriteria assessment were compiled into a user-friendly online tool. Finally, a pilot evaluation was performed to collect feedback about the tool and requirements for user interface design. A detailed explanation of the different steps is presented below: VIEW 4 of 18 Fi 1 Th th d l d t t th li t l Definition of building typologies Reference building models and validation Generation of retrofit measures Multicriteria evaluation Prototype website development Pilot evaluation Figure 1. The methodology used to create the online tool. Figure 1. The methodology used to create the online tool. Buildings 2022, 12, 1513 4 of 18 2.1. Definition of Building Typologies 2.1. Definition of Building Typologies The first step in developing the moisture-safe energy calculator, which is based on a pre- simulated database, is to define building typologies representative of the UK housing stock. The number of reference buildings is directly related to the accuracy of the data generated by the online tool for homeowners; the greater the number of reference buildings is, the more accurate the results are. However, increasing the number of building typologies makes the development of the tool extremely time-consuming, which is an inherent limitation of the pre-simulated database approach. Hence, a compromise should be reached between the number of simulations and the accuracy of the data provided to homeowners. The reference buildings developed by Bennadji et al. [23] as part of the European project Stronghouse and based on the European project EPISCOPE [15] were adopted in this paper to represent the housing stock of the UK for their geometric data, construction, and energy-related properties. gy p p This study considered 20 building typologies split into five construction periods (pre–1919, 1919–1944, 1945–1964, 1965–1980, post–1980) and four building sizes, including single-family house (SFH), terraced house (TH), multi-family house (MFH), and apartment block (AB). The assumptions regarding building typologies in Bennadji et al. [23] took into consideration the current condition of the housing stock as opposed to the EPISCOPE project, where the energy efficiency characteristics of building typologies have been defined for un-modernised conditions. 2.4. Multicriteria Assessment of Renovation Alternatives To provide users with sufficient information to help them proceed in their renovation journey, individual retrofit measures and packages of solutions were evaluated according to the various criteria indicated in PAS 2035. A brief description of the evaluation criteria recommended by PAS 2035 is presented as follows. 2.3. Generation of Retrofit Measures The generated retrofit measures took into consideration three areas of improvement, which were the building envelope, the building services, and renewable energies. The measures targeted a level close to nearly zero building energy requirements, following the concept that if a retrofit measure is implemented, it must be highly energy efficient to avoid missed opportunities, especially in a step-by-step retrofit which represents about 80% of retrofits in reality [28]. In fact, a building that starts with a shallow retrofit will likely never achieve a high level of energy performance in the future, which is called “the locked in effect” [29]. Retrofit measures were evaluated individually and in packages to take into consideration the integrated effect. The saving attributable to a package of improvement measures will be less than the sum of the savings attributable to individual measures. For each housing type, one pre-retrofit simulation was performed, between eleven (for single-family and terraced houses) and five (multifamily houses and apartment blocks) single Energy Efficiency Measures (EEMs), and one application of all EEMs for a total of about 157 simulations. The list of the selected energy improvement measures included exclusively measures that are commonly implemented in UK retrofitting projects. The retrofitting measures were customised to each building typology. For example, an ASHP was simulated for single-family and terraced houses, while a new efficient boiler was simulated for multifamily houses and apartment blocks. The general assumptions of the retrofit measures and the energy simulations are detailed in Supplementary Material S1 (General assumptions of the retrofit measures). 2.2. Creation and Validation of Energy Models of Building Typologies Median SAP ratings were retrieved from the office of national statistics for England and Wales [24], the Scottish house condition survey 2019 [25], and the Northern Ireland house condition survey 2016 [26]. Therefore, to allow the comparison of the calculated data and the statistics, median energy efficiency scores were calculated per dwelling type and per dwelling age. So, for example, the median calculated SAP score for SFH represents the average calculated SAP scores of 5 building types (SFH Pre–1919, SFH 1919–1944, SFH 1945–1964, SFH 1965–1980, and SFH Post–1980). Similarly, the median calculated SAP score for buildings built pre–1919 represents the average calculated SAP scores of 4 building types (SFH Pre–1919, TH Pre–1919, MFH Pre–1919, and AB Pre–1919). The comparison of median SAP scores from statistics and calculated median SAP scores is presented in Table 1. The result of the comparison reveals an overestimation of about ±10 EPC points, which is considered acceptable as indicated in Crawley et al. [27] which compared values from the UK national database of all registered EPCs for properties that have had more than one EPC. Buildings 2022, 12, 1513 5 of 18 Table 1. Comparison of median SAP rating from statistics and calculated median SAP rating. Table 1. Comparison of median SAP rating from statistics and calculated median SAP rating. Median Energy Efficiency Scores (MEES) from Statistics Calculated MEES Dwelling Type England Scotland Wales Northern Ireland UK UK Difference MEES SFH 63 61 62 65 62.75 68 5.25 TH 65 65 62 68 65 69 4 MFH 72 67 58 74 67.75 73 5.25 AB 72 68 70 77 71.75 74 2.25 Dwelling Age Pre–1919 57 56 55 52 55 66 11 1919–1944 57 62 59 61 59.75 70 10.25 1945–1964 64 64 62 64 63.5 70 6.5 1965–1980 63 66 64 67 65 71 6 Post–1980 71 73 73 72 72.25 77 4.75 2.4.3. The Simple Payback Period A simple payback period (in years) is the capital cost of the EEM or a package of EEMs divided by the estimated annual fuel cost savings [22]. 2.4.2. Capital Cost The capital cost took into consideration only the cash flow required to execute the renovation work (Gross Capital Cost), while concepts such as the ongoing need for capital upgrades not related to energy efficiency, the avoided capital costs, the cost of savings through design, and the cost of subsidies and incentives were not considered. Assumptions for the capital costs of retrofit solutions were retrieved from the latest updated assumptions published by the UK Department of Energy & Climate Change about the costs of different energy efficiency interventions in existing homes [30], from the cost analysis report of the Retrofit for the Future programme [31] and from other sources [32,33]. 2.4.1. Annual Energy Cost Saving The annual energy cost savings of the retrofit measures were evaluated using the SAP method. PAS 2035 recommends using Full SAP for retrofit assessments [22]. Retrofit measures were evaluated individually and in packages to take into consideration the integrated effect. The fuel costs embedded in the SAP energy rating were used and the results for each building typology were expressed in GBP/year. Buildings 2022, 12, 1513 6 of 18 2.4.4. Disruption Level The level of disruption to occupants has been recently introduced by the UK standard PAS 2035 as well as the retrofit academy in the retrofit coordinator course as a key element in the evaluation of retrofit solutions [34]. Other scientific studies highlighted the importance of inconvenience and disruption during retrofit activities on the decision of households to invest in energy renovation [35]. Issues such as the duration of the work and the incapacity to occupy the building for a prolonged period are important elements of households’ decision process along with other aspects such as cost and energy savings. Hence, it is important that occupants have a clear idea about how the retrofit work will affect their use of the building. Assumptions regarding the level of disruption of each renovation measures were expressed in a qualitative manner. The score of renovations measures were taken from the work carried out by the experts of the retrofit academy (see Table 2). Table 2. Disruption levels [34]. Disruption Actions Minimal Low-energy lamps, energy-efficient appliances Low Heating control, cavity wall insulation, draught stripping, loft insulation Moderate Replacement boiler, solar water heating, solar PV panels High Replacement windows, whole house ventilation, external wall insulation Significant Ground floor insulation, internal wall insulation, new heating installation Table 2. Disruption levels [34]. Disruption Actions Minimal Low-energy lamps, energy-efficient appliances Low Heating control, cavity wall insulation, draught stripping, loft insulation Moderate Replacement boiler, solar water heating, solar PV panels High Replacement windows, whole house ventilation, external wall insulation Significant Ground floor insulation, internal wall insulation, new heating installation Table 2. Disruption levels [34]. 1. Green icon: means that the solution does not present a risk of moisture. 2.5. Prototype Website Development 2.5. Prototype Website Development The aim of this step is to deploy the pre-simulated retrofit data on a prototype dy- namic website. The content management system Wix was used to developing the Portal. Content management systems such as Wix and WordPress have been widely used in the literature to develop prototype websites. For example, Ali et al. [37] used WordPress for the development of a custom app radiator control system for retrofitting legacy heating systems in existing buildings. Similarly, Carvalho et al. [38] used Wordpress to create a web portal for COVID-19 information in Brazil, which helps users publish, organise, and manage content posted on the web. Merritt & Zhao [39] used WIX in order to develop a website for a prototype supermarket. Authors argued that this method offers an intuitive drag and drop site building which can outline the visual aspects of the site. The aim of this step is to deploy the pre-simulated retrofit data on a prototype dy- namic website. The content management system Wix was used to developing the Portal. Content management systems such as Wix and WordPress have been widely used in the literature to develop prototype websites. For example, Ali et al., [37] used WordPress for the development of a custom app radiator control system for retrofitting legacy heating systems in existing buildings. Similarly, Carvalho et al., [38] used Wordpress to create a web portal for COVID-19 information in Brazil, which helps users publish, organise, and manage content posted on the web. Merritt & Zhao [39] used WIX in order to develop a website for a prototype supermarket. Authors argued that this method offers an intuitive drag and drop site building which can outline the visual aspects of the site. The website front end of the proposed prototype in the paper was developed using the free and code-free website builder Wix. The purpose of selecting this method was that it is an easy-to-use editor, the system is intuitive, accessibility is built into the system, and the drag and drop functions make editing pages or updating content fairly straightforward (Figure 2). The website front end of the proposed prototype in the paper was developed using the free and code-free website builder Wix. 2.4.5. Moisture Risks As mentioned previously, one of the main contributions of this paper is to include an assessment of each suggested retrofit solution in terms of moisture risks. Moisture safe design does not mean that the risks of moisture are all eliminated; it only means that the causes and consequences of moisture are understood, and that appropriate actions are taken to reduce these risks. Often there will be conflicts between the different objectives (energy saving, moisture, etc.). Therefore, a compromise should be reached, and measures to manage the accepted risks should be installed. In some extreme cases, it is even recommended to not insulate because of the high risk of moisture. Moisture risks can be evaluated using dynamic hygrothermal simulation (WUFI), condensation risk analysis (Glaser method), calculation of the properties of thermal bridges and prescriptive guidance [34]. In this paper moisture risks of each solution were evaluated using the UK perspective guidance “Management of moisture in buildings–Code of practice” BS 5020 [36]. For each retrofit solution, the tool presents the results of moisture risks using coloured icons: 1. Green icon: means that the solution does not present a risk of moisture. 1. Green icon: means that the solution does not present a risk of moisture. Buildings 2022, 12, 1513 7 of 18 e risks 7 of 18 e risks 2. Red Icon: means there is a risk, but the situation may not be serious. It is advisable to further investigate the solution by numerical simulations. 2. Red Icon: means there is a risk, but the situation may not be serious. It is advisable to further investigate the solution by numerical simulations. h l f h f l f k d h f The evaluation of each retrofit solution in terms of moisture risks and the information displayed for the user are detailed in Supplementary Material S2 (moisture risks). The evaluation of each retrofit solution in terms of moisture risks and the information displayed for the user are detailed in Supplementary Material S2 (moisture risks). 2.5. Prototype Website Development 2.5. Prototype Website Development The purpose of selecting this method was that it is an easy-to-use editor, the system is intuitive, accessibility is built into the system, and the drag and drop functions make editing pages or updating content fairly straightfor- ward (Figure 2). Figure 2. Wix drag-and-drop functionality. Figure 2. Wix drag-and-drop functionality. The website back end was developed using the content manager Wix functionality. Content collections were created to store information about retrofit measures. The content is stored in a grid layout made up of items (rows) and fields (columns) (Figure 3). 2.6. Pilot Evaluation 2.6. Pilot Evaluation The pilot study involved semi-structured interviews with homeowners that were asked to test the online tool before the interviews. The objectives of the pilot evaluation were to investigate (1) users’ acceptance of and satisfaction with the developed retrofit calculator, (2) effects of the tool on moisture awareness and behaviour, and (3) effects of the tool on the willingness to invest in energy efficiency. The pilot also aimed to collect feedback about the tool and requirements for user interface design. The pilot study involved semi-structured interviews with homeowners that were asked to test the online tool before the interviews. The objectives of the pilot evaluation were to investigate (1) users’ acceptance of and satisfaction with the developed retrofit calculator, (2) effects of the tool on moisture awareness and behaviour, and (3) effects of the tool on the willingness to invest in energy efficiency. The pilot also aimed to collect feedback about the tool and requirements for user interface design. q g Semi-structured interviews have been wildly used in the literature to test pilot new web-based tools [40–42]. The main advantage of conducting interviews is that they pro- vide in-depth information, as they allow the interviewer to expose information regarding the online tool that are unlikely to be captured through questionnaires [43]. Semi-structured interviews have been wildly used in the literature to test pilot new web-based tools [40–42]. The main advantage of conducting interviews is that they provide in-depth information, as they allow the interviewer to expose information regarding the online tool that are unlikely to be captured through questionnaires [43]. Figure 2. Wix drag-and-drop functionality. Figure 2. Wix drag-and-drop functionality. The website back end was developed using the content manager Wix functionality. Content collections were created to store information about retrofit measures. The content is stored in a grid layout made up of items (rows) and fields (columns) (Figure 3). Buildings 2022, 12, 1513 8 of 18 nality. ontent Figure 3. Data entered into Wix Content Manager. Figure 3. Data entered into Wix Content Manager. Figure 3. Data entered into Wix Content Manager. Figure 3. Data entered into Wix Content Manager. 2.6.1. Participants 2.6.1. Participants p The potential testers were recruited through a snowball sampling method, which means the author identified a few testers through convenience sampling who then indi- cated the contact information of other potential testers and so forth. Eligible participants (i) were homeowners; (ii) were located in the UK; (ⅲ) were able to read English profi- ciently, and (ⅳ) were able to give informed consent. A number of studies in the literature have successfully used snowball sampling in order to pilot online tools [43–45]. From May 2022 to July 2022, a sum of 15 homeowners were interviewed. The participants were aged between 30 to 65 years and had a broad range of educational backgrounds. Their proper- ties were built from pre 1919 to post 1980 The potential testers were recruited through a snowball sampling method, which means the author identified a few testers through convenience sampling who then indi- cated the contact information of other potential testers and so forth. Eligible participants (i) were homeowners; (ii) were located in the UK; (iii) were able to read English proficiently, and (iv) were able to give informed consent. A number of studies in the literature have successfully used snowball sampling in order to pilot online tools [43–45]. From May 2022 to July 2022, a sum of 15 homeowners were interviewed. The participants were aged between 30 to 65 years and had a broad range of educational backgrounds. Their properties were built from pre–1919 to post–1980. 2 6 2 Data Collectio a d A alysis 2.6.2. Data Collection and Analysis 2.6.2. Data Collection and Analysis Participants were contacted via email and provided with the following information: (1) an information letter outlining study details and requirements of participants; (2) a link to the web-based tool (https://mseddiki1.wixsite.com/my-site); and (3) a consent form. The interview questions included: “Was the tool easy to use?” and “What did you think of the Participants were contacted via email and provided with the following information: (1) an information letter outlining study details and requirements of participants; (2) a link to the web-based tool (https://mseddiki1.wixsite.com/my-site); and (3) a consent form. The interview questions included: “Was the tool easy to use?” and “What did you think of the amount of information in the tool?” Participants were also asked what they liked or disliked about the website, how it could be improved, and how the tool affected their understanding of energy improvement options and moisture risks in a retrofit project. Buildings 2022, 12, 1513 9 of 18 ners w 9 of 18 ners w Supplementary Material S3 contains the interview guide used in this research. To verify the guideline for the interview, pilot interviews of two homeowners were carried out before the formal semi-structured interview (the pilot interviews were not included in the sum of 15 respondents). The results of the pilot interviews helped to improve the guideline for the interview. i e u o e po e ) e e u o e pi o i e ie e pe o i guideline for the interview. Interviews were conducted and recorded using Zoom and transcribed into documents. Interviews lasted between 30 and 45 minutes. Content analysis usi ware NVivo has been adopted to analyse the qualitative data from interview Interviews were conducted and recorded using Zoom and transcribed into electronic documents. Interviews lasted between 30 and 45 min. Content analysis using the software NVivo has been adopted to analyse the qualitative data from interview transcripts. Content analysis has been commonly used in piloting of tool studies [43,46,47]. Content analysis was chosen as it allows a descriptive approach in both coding of the data and its interpretation of quantitative counts of the codes [48]. Content analysis has been commonly used in piloting of tool studies [43,46,47 analysis was chosen as it allows a descriptive approach in both coding of the d interpretation of quantitative counts of the codes [48]. 3. Prototype Demonstration 3. Prototype Demonstration The user interface is As homeowners are unlik The user interface is crucial, as it is the connection between a person and the system. As homeowners are unlikely to be knowledgeable about moisture risks inherent in retrofit projects, a simple web-based moisture-safe retrofit decision-making tool has been developed for them. Some of the interface forms are shown and explained below. Additional details are available on the tool’s website [49]. The tool interface and procedure for its operation involve three basic steps: y g projects, a simple web-based moisture-safe retrofit decision-making tool has b oped for them. Some of the interface forms are shown and explained below. details are available on the tool’s website [49]. The tool interface and proced operation involve three basic steps: Step 1: Aims to present the tool and educate homeowners on moisture ma Step 1: Aims to present the tool and educate homeowners on moisture management in buildings. p p in buildings. Figure 4 shows “My home page” which is the very first phase of the decisio Figure 4 shows “My home page”, which is the very first phase of the decision process. In this phase, the tool provides information concerning the moisture-safe retrofit calculator, moisture in buildings, the importance of moisture management in a retrofit project, the consequences of moisture unbalance retrofit, and the user interface of the tool. Figure 4 shows My home page , which is the very first phase of the decisio In this phase, the tool provides information concerning the moisture-safe retro tor, moisture in buildings, the importance of moisture management in a retro the consequences of moisture unbalance retrofit, and the user interface of the t Figure 4. My home page. Figure 4. My home page. Step 2: Input the building type. Step 2: Input the building type. In this step users are asked to se Step 2: Input the building type. Step 2: Input the building type. In this step users are asked to se In this step, users are asked to select their dwelling type (Figure 5). The tool auto- matically provides information regarding existing specifications and energy improvement solutions according to the selected building type. The tool considers 20 building typologies split into five construction periods (pre–1919, 1919–1944, 1945–1964, 1965–1980, post–1980) and four building sizes, including single-family houses, terraced houses, multifamily houses, and apartment blocks. Single-family houses encompass detached houses and bungalows. Terraced house includes end and mid terraced houses as well as semi-detached houses. Multifamily house includes converted and low-rise flats. Apartment block includes purpose built and high-rise flats. The reference buildings are based on the European project EPISCOPE [15]. In this step, users are asked to select their dwelling type (Figure 5). The tool automat- ically provides information regarding existing specifications and energy improvement so- lutions according to the selected building type. The tool considers 20 building typologies split into five construction periods (pre–1919, 1919–1944, 1945–1964, 1965–1980, post– 1980) and four building sizes, including single-family houses, terraced houses, multifam- ily houses, and apartment blocks. Single-family houses encompass detached houses and bungalows. Terraced house includes end and mid terraced houses as well as semi-de- tached houses. Multifamily house includes converted and low-rise flats. Apartment block includes purpose built and high-rise flats. The reference buildings are based on the Euro- pean project EPISCOPE [15]. Figure 5. Select your dwelling type page. Figure 5. Select your dwelling type page. Figure 5. Select your dwelling type page. Figure 5. Select your dwelling type page. Step 3: Display the results. Step 3: Display the results. p p y This step shows the output results of the system. The first section, “Existing specifi- cations”, of the results page provides information on the existing specifications of the se- lected building type, which are based on assumptions in Bennadji et al., [23] and based on Loga et al., [15]. The displayed data (Figure 6) includes property size, type of windows, walls, roof, floor, heating system, ventilation, airtightness, final energy consumption, and Energy Performance Certificate (EPC). p p y This step shows the output results of the system. The first section, “Existing speci- fications”, of the results page provides information on the existing specifications of the selected building type, which are based on assumptions in Bennadji et al. Figure 4. My home pag Figure 4. My home page. Buildings 2022, 12, 1513 Buildings 2022, 12, x FOR 10 of 18 10 of 18 10 of 18 10 of 18 Step 2: Input the building type. Step 2: Input the building type. In this step users are asked to se [23] and based on Loga et al. [15]. The displayed data (Figure 6) includes property size, type of windows, walls, roof, floor, heating system, ventilation, airtightness, final energy consumption, and Energy Performance Certificate (EPC). gy ( ) gy The second section, “Your suggested energy efficiency home improvements”, provides a list of suggested energy efficiency home improvements (Figure 7). 11 of 18 11 of 1 Buildings 2022, 12, 1513 ildings 2022, 12, x FOR PE Figure 6. Existing specifications section. The second section, “Your suggested energy efficiency home imp vides a list of suggested energy efficiency home improvements (Figure Figure 6. Existing specifications section. Figure 6. Existing specifications section. The second section, “Your suggested energy efficiency home improv vides a list of suggested energy efficiency home improvements (Figure 7). Figure 7. Your suggested energy efficiency home improvements section. Figure 6. Existing specifications section. The second section, “Your suggested energy efficiency home imp Figure 6. Existing specifications section. Figure 6. Existing specifications section. vides a list of suggested energy efficie Figure 6. Existing specifications section. Figure 6. Existing specifications section. vides a list of suggested energy efficie Figure 6. Existing specifications section. Figure 6. Existing specifications section. vides a list of suggested energy efficien g g p The second section, “Your suggested energy efficiency home imp vides a list of suggested energy efficiency home improvements (Figure Figure 7. Your suggested energy efficiency home improvements section. The second section, “Your suggested energy efficien Figure 7. Your suggested energy efficiency home improvements section. Buildings 2022, 12, 1513 12 of 18 12 of 18 Each solution is evaluated individually in terms of estimated cost, yearly savings, payback period, disruption, and risk of moisture. In addition, users can click the “i” icon to learn more about moisture risks related to each solution (Figure 8). Information includes simplified explanations of moisture risks as well as mitigation strategies. Each solution is evaluated individually in terms of estimated cost, yearly savings, payback period, disruption, and risk of moisture. In addition, users can click the “i” icon to learn more about moisture risks related to each solution (Figure 8). Information in- cludes simplified explanations of moisture risks as well as mitigation strategies. Each solution is evaluated individually in terms of estimated cost, yearly savings, payback period, disruption, and risk of moisture. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. In the third section, “Upgrade recommendation summary”, the upgrade recommen- dation summary (Figure 9) shows the effect of undertaking all of the improvement measures listed in your suggested energy efficiency home improvements table. Again, the package of solutions is evaluated in terms of estimated cost, yearly savings, payback pe- riod, disruption, and risk of moisture. In the third section, “Upgrade recommendation summary”, the upgrade recommenda- tion summary (Figure 9) shows the effect of undertaking all of the improvement measures listed in your suggested energy efficiency home improvements table. Again, the package of solutions is evaluated in terms of estimated cost, yearly savings, payback period, disruption, and risk of moisture. In the third section, Upgrade recommendation summary , the upgrade recommen- dation summary (Figure 9) shows the effect of undertaking all of the improvement measures listed in your suggested energy efficiency home improvements table. Again, the package of solutions is evaluated in terms of estimated cost, yearly savings, payback pe- riod, disruption, and risk of moisture. Step 2: Input the building type. Step 2: Input the building type. In this step users are asked to se In addition, users can click the “i” icon to learn more about moisture risks related to each solution (Figure 8). Information in- cludes simplified explanations of moisture risks as well as mitigation strategies. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. 4.1. User Acceptance and Satisfaction The pilot-testing phase revealed that the tool was positively received, with all partic- ipants reporting that the website was easy to use and easy to navigate. The majority of participants found that they could understand the information. Participants highlighted the importance of a retrofit tool to combine text explanations with visual illustrations (P1: “you can better understand with images”). “These findings are in line with those by Kok and Barendregt [50]. Participants agreed that data entry was easy and not too time-consuming. y g Preferably, online retrofit tools should not require users to enter technical details (P3: “I was thinking maybe I need to measure space in my home [ . . . ] what kind of windows do I have, or what sort of walls, do I have, so the website did that, for me, by just saying just give us what type of property and we’ll do the rest which is a relief because you don’t want obviously to overload the user with the burden of finding out all these technical details”). In fact, it is argued that many people prefer a quick-test calculator over a more comprehensive version [51]. Participants indicated that the tool would have been more accessible if it had been adapted to phones’ displays (P7: “I have tried to open the website on my mobile phone, but it didn’t work”). Indeed, scholars have argued that mobile-like advice tool should be used in energy retrofit communication [44]. Moreover, most participants felt the amount of information was sufficient and that no parts needed to be left out or removed. Participants liked to see the multicriteria evaluation of the retrofit solutions (P11: “the breakdown and the detail provided in terms of the estimated cost and yearly savings and the payback period that was really kind of very helpful in kind of giving me kind of understanding of options going forward”). Participants also appreciated the possibility to click “i” icon to learn more about moisture risks related to each solution (P3: “very much appreciated also to have the additional information next to each of the recommendations, so if I wanted to learn more about each one of the types of improvement, I can go to the to the relevant sites”). They liked that the tool indicates how to find accredited professionals (P9: “The feedback at the end and how to contact a contractor make things easy”). Figure 9. Upgrade recommendation summary. Figure 9. Upgrade recommendation summary. Buildings 2022, 12, 1513 13 of 18 13 of 18 The fourth section, “Your step-by-step retrofit plan”, provides advice regarding the sequencing of the renovation work in the case where the package of solutions cannot be implemented at once (lack of time, money, too much inconvenience, or any other reason). This section introduces the concept of a “medium-term improvement plan” according to the British standard for domestic retrofitting PAS2035. The tool only aims to raise homeowners’ awareness on energy improvement options and moisture risks related to a retrofit project. The results presented by the tool are only estimates and not valid as an actual EPC. To continue their renovation journey, homeowners would have to contact an accredited retrofit coordinator. Hence, the fifth section, “What’s Next?” provides useful links to help homeowners find an accredited retrofit coordinator, determine how they can pay for improvements, and explore hints and tips on dealing with damp and condensation. 4. Discussion This article describes the development and pilot testing of a web-based moisture-safe retrofit decision-making tool for homeowners. The discussion section is organised into four sections, including the three focal points of this study: user acceptance and satisfaction, increased awareness about moisture, willingness to pursue a renovation project, and the study’s limitations. We illustrate our findings with representative quotes from participants. 4.1. User Acceptance and Satisfaction All participants reported that they would recommend the website to other homeowners. This is in line with the study by Collins et al. [52] in which more than 66% of respondents recommended a footprint calculator to others. Buildings 2022, 12, 1513 14 of 18 4.3. Willingness to Pursue a Renovation Project Most participants reported that using the tool did not increase their willingness to pursue a renovation project. This disagrees with another study in which homeowners evaluated a green home decision-making tool, and it has been found that the tool increased homeowners’ willingness to pursue a green renovation [44]. Many participants reported feeling discouraged from investing in energy efficiency measures because of high invest- ment costs and long payback periods of some measures. The discouragement was not related to an increased awareness of moisture issues. P8 said, “holy crap thirty-two grands for windows wow”. P3 said, “discouraged is maybe the word, because obviously of the esti- mated cost and the payback period kind of It makes you think whether it is worth it or not”). For instance, installing solid floor with insulation beneath the floor might show poor return of investment with long payback periods for up to 113 years which exceeds the estimated lifespan of most construction products and buildings themselves. This agrees with a study conducted by Alabid et al. [54] that argued that long payback periods represent one of the main barriers to the implementation of a deep retrofitting approach. The Association for Environment Conscious Building (AECB) argued that to encourage homeowners to invest in a deep retrofit, economic decision-making should not focus only on payback periods and should consider co-benefits associated with improving the energy performance of existing buildings including upgraded health, comfort and perceived wellbeing combined with grants or financial incentives, lower maintenance and running costs, and higher energy prices [55]. Therefore, it may be helpful for online retrofit tools to include co-benefits associated with a retrofit in the financial evaluation in order to reassure homeowners about their investment. Finally, online retrofit tools should not present the users only with the best energy efficiency solutions, which are generally expensive for homeowners. Tools should instead provide users the opportunity to select between a range of retrofit measures (e.g., various thicknesses of insulation, different window types, etc.) from the most efficient to the least efficient solutions in order not to scare homeowners with high investment costs of measure targeting high energy standards (P8: “I think it probably would put me off doing anything, right now, because it it’s giving you the most expensive scenario”). 4.2. Awareness about Moisture Risks 4.2. Awareness about Moisture Risks Most participants indicated that the tool improved their awareness of moisture risks in a retrofit project. This is in line with the findings of Dreijerink and Paradies [53] that online calculators increased participants’ awareness of environmental issues. Participants were made aware of different consequences of moisture-imbalanced retrofits (P13: “I was really surprised to know that renovation solutions can lead to dampness”). In that sense, the tool was considered an eye-opener. The new awareness of moisture issues was scary for a few participants (P12: “It was kind of scary for me”). Only one participant said the tool had not increased his awareness (P3: “I would say not by much because obviously here my situation is a little bit [ . . . ] I had kind of some pre knowledge of some of the options that were available to me”). By educating homeowners on moisture risk issues such as damp and mould, the developed tool is expected to protect homeowners’ health. 5. Conclusions While there are several online tools available to inform homeowners about the benefits of retrofitting, to the best of our knowledge, this is the first study that aims to develop and evaluate an online tool that aspires to raise homeowners’ awareness about moisture risks in a retrofit project. Furthermore, the proposed paper extends existing findings by shedding light on the experiences of retrofit calculator users. The paper focuses on the (1) users’ acceptance of and satisfaction with the developed retrofit calculator, (2) effects of the tool on moisture awareness and behaviour, and (3) effects of the tool on the willingness to invest in energy efficiency. The results of the pilot study demonstrate that the developed web-based tool has been well received by homeowners. The tool significantly increased participants’ awareness of moisture risks related to a retrofit project. Most participants considered the tool an eye-opener, while few of them found it scary. However, another important finding is that the tool did not result in an increased willingness to invest in energy efficiency measures. The discouragement was not related to an increased awareness of moisture issues and was linked to high investment costs and long payback periods of some retrofit measures. The pilot study also generated valuable insights on how to improve the design and development of online retrofit calculators to stimulate the adoption of energy efficiency measures. Future improvements would be to: 1. Include co-benefits associated with a retrofit in the financial evaluation, which will enable retrofit tools to make a stronger case for investment in retrofitting. The co- benefits that may be quantified are the Net Present Value (NPV) calculation, including available grants, operational and maintenance cost savings, carbon savings, thermal comfort, and reduced incidence of asthma. 2. Provide users with the opportunity to select between a range of retrofit measures (e.g., various thicknesses of insulation, different window types, etc.) from the most efficient to the least efficient solutions in order not to scare homeowners with high investment costs of measures targeting high energy standards. g g g gy 3. Use simple language, a clear design, and use graphics to clarify information. 4. Facilitate easy and quick data entry without asking users for technical details. This can be done by using automated data collection. 5. Use a responsive layout to adapt to various screen sizes. 4.4. Limitations Although the developed online tool aims to increase homeowners’ awareness regard- ing moisture issues related to a retrofit project, homeowners cannot rely on decision aids to replace a retrofit coordinator whose job is to oversee the management and design of all retrofit measures [56]. Risks associated with domestic retrofit comprises a much broader range of issues than those addressed by the developed tool, including technical risk such as putting the correct package of improvements in place, managing the interaction between measures, and process risks such as assessing existing dwelling(s) adequately and ensuring that appropriate retrofit designs are developed [34]. Therefore, while the developed tool provides useful and relevant information for homeowners, it is not designed to replace consultation with a retrofit coordinator. Buildings 2022, 12, 1513 15 of 18 15 of 18 The proposed tool in its actual form has several limitations. It currently considers only a limited number of retrofit measures. A future improvement could be to allow users to select between a range of retrofit measures (e.g., various thicknesses of insulation, different window types, etc.) from the most efficient to the least efficient solutions. Another improvement could be to filter different retrofit options according to budget limitations. Furthermore, to ensure quick and easy data entry, the developed tool is based on a pre- simulated database that includes the most common building typologies in the UK. The user can get an idea of energy-saving options only by indicating the building typology. However, the shortcoming of using a pre-simulated database is that the accuracy of the result is limited. In fact, every house in the UK is unique (occupation pattern, history of modifications, location, etc.). Therefore, it is impossible to generate accurate results for every homeowner based on a pre-simulated database and the displayed information for the users could be significantly different from their actual building data. An alternative way to reach a balance between ease of use and precision of the results is to use automated data collection, as suggested by Bekaroo et al. [57] and Guzman et al. [58]. The future version of the tool could be connected to national EPC databases to collect users’ data automatically. Moreover, the evaluation provided valuable feedback, which will lead to the future development of the proposed tool. Finally, given the small pilot nature of the study, the generalisability of study findings is limited. Further research with larger sample size is necessary. 5. Conclusions For future works, the recommendations proposed in this study will be implemented in the future version of the developed tool. Furthermore, large-scale deployment of the Buildings 2022, 12, 1513 16 of 18 proposed tool is envisaged while also planning for feedback from the larger audience. This will also enable a comparison of usability, awareness, and willingness to pursue a renovation project based on the demographic details of the participants. proposed tool is envisaged while also planning for feedback from the larger audience. This will also enable a comparison of usability, awareness, and willingness to pursue a renovation project based on the demographic details of the participants. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/buildings12101513/s1, Supplementary Material S1: General assumptions of the retrofit measures; supplementary Material S2: Moisture risks evaluation; Supple- mentary Material S3: Interview guide. Author Contributions: Conceptualization, M.S., A.B., J.A., D.G. and G.D.; methodology, M.S., A.B. and J.A.; software, M.S., A.B. and J.A.; validation, M.S., A.B. and J.A.; writing—original draft preparation, M.S., A.B. and J.A.; writing—review and editing, M.S., A.B. and J.A.; supervision, project administration, and funding acquisition, A.B., D.G. and G.D. All authors have read and agreed to the published version of the manuscript. Funding: This research was carried out within Stronghouse project and was funded by Interreg North Sea region programme grant number [J-NO: 38-2-15-19]. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We would like to thank all partners from the Stronghouse project for their continuous and rich discussions throughout the development of this research. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. National-Statistics 2020 UK Greenhouse Gas Emissions, Provisional Figures. 2020. Available online: https://assets.publishing. service.gov.uk/government/uploads/system/uploads/attachment_data/file/972583/2020_Provisional_emissions_statistics_ report.pdf (accessed on 12 February 2022). p p y 2. 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Health Sci. 2013, 15, 398–405. [CrossRef] p y 49. Moisture Safe Retrofit Calculator. Available online: https://mseddiki1.wixsite.com/my-site (accessed 50. Kok, A.L.; Barendregt, W. Understanding the adoption, use, and effects of ecological footprint calculators among Dutch citizens. J. Clean Prod. 2021, 326, 129341. [CrossRef] J 51. Gram-Hanssen, K.; Christensen, T.H. Carbon calculators as a tool for a low-carbon everyday life? Sustain. Sci. Pract. Policy 2012, 8, 19–30. [CrossRef] 52. Collins, A.; Galli, A.; Hipwood, T.; Murthy, A. Living within a One Planet reality: The contribution of personal Footprint calculators. Environ. Res. References Palmer, J.; Livingstone, M.; Adams, A. What Does It Cost to Retrofit Homes? Updating the Cost Assumptions for BEIS’s Energy Efficiency Modelling; CAR: London, UK, 2017. g 31. GOV.UK Retrofit for the Future: Analysis of Cost Data. 2014. Available online: https://assets.publishing.service.gov.uk/ government/uploads/system/uploads/attachment_data/file/669109/Retrofit_for_the_Future_-_analysis_of_cost_data_ report_2014.pdf (accessed on 11 March 2022). p p ( ) 32. Solarguide. How Much Does It Cost to Install Solar Panels in the UK? Available online: https://www.solarguide.co.uk/how- much-does-it-cost-to-install-solar-panels#/contact-details (accessed on 11 April 2022). 33. Checkatrade. Door Installation Cost. Available online: https://www.checkatrade.com/blog/cost-guides/door-installation-costs/ (accessed on 15 April 2022). 34. Retrofit-Academy Level 5 Diploma in Retrofit Coordination & Risk Management. 2022. Available online /? lid EAI IQ bChMI GI IV 7tCh2 AI5EAAYASAAE J j D B E ( d 15 J 34. Retrofit-Academy Level 5 Diploma in Retrofit Coordination & Risk Management. 2022. Available online org/?gclid=EAIaIQobChMIv-GIrsao-gIVwp7tCh2yzAI5EAAYASAAEgJxj_D_BwE (accessed on 15 Jan y p g p // gclid=EAIaIQobChMIv-GIrsao-gIVwp7tCh2yzAI5EAAYASAAEgJxj_D_BwE (accessed on 15 January 2022 g g g p y gJ j ( J y ) 35. Karvonen, A. Towards systemic domestic retrofit: A social practices approach. Build Res. Inf. 2013, 41, 563–574. [CrossRef] y p pp f 36. Sanders, C.; Glenfeulan, B.S. 5250: 2021 Management of Moisture in Buildings–Code of Practice. 2021. Available online: https://www.bsigroup.com/en-GB/blog/Built-Environment-Blog/bs-5250-2021-management-of-moisture-in-buildings- -code-of-practice/ (accessed on 17 January 2022). p y 37. Ali, A.S.; Riley, C.; Acton, E.; Ali, A.; Heidarinejad, M.; Stephens, B. Development and evaluation of an automatic steam radiator control system for retrofitting legacy heating systems in existing buildings. Energy Build. 2021, 251, 111344. system for retrofitting legacy heating systems in existing buildings. Energy Build. 2021, 251, 111344. 38. Carvalho, I.; Bernardi, F.A.; Neiva, M.B.; Lima, V.C.; de Oliveira, L.L.; Miyoshi, N.S.B.; Sanches, T.L.M.; Barbosa-Junior, F.; Crepaldi, N.Y.; Yamada, D.B. COVID-19 BR: A web portal for COVID-19 information in Brazil. Procedia Comput. Sci. 2022, 196, 525–532. [CrossRef] 39. Merritt, K.; Zhao, S. An innovative reflection based on critically applying ux design principles. J. Open Innov. Technol. Mark. Complex. 2021, 7, 129. [CrossRef] 40. Wakefield, C.E.; Watts, K.J.; Meiser, B.; Sansom-Daly, U.; Barratt, A.; Mann, G.J.; Lobb, E.A.; Gaff, C.L.; Howard, K.; Patel, M.I. Development and pilot testing of an online screening decision aid for men with a family history of prostate cancer. Patient Educ. Couns. 2011, 83, 64–72. [CrossRef] 18 of 18 18 of 18 Buildings 2022, 12, 1513 41. Habes, E.; Jepma, P.; Parlevliet, J.; Bakker, A.; Buurman, B. y pp 58. Guzman, L.; Makonin, S.; Clapp, A. CarbonKit: Designing a personal carbon tracking platform. In Proceedings of the Fourth International Workshop on Social Sensing, Montreal, QC, Canada, 15 April 2019; pp. 24–29. References Lett. 2020, 15, 025008. [CrossRef] 53. Dreijerink, L.; Paradies, G. How to Reduce Individual Environmental Impact? A Literature review into the Effects and Behavioral Change Potential of Carbon Footprint Calculators. 2020. Available online: https://repository.tno.nl/islandora/object/uuid: 82bec847-f0f7-4b83-bcef-af92b255b525 (accessed on 12 May 2022). ( y ) 54. Alabid, J.; Bennadji, A.; Seddiki, M. A review on the energy retrofit policies and improvements of the UK existing buildings, challenges and benefits. Renew. Sustain. Energy Rev. 2022, 159, 112161. [CrossRef] 55. AECB AECB CarbonLite Retrofit Training Course. 2022. Available online: https://aecb.net/the-aecb-carbonlite-retrofit-online- training-course/ (accessed on 5 January 2022). 56. Edwards, J. Retrofitting traditional buildings for improved energy performance in the context of BSI PAS 2035. J. Build. Surv. Apprais. Valuat. 2021, 9, 294–304. 57. Bekaroo, G.; Roopowa, D.; Bokhoree, C. Mobile-based Carbon footprint calculation: Insights from a usability study. In Proceedings of the 2019 Conference on Next Generation Computing Applications (NextComp), Mauritius, Madagascar, 19–21 September 2019; IEEE: Piscataway, NJ, USA, 2019; pp. 1–6. y pp 58. Guzman, L.; Makonin, S.; Clapp, A. CarbonKit: Designing a personal carbon tracking platform. In Proceedings of the Fourth International Workshop on Social Sensing, Montreal, QC, Canada, 15 April 2019; pp. 24–29.
https://openalex.org/W4361016044
https://wnus.usz.edu.pl/cto/file/article/view/20046.pdf
Italian
null
SYNODALITY AND INTERCOMMUNION ACCORDING TO DUMITRU STĂNILOAE
Colloquia Theologica Ottoniana/Colloquia Theologica Ottoniania
2,022
cc-by-sa
11,744
COLLOQUIA THEOLOGICA OTTONIANA 38 (2022) s. 9–30 DOI: 10.18276/cto.2022.38-01 ISSN (print) 1731-0555 | ISSN (online) 2353-2998 ZOLTÁN JÓZSEF BARA Universitatea UBB Cluj Napoca, Romania | Facultatea de teologie romano catolică ORCID: 0000-0002-1254-8458 Introduzione La Chiesa Ortodossa Romena, come pure la sua teologia, rappresentano una sintesi tra Oriente e Occidente, grazie al carattere specifico del popolo romeno, che ha un’origine latina, ma vive una spiritualità bizantina. “Dumitru Stăniloae (1903–1993) è ritenuto insieme con Romano Guardini, Paul Tillich, Karl Barth, Karl Rahner, Yves Congar, Nikos Nissiotis ed altri, tra i più rinomati teologi del ventesimo secolo”1. L’opera di riflessione teologica e filosofica di Dumitru Stăniloae, elaborata durante i sei decenni di vita accademica e pubblicistica svolta nei due centri dell’Orto­ dossia romena, Sibiu e Bucarest, ammonta a circa 20 libri originali, 1300 arti- coli e 30 traduzioni dai Padri della Chiesa e dai grandi teologi contemporanei2. Nell’opera di Dumitru Stăniloae esiste una grande varietà di temi e problemi ecu- menici, in cui egli ha assunto delle posizioni originali, a volte radicali, ma sempre favorevoli al dialogo3. In questo brano vorrei presentare due temi che, in un certo senso, sintetizzano la visione ecumenico originale di Stăniloae sull’unione delle Chiese. Si tratta del tema della cattolicità o sinodalità aperta4 e quello dell’interco- munione eucaristica come tipologia ecumenica5. Summary In Dumitru Stăniloae’s work, he takes original, albeit sometimes radical, positions on a great variety of ecumenical themes and problems; however, Stăniloae is always in favor of dialogue. Following Stăniloae’s arguments, I will present two themes that summarize Stăniloae’s original ecumenical vision on the union of the Churches: the theme of catholicity or open synodality, and Eucharistic intercommunion. Stăniloae suggests an open synodality or catholicity as a dogmatic and spiritual opening of the Christian Churches to rediscover their unity. At the basis of this open catholicity is the Sacred Scripture where there are diverse traditions and meanings – differences that come from the diversity of God’s actions in the history of salvation. The Catholic or synodal approach can become, in all the senses of the Scripture, the meeting point for all Christians. On the other hand, in Stăniloae’s vision, Eucharistic intercommunion must be a full Eucharistic communion. Orthodoxy considers the unity of the Church as deeply and ontologically anchored in Christ, and it considers the Eucharist as the mean that sustains this unity. This unity can- not bring the Eucharist down to the level of being psychologically and physically lax among Christians. Indeed, without the unity of faith, and without communion with the body and blood of Christ, the Church could not exist in the full sense of the word; just as without unity in faith and without the Church, Eucharistic communion cannot be achieved. Eucharistic intercommunion makes a separation between the unity of faith and the Church, inconceivable because it leads to the dissolution of the Church, of the unity of faith, and of the Eucharist itself. Communion with the body and blood of Christ cannot be separated from unity in faith or from the conviction that the Church represents a profound ontological unity of the faithful in Christ and among them. Keywords: Dumitru Stăniloae, ecumenism, open catholicity (sinodality), union of Churches, eucharistic inter-communion 10 Zoltán József Bara 1 Cf. Theologen des 20. Jahrhunderts. Eine Einführung, a cura di P. Neuner, G. Wenz, Darm stadt 2002, p. 145–156. 2 Z. Bara, Un approccio ortodosso ecclesiologico alla dimensione sacramentale della Chiesa: la Chiesa, Sacramento di Cristo, secondo Dumitru Stăniloae, in: Sakramenty nowym wyzwa- niem dla Kościoła Katolickiego, a cura di J. Radkiewicz, Szczecin 2019, p. 155–156. 4 sobornicitatea deschisă 1. La sinodalità aperta Il tema della sinodalità aperta è stato descritto da Stăniloae in un articolo pubblicato nel 19716, in cui voleva sottolineare la necessità dell’apertura dogma- tica e  spirituale delle Chiese cristiane, per ritrovare la loro unità. Il progresso del mondo – secondo Stăniloae – è anche un progresso della Chiesa e un progresso 2 Z. Bara, Un approccio ortodosso ecclesiologico alla dimensione sacramentale della Chiesa: la Chiesa, Sacramento di Cristo, secondo Dumitru Stăniloae, in: Sakramenty nowym wyzwa- niem dla Kościoła Katolickiego, a cura di J. Radkiewicz, Szczecin 2019, p. 155–156. 3 M. Stavrou, Teologia cristiana ortodossa, in: Dizionario del sapere storico-religioso del Nove- cento, Bologna 2010, p. 1514; G. R. Roberson, Ecumenism in the Thought of Dumitru Stăniloae, a cura di A. Melloni, New York 1999, p. 51. 4 sobornicitatea deschisă 5 N. Moşoiu, “Sobornicitatea deschisă” ca tipologie ecumenică sau De la teologia confesională la teo- logia ecumenică în perspectiva viziunii despre “sobornicitatea deschisă”, “Revista Teologică” 4 (1997), p. 87–117. 5 N. Moşoiu, “Sobornicitatea deschisă” ca tipologie ecumenică sau De la teologia confesională la teo- logia ecumenică în perspectiva viziunii despre “sobornicitatea deschisă”, “Revista Teologică” 4 (1997), p. 87–117. 6 D. Stăniloae, Sobornicitate deschisă, “Ortodoxia” 23 (1971) 2, p. 165–180. Cf. V. Coman, “Open Sobornicity” and “Receptive Ecumenism”: Fruitful Models of Ecumenical Interaction, in: Just Do It? Recognition and Reception in Ecumenical Relations: Proceedings of the 19th Academic Consul- tation of the Societas Oecumenica, a cura di D. Heller, M. Hietamaki, Leipzig 2018, p. 241–251; V. Coman, Le Saint Esprit comme liaison de l’amour éternel entre le Père et le Fils: un cas de ‘sobor- nicité ouverte’ dans la théologie orthodoxe, “Irénikon” 89 (2016) 1, p. 25–51; R. Bordeianu, La sinodalità e l’intercomunione in Dumitru Stăniloae 11 del cristianesimo verso la Chiesa Una e Cattolica. L’arricchimento spirituale e l’ascensione del mondo verso i veri valori porta anche ad un arricchimento spi- rituale, ad un’amplificazione e ad una scoperta del contenuto spirituale che è più propriamente della Chiesa. E se la Chiesa deve comprendere tutta la verità, tutta la realtà di Dio, essa non si può chiudere ai valori che, nel suo continuo sforzo, l’umanità svela, aiutata anche dall’opera di Dio7. Punto di partenza quindi per l’approfondimento di questo tema lo rappresenta il documento Scrittura e Tradizione emanato dalla Comissione Fede e Costituzione del Consiglio Ecumenico delle Chiese8. (In) Voluntary Ecumenism: Dumitru Stăniloae’s Interaction with the West as Open Soborni- city, in: Orthodox Constructions of the West, a cura di E.G. Demacopoulos, A. Papanikolaou, New York 2013, p. 240–253; L. Turcescu, Eucharistic Ecclesiology or Open Sobornicity?, in: Dumi- tru Stăniloae: Tradition and Modernity in Theology, a cura di L. Turescu, Palm Beach 2002, p. 83–103. 9 Nouveauté dans l’oecumenisme, Taizé 1968, p. 58. 10 Ibidem. 7 “Progresul lumii este astfel și un progres al Bisericii și un progres al creștinismului spre Bise- rica cea una și sobornicească. Îmbogățirea spirituală și înălțarea lumii spre valorile adevărate, prilejuiește și o îmbogățire spirituală, o amplificare și o descoperire a conținutului spiritual cel mai propriu al Bisericii. Și dacă Biserica trebuie să cuprindă tot adevărul, să înțeleagă toată rea- litatea lui Dumnezeu, ea nu se poate închide valorilor pe care le dezvăluie omenirea în efortul ei continuu, ajutată și ea prin lucrarea lui Dumnezeu”. Cf. D. Stăniloae, Coordonatele ecumeni- smului din punct de vedere ortodox, “Ortodoxia” 19 (1967) 4, p. 540. Cf. C. Sonea, Teologia dia- logului intercreștin. O introducere, Cluj Napoca 2017, p. 273–278. 8 Questo documento era difatti il risultato delle discussioni ecumeniche tenute tra diversi gruppi di teologi, in seguito alla decisione presa all’incontro di Aarhus nel 1964, risultato che è stato presentato all’incontro di Bristol nel 1967. Oltre al tema ricordato, il documento include anche altri tre temi: Dio nella natura e nella storia, l’Eucaristia e la Chiesa e il popolo ebreo. (In) Voluntary Ecumenism: Dumitru Stăniloae’s Interaction with the West as Open Soborni- city, in: Orthodox Constructions of the West, a cura di E.G. Demacopoulos, A. Papanikolaou, y f p p New York 2013, p. 240–253; L. Turcescu, Eucharistic Ecclesiology or Open Sobornicity?, in: Dumi- tru Stăniloae: Tradition and Modernity in Theology, a cura di L. Turescu, Palm Beach 2002, p. 83–103. 11 “Sobornicitatea deschisă are o perspectivă profetică, deoarece insistă asupra însemnătăţii uni- tăţii creştinilor pentru reconcilierea lumii, fapt realizabil în măsura trecerii lui Hristos pascal în viaţa umană sub “forma Bisericii”. Cf. D. Stăniloae, Dogmatica I, București 2003, p. 61; ibidem, Dogmatica II, p. 206. 12 “E un mister această ținere la un loc a unor aspecte contradictorii. Iar unitatea părților are un sens cu mult mai bogat decât suma sensurilor părților, un sens inepuizabil rațional. Rațiunea are totuși un rol în sesizarea aspectelor care sunt împletite în această unitate, făcând ca să nu fie redus în mod simplist la unul singur, cu negarea celorlalte”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 167. 1. La sinodalità aperta Questo documento, dopo aver notato che nella Bibbia c’è una unità nella diversità, afferma che la diversità delle tradizioni delle Chiese attuali può essere ridotta alla diversità delle tradizioni esistenti nella Sacra Scrittura, per cui la ricerca dell’unità tra le diverse tradizioni della Chiesa dovrà appropriarsi dell’unità del Vangelo, così come la riflette la pluralità delle diverse tradizioni bibliche9. Il documento sostiene inoltre che la diversità delle tradizioni nella Bibbia riflette difatti la diversità delle azioni di Dio nella storia umana, come pure la diversità delle risposte umane a queste azioni. In questo senso il documento raccomanda agli esegeti di non attacarsi ad una sola riflessione biblica, anche se questa sembra centrale, perché tale scelta li porterebbe a non comprendere più la diversità di questa ricchezza10. Secondo Stăniloae, questa raccomandazione vale non solo per gli esegeti, ma anche per le Chiese cristiane, perché molte delle divisioni apparse in seno al cristianesimo sono dovute ad un attaccamento unilaterale a certe affermazioni 12 Zoltán József Bara bibliche, ignorando le altre, che non sono meno importanti. Perciò, accettare la diversità dei sensi presenti nella Scrittura, può diventare una via che porta alla vera cattolicità (sobornicitate) della Chiesa e quindi all’avvicinamento ecumenico, tanto desiderato dal mondo cristiano11. L’approccio unilaterale alla diversità dei sensi presenti nella Sacra Scrittura, osserva il teologo romeno, si è manifestato fin dall’inizio nel cristianesimo, e questo ha portato a delle dolorose divisioni. Di questo peccato si sono fatte colpevoli, per esempio, le eresie antitrinitarie, le quali riducevano sia la divinità all’unità di una sola persona, sia scioglievano l’unità in tre dei. Di fronte a queste eresie, la Chiesa, tenendo conto delle espressioni bibliche, ha affermato sia l’unità d’essere della divinità, che la trinità delle persone. Quindi, mentre le eresie si lasciavano portare da un giudizio semplicistico, la Chiesa era animata dalla realtà complessa, in cui sono uniti aspetti diversi, complementari e a volte anche contraddittori. La realtà che guida la Chiesa, ha perciò un carattere misterioso, in quanto è un mistero questo tenere insieme aspetti contraddittori, e l’unità delle parti ha un senso molto più ricco che la somma dei sensi delle parti, un senso inesauribile. La ragione ha comunque un ruolo nella percezione degli aspetti che sono impliciti in questa unità, facendo sì che essi non siano ridotti in modo semplicistico ad un solo, negando tutti gli altri12. 13 “Nu s-a plasat pe o altă poziție unilaterală și contradictorie față de poziția unilaterală a unei erezii sau alteia, ci s-a menținut pe o poziție de largă îmbrățișare a tuturor aspectelor credinței, inclusiv a celor afirmate de erezii. Erezia își vedea în învățătura Bisericii și aspectul susținut de ea, deși încadrat în toate celelalte aspecte ale învățăturii creștine. Încât, îndată ce se renăștea în ea tendința spre plenitudine, ajungea vrând-nevrând la întâlnirea cu Biserica în învățătura pe care o păstrase aceasta. Iar tendința spre plenitudine se naște aproape firesc în erezie, după perioada de afirmare inițială a unei teze unilaterale din opoziția pasionată față de Bise- rică și față de plenitudinea susținută de ea”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 167–168. 1. La sinodalità aperta In questo senso, è interessante notare che, nell’Oriente cristiano, tutte le eresie, a eccezione di quelle cristologiche, sono state riassorbite nella grande Chiesa, e questo è dovuto, secondo Stăniloae, non solo all’aiuto dato dall’imperatore bizantino nel riportare i cristiani separati all’interno della Chiesa, ma anche al fatto che questa non si è situata su un’altra posizione unilaterale e contraddittoria nei confronti della posizione unilaterale delle eresie, ma si è mantenuta su una posizione che abbracciava tutti gli aspetti della fede, inclusivamente quelli affermati dalle eresie. L’eresia vedeva nella dottrina della Chiesa anche l’aspetto sostenuto da essa, anche se inquadrato in tutti gli altri aspetti della dottrina cristiana. Tanto che, una volta che nasceva dentro di essa la tendenza verso la pienezza, arrivava, volendo o no, all’incontro con la dottrina che la Chiesa aveva conservato. E la tendenza verso la pienezza nasce quasi naturalmente nell’eresia, dopo il periodo iniziale La sinodalità e l’intercomunione in Dumitru Stăniloae 13 d’affermazione di una tesi unilaterale, a causa di un’opposizione passionale verso la Chiesa e verso la pienezza sostenuta da essa13. Un caso particolare lo rappresenta lo scisma tra la Chiesa d’Oriente e quella dell’Occidente, consumatasi nel 1054, perché allora la divisione non è stata più provocata, afferma Stăniloae, dalla negazione di un aspetto dottrinale, ma dall’accen­ tuazione esagerata da parte della Chiesa occidentale di un punto dottrinale relativo all’unità della Chiesa, accentuazione che ha portato alla subordinazione e all’inter- pretazione dell’intera dottrina alla luce di questo punto. Perciò, l’unità della Chiesa non era più vista essenzialmente come un’unità d’equilibrio, come un’unità dei contrari, ma come un’unità in cui una parte viene innalzata alla dignità di centro onnipotente, che soffoca sempre di più tutte le altri parti componenti, che sempli- fica la complessità, uniformizza la diversità, portando ad un impoverimento della vita della Chiesa in Dio, impoverimento uguale a quello provocato dalle eresie, che negavano parti essenziali della dottrina cristiana14. Pur ammettendo che il canone 28 del Concilio di Calcedonia aveva stabilito una gradazione delle prime cinque sedi episcopali, in base alla loro origine apostolica e all’importanza politica delle loro città e che, secondo questa gradazione, il vescovo di Roma era il primo, il teologo romeno sottolinea che questa gradazione era soltanto presidenziale, rappresentando una certa autorità morale dei loro vescovi. 14 “Nu ca o unitate de echilibru, ca o unitate a contrariilor, ci ca o unitate în care o parte e ridi- cată la rang de centru atotputernic care sufocă tot mai mult celelalte părți componente, simpli- fică complexitatea, uniformizează varietatea, ducând la o sărăcire a vieții Bisericii în Dumne- zeu, egală cu cea pe care o provocau ereziile care negau părți esențiale ale învățăturii creștine”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 68. 15 “Despărțirea Apusului creștin de Biserica răsăriteană, care a rămas în complexitatea echili- brată a învățăturii creștine. Scaunul Romei a suprimat în Apus prin aceasta desfășurarea liberă a vieții în Dumnezeu, sau posibilitatea ei nestingherită de îmbogățire spirituală, implicit posi- bilitatea de creștere neîmpiedicată a umanului”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 169. 16 Infatti, i sacramenti sono “gli atti parziali per mezzo dei quali si offre a ciascuno, personalmente, la vita di Cristo dalla Chiesa”. In questo senso, è giusto affermare che “per mezzo dei sacramenti cresce e si nutre la Chiesa, cioè il Corpo mistico del Signore, che non è altro che l’allargamento e il prolungamento dell’Incarnazione di Cristo”. D. Stăniloae, Sinteză eclesiologică, “Studii Teo- logice” 7 (1955), 5–6, p. 267–284, 268. D. Stăniloae, Transparenţa Bisericii în viaţa sacramen- tală, “Ortodoxia” 22 (1970) 4, p. 501–516. Cf. Z. Bara, La realtà mistica della chiesa nella rifles- sione ortodossa di Dumitru Stăniloae, “Studia Theologia Catholica Latina” 66 (2021) 2, p. 83–84. 1. La sinodalità aperta I vescovi di queste sedi primaziali, grazie alla loro autorità morale, svolgevano quindi un servizio a favore dell’unità della Chiesa, ma senza restringere la sua libertà, in quanto l’unità si manteneva prima di tutto, mediante la forza dell’amore e della stessa fede, per l’opera dello Spirito Santo. Purtroppo, aggiunge lui, la sede romana, accentuando la sua autorità esclusiva e dittatoriale, dopo aver soppresso la libertà della Chiesa Occidentale, ha cercato di sopprimere anche la libertà della Chiesa Orientale, fatto che ha avuto come conseguenza la separazione dell’Occidente cristiano dalla Chiesa orientale, che è rimasta nella complessità equilibrata della dottrina cristiana. La Sede 14 Zoltán József Bara romana ha soppresso in Occidente lo sviluppo libero della vita in Dio, ossia la sua possibilità di arricchimento spirituale e, implicitamente, la possibilità di crescita senza restrizioni dell’umanità15. L’opzione della sede romana a favore di un’autorità contraria alla libertà ha portato poi, continua Stăniloae, ad una nuova divisione nel seno del cristiane- simo, divisione provocata dalla Riforma protestante del XVI secolo. Se nel 1054 si aveva scelto l’unità della Chiesa contro la sua libertà, adesso veniva contestata la Chiesa stessa, in quanto contraria alla libertà, affermandosi così una fede cri- stiana individuale. Dal complesso bipolare Chiesa – individuo, il protestantesimo aveva optato per l’individuo, in quanto il cattolicesimo aveva accentuato l’unità della Chiesa contro la libertà dell’individuo. Insieme a questo però, sono state contestate tutte le strutture che sostengono la comunione della Chiesa con Dio: i sacramenti, la partecipazione alla grazia divina, la santità, la comunione nella fede e nell’amore.16 La Riforma ha contestato queste strutture ecclesiali, perché prima di tutto contestava la struttura gerarchica, per mezzo di cui si mantenevano le altre strutture. Per legittimare la sua autorità quindi, scrive Stăniloae, la Sede romana aveva scelto dalla complessità contraddittoria dei testi biblici, quelli che sembra- vano confermare l’idea di un primato di Pietro; la Riforma invece, per legittimare il rifiuto di ogni struttura ecclesiale, quelli che affermavano il sacerdozio generale dei fedeli, la dimensione fraterna della comunità cristiana e la diversità dei ministeri. La situazione era diventata tanto più senza via d’uscita, quanto ambedue le parti, il cattolicesimo e il protestantesimo, rifiutando la complessità contraddittoria dei testi scritturistici, definivano ciascuna se stessa in contrapposizione con l’altra. Lo spirito occidentale era diventato uno spirito di dure alternative, taglienti, abituato con le semplificazioni di un razionalismo semplicistico, inferiore. 17 “Spiritul occidental devenise un spirit de alternative dure, tranșante, obișnuit cu simplificările unui raționalism simplist, inferior. Pierduse suplețea unei rațiuni superioare care se orientează după complexitatea suplă, echilibrată a realității, exprimată de Evanghelie, și a unei vieți spirituale bogate și autentice... A înflorit teologia produsă de rațiune ca o valoare in sine, independentă de viața complexă a Bisericii; a înflorit o organizație juridică clădită... din câteva principii rațion- ale, organizație care și-a aservit ei și teologia”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 170. 18 A questo proposito, Stăniloae non è sicuro sulla provenienza di questa tendenza del cristia- nesimo occidentale, cioè se è il risultato del suo incontro reale con l’Ortodossia, oppure se è nato da sé. Comunque, egli è certo che essa porta in modo naturale verso l’Ortodossia. Inol- tre, egli è contento di notare che oggi sempre più cristiani occidentali guardano con più inte- resse la Chiesa Orientale, interesse che non è una semplice curiosità intellettuale, ma l’espres- sione della convinzione che l’Ortodossia potrebbe aiutare al rinnovamento del cristianesimo in genere. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 171. 19 “Fără Ortodoxie creștinismul occidental nu se poate mișca decât de la o extremitate la alta... Refacerea unității e pentru creștinismul occidental o chestiune de ieșire din planul alternati- velor exclusiviste. El trebuie să regăsească spiritul Ortodoxiei care nu se opune uneia sau alteia din alternative, ci îmbrățișează în învățătura și în echilibrul ei și punctele afirmate de ambele forme al creștinismului apusean”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 171. 1. La sinodalità aperta Aveva perso l’agilità di una ragione superiore che si orienta secondo la complessità equi- librata della realtà, espressa dal Vangelo, come pure di una vita spirituale ricca ed autentica [...] Ha fatto fiorire una teologia prodotta dalla ragione come valore in sé, La sinodalità e l’intercomunione in Dumitru Stăniloae 15 indipendente dalla vita complessa della Chiesa; ha fatto fiorire un’organizzazione giuridica, costruita anche essa... su alcuni principi razionali, su una organizzazione che ha asservito anche la teologia17. Oggi però, osserva lui, in Occidente si nota il risveglio del desiderio verso la complessità della dottrina e della vita cristiana, e questa tendenza va incontro all’Ortodossia, che ha conservato la pienezza e l’equilibrio della vera spiritualità18. Senza l’Ortodossia, il cristianesimo occidentale non si può muovere che da un’e- stremità all’altra [...] Il rifacimento dell’unità è per il cristianesimo occidentale il problema d’uscire dal piano delle alternative esclusivistiche. Esso deve ritrovare lo spirito dell’Ortodossia, che non si oppone ad una o all’altra di queste alternative, ma abbraccia, nella sua dottrina e nel suo equilibrio, anche i punti affermati da ambedue le forme del cristianesimo occidentale19. L’affermazione che l’unità delle Chiese cristiane si deve realizzare sotto il segno dell’Ortodossia, non significa certo, riconosce Stăniloae, che la Chiesa Ortodossa ha attualizzato finora, in modo soddisfacente, nel piano della spiritualità e della vita dei fedeli, tutti i valori potenziali compresi nel tesoro dell’Ortodossia. Perciò anch’essa ha bisogno di aprirsi ai valori spirituali attualizzati dai cristiani occi- dentali, come, per esempio, il valore delle forme esterne dell’unità della Chiesa e il valore della Sacra Scrittura. Forse in Oriente, dopo un certo tempo, l’unità cristiana non è stata più vissuta, con tutta la sua intensità, come all’inizio della Chiesa, sotto la forma superiore della comunità universale tra le Chiese locali, e allora all’Occidente è sembrato necessario, forse a causa di speciali circostanze, di vivere questa unità in un modo più pieno, senza rendersi conto che con il tempo 16 Zoltán József Bara Zoltán József Bara questa unità sarebbe stata pagata con un sempre più grande sacrificio. D’altra parte, l’apparizione del cattolicesimo, quale affermazione giuridico-centrale dell’unità del cristianesimo, ha portato praticamente gli ortodossi ad accentuare sempre più l’indipendenza delle Chiese locali quali forme per mantenere la loro unità. 20 “Poate în Răsărit nu s-a mai trăit, începând de la o vreme, cu toată intensitatea unitatea creștină ca la începutul Bisericii, sub forma superioară a comunității universale între Bisericile locale, și atunci Occidentului i-a apărut necesar, poate din cauza unor împrejurări speciale, să trăiască această unitate într-un mod mai deplin, fără să-și dea seama că această unitate avea să fie plătită cu vremea cu un tot mai mare sacrificiu. Pe de altă parte, apariția catolicismului ca afirmare juridic-centralistă a unității creștinismului, a făcut și pe ortodocși ca în practică să accen- tueze mai mult independența Bisericilor lor locale decât formele de menținere a unității lor. Iar când a apărut protestantismul, a preluat în practică de la catolicism un fel de antireformă, accentuând Tradiția în dauna Sfintei Scripturi și importanța actelor obiective ale Tainelor și a ierarhiei în dauna trăirii personale și în comuniunea legăturii cu Dumnezeu”. Cf. D. Stăni­ loae, Sobornicitate deschisă..., p. 171. 21 “Ea trebuie să fie soborul a toată lumea, în care toți creștinii își aduc înțelegerea întregii realități divine revelate și a întregii realități umane văzute în lumina revelației integrale, pentru a o împ- ărtăși tuturor și pentru ca fiecare să se împărtășească de înțelegerea tuturor”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 172. 1. La sinodalità aperta Quando è apparso il protestantesimo, [la Chiesa Ortodossa] ha preso praticamente dal cat- tolicesimo un certo tipo di controriforma, accentuando la Tradizione a scapito della Sacra Scrittura e l’importanza degli atti oggettivi dei Sacramenti e della gerarchia a scapito del vivere personale la comunione con Dio20. La cattolicità-sinodale della Chiesa rappresenta l’universalità della fede cristiana vissuta sotto la forma di comunione. In questo senso, scrive Stăniloae, essa significa non solo l’unità universale dei cristiani in forma di comunione, ma anche l’unità onnicomprensiva della dottrina cristiana, vissuta dalla comunità universale e libera dei cristiani. Perciò essa equivale all’universalità degli aspetti della Rivelazione, percepiti dalla totalità delle prospettive umane e diventati il bene comune della comunità umana universale. Essa dev’essere l’assemblea (soborul) di tutto il mondo, in cui tutti i cristiani portano la loro comprensione della realtà divina rivelata e dell’intera realtà umana vista alla luce della Rivelazione integrale, per comunicarla a tutti e perché ciascuno partecipi alla comprensione di tutti21. Le Chiese cristiane sono chiamate quindi ad attualizzare la comprensione dell’intera realtà divina e umana, riflessa e presente in una forma concentrata nel Vangelo. Ma come si potrebbe inquadrare l’esperienza cattolica e protestante in quella ortodossa? Secondo Stăniloae, per dare una risposta soddisfacente a questa domanda, bisogna trovare anzitutto la spiegazione della diversità spesso contrad- ditoria delle affermazioni bibliche. Il documento sopra ricordato della Comissione Fede e Costituzione aveva detto che questa diversità proviene dalla diversità delle 21 “Ea trebuie să fie soborul a toată lumea, în care toți creștinii își aduc înțelegerea întregii realități divine revelate și a întregii realități umane văzute în lumina revelației integrale, pentru a o împ- ărtăși tuturor și pentru ca fiecare să se împărtășească de înțelegerea tuturor”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 172. 21 “Ea trebuie să fie soborul a toată lumea, în care toți creștinii își aduc înțelegerea întregii realități divine revelate și a întregii realități umane văzute în lumina revelației integrale, pentru a o împ- ărtăși tuturor și pentru ca fiecare să se împărtășească de înțelegerea tuturor”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 172. La sinodalità e l’intercomunione in Dumitru Stăniloae 17 azioni di Dio e dalla diversità delle risposte umane ad esse. 23 “Dumnezeu se face cunoscut și lucrează prin acte, cuvinte și imagini mereu diferite, pentru că nu-și epuizează ființa în nici una din ele, dar prin toate se revelează. Pe nici unul din aceste moduri nu trebuie să-l disprețuim, o dată ce Dumnezeu a găsit potrivit să-l folosească, dar nu trebuie să socotim pe nici unul și nici pe toate cele din Revelație și din istoria evlaviei de până acum ca exprimând integral pe Dumnezeu, ca excluzând pentru viitor alte moduri posibile de a exprima pe Dumnezeu, chiar dacă cadrele adevărului divin au fost descoperite în totalita- tea Revelației”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 22 “Care ne oferă revelația sa prin Biblie ca ceva unitar cu toată diversitatea prezentă în ea, care ni se prezintă general obligatorie de crezut în tot ansamblul ei și nu se vrea obligatorie pentru unii în unele părți, pentru alții în altele”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 172. 22 “Care ne oferă revelația sa prin Biblie ca ceva unitar cu toată diversitatea prezentă în ea, care ni se prezintă general obligatorie de crezut în tot ansamblul ei și nu se vrea obligatorie pentru unii în unele părți, pentru alții în altele”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 172. 23 “Dumnezeu se face cunoscut și lucrează prin acte, cuvinte și imagini mereu diferite, pentru că nu-și epuizează ființa în nici una din ele, dar prin toate se revelează. Pe nici unul din aceste moduri nu trebuie să-l disprețuim, o dată ce Dumnezeu a găsit potrivit să-l folosească, dar nu trebuie să socotim pe nici unul și nici pe toate cele din Revelație și din istoria evlaviei de până acum ca exprimând integral pe Dumnezeu, ca excluzând pentru viitor alte moduri posibile de a exprima pe Dumnezeu, chiar dacă cadrele adevărului divin au fost descoperite în totalita- tea Revelației”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 1. La sinodalità aperta Ma se questo fosse vero, allora si potrebbe dire, scrive Stăniloae, che la diversità delle situazioni in cui vivono oggi i cristiani, giustificherebbe la persistenza nella loro diversità, ogni gruppo attaccandosi a quelle azioni divine che corrispondono alla loro situazione storica specifica. Questa conclusione non corrisponde però alla volontà di Dio, che ci offre la sua Rivelazione mediante la Bibbia come qualcosa di unitario, nono- stante la diversità presente in essa, che ci si presenta generalmente obbligatoria da credere nel suo insieme e che non si vuole obbligatoria per alcuni in alcune delle sue parti, mentre per gli altri in altri parti22. Nella diversità delle sue azioni e delle sue parole, Dio persegue un unico piano con l’intera umanità, e questo piano consiste nella ricapitolazione degli uomini nella ricchezza infinita della sua vita e della sua verità, rivelate nella persona di Gesù Cristo. Perciò, tutti i cristiani devono superare la diversità dei modi in cui Dio si rivela e in cui essi lo comprendono, per arrivare alla sua unità integrale, che è al di là di questi modi diversi di Rivelazione e di comprensione. Dio si fa conoscere e agisce attraverso atti, parole e immagini sempre diversi, perché il suo essere non si esaurisce in nessuno di questi, ma si rivela in tutti. Non dobbiamo disprezzare nessuno di questi modi, se Dio ha trovato conveniente di utilizzarli. Così pure non dobbiamo considerare che uno o tutti i modi della Rivelazione e della storia della spiritualità sviluppatasi finora, esprimono integralmente Dio, o escludono per il futuro altri modi possibili per esprimere Dio, anche se il quadro della verità divina è stato svelato nella totalità dalla Rivelazione23. Di conseguenza, le Chiese cristiane non si devono attaccare ai modi della Rive- lazione divina come alla realtà ultima, perché questi rappresentano solo la realtà penultima; oltre a questo, bisogna ammettere anche la possibilità di nuovi modi per esprimere Dio. Questo doppio atteggiamento della coscienza cristiana, cioè, da una lato, riconoscere il valore dei modi utilizzati finora per esprimere Dio e, dall’altra parte, il loro valore relativo o incompiuto, può diventare, secondo Stăniloae, un importante aiuto per ritrovare la strada dell’unità tra i cristiani. 23 “Dumnezeu se face cunoscut și lucrează prin acte, cuvinte și imagini mereu diferite, pentru că nu-și epuizează ființa în nici una din ele, dar prin toate se revelează. 24 “A rămâne numai la unele moduri ale exprimării lui Dumnezeu ca la ultima și exclusiva reali- tate înseamnă a nu trăi prezența lui decât într-un mod mărginit, sau poate deloc, și a absolutiza diversitatea noastră, nevăzând prin această diversitate pe Același Unic Dumnezeu, cu atât mai bogat cu cât îl trăim prin mai multe moduri de revelare și exprimare. Iar aceasta ne menține în dezbinare, socotind fiecare că prin modul său de a trăi și exprima pe Dumnezeu îl trăiește și-l exprimă pe Dumnezeu în întregime, și acest mod al său e unicul mod de a trăi și exprima pe Dumnezeu”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 1. La sinodalità aperta Pe nici unul din aceste moduri nu trebuie să-l disprețuim, o dată ce Dumnezeu a găsit potrivit să-l folosească, dar nu trebuie să socotim pe nici unul și nici pe toate cele din Revelație și din istoria evlaviei de până acum ca exprimând integral pe Dumnezeu, ca excluzând pentru viitor alte moduri posibile de a exprima pe Dumnezeu, chiar dacă cadrele adevărului divin au fost descoperite în totalita- tea Revelației”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 18 Zoltán József Bara Zoltán József Bara Rimanere attaccati solo ad alcuni modi che esprimono Dio, come alla realtà ultima ed esclusiva, significa vivere la sua presenza in un modo limitato, o forse affatto, e assolutizzare la nostra diversità, non vedendo più per questa diversità lo stesso Unico Dio, tanto più ricco, quanto lo viviamo in più modi di rivelazione e di espressione. E questo ci mantiene in disunione perché ognuno ritiene che il suo modo sia l’unico per vivere ed esprimere integralmente Dio24. Grazie al suo approccio cattolico-sinodale alla pienezza della Rivelazione, l’Orto­ dossia, afferma Stăniloae, è tutelata dall’attaccamento rigido ad un certo mezzo della Rivelazione. Ciò non significa però, che i suoi fedeli vivano praticamente questo movimento di trascendere la realtà penultima, per arrivare alla realtà ultima. Oltre ad essere tutelata da questo attaccamento rigido, l’Ortodossia ha la possibilità di inquadrare nel suo modo di vivere anche gli aspetti accentuati dalla Chiesa Cattolica e da quella Protestante. L’Ortodossia può dare un’importanza molto più grande alla Scrittura e alla fede, che nel passato, senza darle un valore assoluto. Essa può dare una più grande attenzione che nel passato, all’unità ecclesiale e alla sua orga- nizzazione, riattivando l’autorità morale e il ruolo presidenziale delle antiche sedi patriarcali, senza accordare un valore supremo e assolutista alla sede romana25. Per evitare l’attacamento rigido ad alcuni mezzi della Rivelazione come alla realtà ultima, di grande aiuto sono anche le critiche reciproche che si fanno le Chiese cristiane. 25 “Ortodoxia poate să dea o importanță foarte mare Scripturii și credinței - mai mare decât s-a dat în trecut - fără să le dea o valoare exclusivă. Ea poate acorda o mai mare atenție unității biseri- cești și organizării decât i-a dat în trecut, reactivând autoritatea morală și rolul prezidențial al vechilor scaune patriarhale, fără să acorde o valoare suprempă și absolutistă scaunului roman”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 175. 26 “Critica romano-cattolicismului ne poate fi de folos pentru ca să întărim mai mult unitatea noa- stră, dar nu prin dependența de un centru exterior, ci printr-o ancorare mai profundă a tuturor în Dumnezeu, Cel ma presus de toate modurile în care ni se revelează și în care îl adorăm. Cri- tica protestantismului ne poate îndemna ca, afirmând valoarea tuturor modurilor de revelare și de închinare a lui Dumnezeu..., să le socotim numai mijloace transparente pentru Dumnezeu Cel mai presus de ele, trepte de pe care să contemplăm pe Dumnezeu în bogăția lui nesfârșită”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 176. 1. La sinodalità aperta L’approccio cattolico-sinodale alla totalità dei sensi della Rivelazione implica, sottolinea infine Stăniloae, anche un certo pluralismo teologico. Il pluralismo teologico si basa sulla convinzione che ogni teologia rappresenta la contempla- zione del mistero apofatico di Dio, da un certo punto di vista. In questo modo si realizza una comprensione sinfonica di tutte queste prospettive teologiche e, quindi, una comprensione comune, che è più corrispondente alla realtà divina. 1. La sinodalità aperta Per esempio, alla critica fatta dal cattolicesimo che l’Ortodos- sia ha indebolito l’unità al suo interno, oppure a quella del protestantesimo, che essa è troppo attaccata alle forme esterne, egli risponde: La critica del romano- -cattolicesimo ci può essere utile per confermare di più la nostra unità, però non per una dipendenza da un centro esteriore, ma per un ancoraggio più profondo di tutte le cose in Dio, il quale è al di là di tutti i modi in cui ci si rivela e in cui noi lo adoriamo. La critica del protestantesimo, oltre all’affermazione del valore di tutti i modi di rivelazione e di adorazione di Dio, ci può suggerire di considerarli La sinodalità e l’intercomunione in Dumitru Stăniloae 19 soltanto come mezzi trasparenti per il Dio, che è al di là di essi, come gradini dai quali possiamo contemplare Dio nella sua ricchezza infinita26. L’approccio cattolico-sinodale alla totalità dei sensi della Rivelazione implica, sottolinea infine Stăniloae, anche un certo pluralismo teologico. Il pluralismo teologico si basa sulla convinzione che ogni teologia rappresenta la contempla- zione del mistero apofatico di Dio, da un certo punto di vista. In questo modo si realizza una comprensione sinfonica di tutte queste prospettive teologiche e, quindi, una comprensione comune, che è più corrispondente alla realtà divina. Il mistero infinito di Dio infatti, non può essere esaurito dai concetti e dalle com- prensioni umane cristiane, ma viene percepito, sotto diversi aspetti, non solo dalle singole persone, ma anche dalle diverse epoche storiche, come pure dalle diverse comunità cristiane, nazionali e geografiche. Tutte queste diverse percezioni, che si condizionano reciprocamente, ma senza uniformarsi, producono poi un adegua- mento del linguaggio cristiano ai modi di percezione del mistero divino rivelato. Così si realizza un arricchimento della comprensione umana, pur rimanendo in continuità e all’interno della stessa fede apostolica. Secondo Stăniloae, dunque, nella prospettiva della cattolicità aperta, vengono valorizzate non solo le immagini e le forme ecclesiali, ma anche quelle terrene. Le parole, le azioni e le immagini utilizzate dalla Rivelazione sottolineano l’importanza che hanno le immagini, le forme, i concetti e le parole umane nel conoscere Dio e la sua opera nel mondo, come pure nel comprendere il contenuto profondo della Scrittura. Alla luce della Rivelazione inoltre, anche l’uomo appare in tutta la sua ampiezza, in un continuo movimento di attualizzazione e di superamento, quale immagine espressiva di Dio. 27 D. Stăniloae, Iubire și adevăr: pentru o depășire a dilemei ecumenismului contemporan pe mar- ginea întâlnirii ecumeniste de la Taizé, “Ortodoxia” 19 (1967) 2, p. 288–290. Il tema di questo incontro, a cui avevano preso parte, oltre ai circa 1200 giovani, anche rappresentanti della Chiesa Cattolica, Ortodossa e Protestante, è stato dedicato alla riconciliazione tra le Chiese cristiane. Alla fine dell’incontro, i giovani chiesero alle autorità ecclesiali e ai teologi delle rispettive Chiese di trovare una soluzione positiva alle divergenze dogmatiche che impediscono la comune par- tecipazione al banchetto eucaristico. Nella sua risposta, il cardinale Bea, allora presidente del Secretariato per l’unità dei cristiani, ha affermato che la communicatio in sacris è strettamente collegata all’unità nella fede e alla Chiesa, in quanto i sacramenti sono dentro la Chiesa e non fuori di essa. Per questo motivo, la communicatio in sacris si può fare soltanto in casi eccezionali. Prendendo spunto da questa dichiarazione, Stăniloae espone di seguito la posizione ortodossa relativa al tema del’intercomunione, sottolineando soprattutto il legame tra l’amore e la verità. 29 “Este produsul celui mai patent spirit relativist și sincretist, al unei diplomații și porniri senti- mentale tranzacționiste pe tema învățăturii despre Euharistie. Ea reflectă importanța margi- nală, secundară acordată acestei învățături și practici în formațiile creștine de unde a apărut” – D. Stăniloae, În problema intercomuniunii..., p. 561. 28 D. Stăniloae, În problema intercomuniunii, “Ortodoxia” 4 (1971), p. 561–584. 30 “Poți oare să fii în Hristos pe de o parte unit, pe de alta neunit cu alții? Poți să cugeți în pun- cte importante altfel decât alții, aflându-te cu ei în același Hristos? Sau, poți să te afli deplin în Hristos cu cei ce cugetă în multe privințe altfel decât tine despre El? În ce mai constă comu- niunea cu ei în Hristos, o dată ce nu ai aceeași cugetare despre El cu cei cu care afirmi că ești în El?”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 562. “Noi non possiamo avere accesso al Padre se non nello stato di sacrificio puro; ma non perveniamo a tale stato se non mediante l’imprimersi in noi del sacrificio puro del Cristo, che ci permette di darci totalmente al Padre… Così la comunione eucaristica spesso ripetuta nella vita terrena e l’ascesa spirituale realizzata dalla nostra offerta al Cristo sempre più profonda e ininterrotta, nutrita dalla comunione eucari- stica, ci conducono verso la comunione perfetta e pienamente svelata del Cristo nella vita futura, dove la distinzione fra la comunione eucaristica con il Cristo e l’apice della nostra vita in lui ces- serà interamente”. Cf. D. Stăniloae, La Liturgie de la communauté el la Liturgie intèrieure dans la vision philocalique (Gestes et paroles dans les diverses familles liturgiques. Confèrences Saint Serge XXV-c semaine d’étude, juillet 1977), Roma 1978, p. 270. 2. L’intercomunione eucaristica Il secondo tema che sintetizza la visione ecumenica del nostro autore riguarda l’intercomunione eucaristica. Pur riconoscendo che il movimento ecumenico ha il grande merito di aver riproposto il problema dell’unità tra le Chiese cristiane e di aver stimolato il dialogo tra esse, Stăniloae sottolinea che, a volte, questo desiderio verso l’unità ha generato in molti cristiani sia un entusiasmo facile, secondo cui le differenze dottrinali tra le Chiese possono essere superate mediante 20 Zoltán József Bara il calore sentimentale dell’amore, sia uno spirito diplomatico, secondo cui l’unità tra le Chiese può essere realizzata mediante un compromesso tra gli aspetti divergenti. Un caso significativo in questo senso è rappresentato appunto dall’intercomunione eucaristica. Su questo tema, Stăniloae si è espresso inizialmente in un’articolo pubblicato nel 196727, in cui commenta il tema dell’incontro internazionale della gioventù (2–5 ottobre 1966) promosso dalla Comunità di Taizé. Uno studio più approfondito su questo argomento è apparso nel 197128, in cui espone i motivi per cui la Chiesa Ortodossa non può accettare, nello stadio attuale, la comunione eucaristica con le altre Chiese cristiane. La tendenza verso l’intercomunione eucaristica rappresenta per Stăniloae il pro- dotto del più chiaro spirito relativistico e sincretista, di una diplomazia e di una mossa sentimentale transazionista sul tema della dottrina eucaristica. Essa riflette l’importanza marginale, secondaria accordata a questa dottrina e pratica nelle formazioni cristiane dov’è apparsa29. Prodotto di uno spirito di transazione, il concetto di intercomunione eucaristica esprime, anzitutto, una grave contraddizione logica. Infatti, quale comunione eucaristica, l’intercomunione dovrebbe essere una comunione piena; quale interco- munione, però, essa è qualcosa di meno della comunione eucaristica piena, per cui non può essere considerata come comunione eucaristica, perché i gruppi che sono o vogliono essere in intercomunione eucaristica, non sono e non saranno in comu- nione totale, così come viene concepita la comunione eucaristica. Puoi forse essere, da una parte, unito in Cristo e, dall’altra parte, disunito con gli altri? Puoi pensare in punti importanti diversamente dagli altri, essendo insieme con loro nello stesso La sinodalità e l’intercomunione in Dumitru Stăniloae 21 Cristo? Oppure, puoi essere pienamente in Cristo con quelli che pensano di lui in molti aspetti diversamente da te? 31 “Prin Euharistie suntem adunați în mod real în El, că El ni se face prin Euharistie fundamen- tul comun în care viețuim și factorul care crează în toți creștinii această simțire”. Cf. D. Stăni­ loae, În problema intercomuniunii..., p. 562. 2. L’intercomunione eucaristica In che cosa consisterebbe la comunione con essi in Cristo, dato che non hai la stessa concezione su di lui con quelli che affermi che sei in lui?30 Una unione con Cristo, nonostante la differenza di concezione su di lui, si potrebbe ammettere, afferma Stăniloae, soltanto nel caso di una comunione metanoetica, vissuta nel sentimento. Ma anche in questo caso si presuppone la fede comune in qualcosa di essenziale relativo a Cristo, la fede cioè che mediante l’Eucaristia siamo radunati realmente in lui, che, per l’Eucaristia, egli diventa il fondamento comune in cui viviamo e il fattore che crea in tutti i cristiani questo sentimento31. Questo tipo d’unità però, non si trova in quelli che tendono verso l’intercomunione, in quanto manca il fondamento di una comunione nella fede. L’intercomunione che si desidera realizzare in questo senso è piuttosto una comunione formale, che vuole far convivere la comunione eucaristica con la non comunione cosciente e volontaria in molti punti essenziali della fede. Perciò, la mossa sentimentale e diplomatica, che ha lanciato l’idea e la pratica dell’intercomunione, ha creato una formula ibrida, contradditoria in se stessa, che può essere accettata soltanto da una coscienza addormentata, che non pensa fino in fondo al senso dell’atto che compie. Per una coscienza sveglia, invece, una comunione eucaristica reale e durabile non è pensabile senza la comunione nella fede. Privi di questa coscienza, i sostenitori dell’idea di intercomunione esprimono, quindi, un certo disprezzo, sia nei confronti del tesoro della fede, sia nei confronti dell’Eucaristia. Infatti, se considerano che l’intercomunione eucaristica è una comunione piena, essi mettono l’Eucaristia al di sopra di ogni cosa e disprezzano la comunione nei punti importanti della 22 Zoltán József Bara fede. Ma siccome tra i punti di fede diversi fa parte anche l’Eucaristia, sulla quale le Chiese hanno delle concezioni molto divergenti, si può dire piuttosto che questa alternativa rappresenta un atteggiamento relativistico non solo riguardo al tesoro della fede, ma anche nei confronti della dottrina eucaristica della propria Chiesa32. Se invece, ammettono che per l’intercomunione non si vuole realizzare una comunione ontologica in Cristo, ma soltanto una comunione psicologica parziale, quale mezzo per l’avvicinamento dottrinale tra le Chiese, essi disprezzano l’Euca- ristia, mentre rispettano il tesoro della fede proprio di ogni Chiesa, con tutte le sue differenze, dato che non possono rinunciare a queste differenze. 32 “Pun Euharistia mai presus de orice și disprețuiesc comuniunea în puncte importante ale cre- dinței... Dar întrucât dintre punctele de credință deosebite face parte și Eucaristia, despre care Bisericile au concepții foarte deosebite, se poate spune mai degrabă că această alternativă repre- zintă o atitudine relativistă nu numai față de tezaurul de credință, ci și față de învățătura despre Euharistie a propriei Biserici”. D. Stăniloae, În problema intercomuniunii..., p. 563. 33 “Ortodoxia, considerând Biserica drept o unitate profund și ontologic ancorată în Hristos, iar Euharistia ca mijloc culminant de susținere continuă a acestei unități, nu poate coborî Euha- ristia la nivelul unui mijloc de unitate laxă, psihologică și superficială între creștini. Aceasta ar avea repercusiuni pentru însăși sensul ei despre Biserică... Căci ar fi greu să conceapă și să practice Euharistia în două sensuri, ca având două roluri: într-unul când e primită împreună cu membrii Bisericii și într-altul când e primită cu cei ce nu sunt membrii Bisericii Ortodoxe”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 564. 32 “Pun Euharistia mai presus de orice și disprețuiesc comuniunea în puncte importante ale cre- dinței... Dar întrucât dintre punctele de credință deosebite face parte și Eucaristia, despre care Bisericile au concepții foarte deosebite, se poate spune mai degrabă că această alternativă repre- zintă o atitudine relativistă nu numai față de tezaurul de credință, ci și față de învățătura despre Euharistie a propriei Biserici”. D. Stăniloae, În problema intercomuniunii..., p. 563. 33 “Ortodoxia, considerând Biserica drept o unitate profund și ontologic ancorată în Hristos, iar Euharistia ca mijloc culminant de susținere continuă a acestei unități, nu poate coborî Euha- ristia la nivelul unui mijloc de unitate laxă, psihologică și superficială între creștini. Aceasta ar avea repercusiuni pentru însăși sensul ei despre Biserică... Căci ar fi greu să conceapă și să practice Euharistia în două sensuri, ca având două roluri: într-unul când e primită împreună cu membrii Bisericii și într-altul când e primită cu cei ce nu sunt membrii Bisericii Ortodoxe”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 564. 34 Stăniloae si è espresso in termini chiari contro l’intercomunione in assenza di unità dottrinale: “Non posso capire come la comunione alla santa eucaristia possa compensare in qualche modo la non comunione nella fede”. Ha sostenuto che l’ortodossia non è risultato dell’eucaristia, ma che la vera eucaristia è risultato dell’ortodossia, cosicché la comunione eucaristica può aver luogo soltanto in un contesto in cui si condivide la stessa fede. Cf. R. Bordeianu, Ospitalità eucari- stica: un’analisi fenomenologica della recente teologia ortodossa, in: Il dono dell’ospitalità. Atti del XXV Convegno ecumenico internazionale di spiritualità ortodossa (Bose, 6–9 settembre 2017), a cura di L. Chremaschi, L.D’Ayala Valva, A. Mainardi, Magnano (BI) 2018, p. qui 271–272. Le parole di Stăniloae in: T. Ware, Church and Eucharist, Communion and Intercommunion, “Sobornost” 7 (1978), p. 558. 2. L’intercomunione eucaristica Alla base di questo atteggiamento si trova difatti una diversa concezione sull’unità della Chiesa, come pure sulla natura stessa della Chiesa, concezione che l’Ortodossia non può accettare. L’Ortodossia, considerando la Chiesa come un’unità profondamente e ontologi- camente ancorata in Cristo, e l’Eucaristia come il mezzo culminante che sostiene quest’unità, non può far scendere l’Eucaristia al livello di un mezzo di unità lassa, psicologica e superficiale tra i cristiani. Questo avrebbe delle ripercussioni sulla sua stessa concezione sulla Chiesa... Perché sarebbe difficile concepire e praticare l’Eucaristia in due sensi, o come avente due ruoli: il primo quando è ricevuta insieme con i membri della Chiesa, il secondo quando è ricevuta insieme con quelli che non sono membri della Chiesa Ortodossa33. Nella concezione della Chiesa Ortodossa, sottolinea lui, l’unità della fede e la convinzione che nell’Eucaristia ci si comunica al Corpo e al Sangue di Cristo sono strettamente collegate con la convinzione che la Chiesa rappresenta un’unità ontologica profonda dei fedeli con Cristo e tra di loro. Infatti, senza l’unità della fede e senza la comunione con il Corpo e il Sangue di Cristo, non potrebbe esi- stere la Chiesa nel senso pieno della parola, così come senza l’unità nella fede e senza la Chiesa, non si può realizzare la comunione eucaristica. Nello stesso modo, senza la Chiesa e senza la comunione eucaristica, l’unità della fede non è durabile. Perciò, per la Chiesa Ortodossa è inconcepibile un’intercomunione eucaristica, La sinodalità e l’intercomunione in Dumitru Stăniloae 23 che separa l’unità della fede dalla Chiesa, perché porta alla dissoluzione della Chiesa, dell’unità della fede e dell’Eucaristia34. Tenendo conto di queste tre coordinate essenziali della vera e piena comunione tra i cristiani in Cristo, Stăniloae esprime il suo disaccordo nei confronti di quei teologi ortodossi, che, sotto l’influsso del movimento ecumenico, inclinano ad ammettere che la Chiesa Ortodossa può dare, in certe circostanze, l’Eucaristia ai membri della Chiesa Romano-Cattolica, della Chiesa Vecchio-Cattolica e delle Chiese Antiche Orientali, in virtù del principio dell’economia, perché, secondo lui, ciò significa un’interpretazione errata della nozione e della pratica dell’economia. Nella con- cezione ortodossa, difatti, l’economia significa il riconoscimento dei sacramenti (Battesimo, Confermazione, Matrimonio e Ordine) ricevuti da qualcuno fuori della Chiesa Ortodossa, nel momento in cui diventa membro di questa Chiesa, e non l’amministrazione di un sacramento a qualcuno che continua a rimanere membro di un’altra Chiesa. 35 “În fața unei atitudini favorabile față de intercomunione e cazul să ne întrebăm: membrii unei Biserici care se împărtășesc de Tainele altei Biserici, cărei Biserici aparțin?... Ei nu mai aparțin deplin nici Bisericii lor, o dată ce primesc Euharistia de la altă Biserică, nici celei de la care pri- mesc uneori Euharistia, pentru că nu au credința ei. Ei plutesc oarecum în spațiul spiritual 2. L’intercomunione eucaristica Perciò, la comunione sacramentale della rispettiva persona con i membri della Chiesa Ortodossa ha come fondamento l’avvenuta comunione nella fede con questi. In questo modo non si rompe il legame tra i sacramenti, la Chiesa e l’unità di fede, come avviene invece nel caso dell’intercomunione. Di fronte ad un atteggiamento favorevole verso l’intercomunione, è il caso di chiederci: i membri di una Chiesa che partecipano ai sacramenti di un’altra Chiesa, a quale Chiesa appartengono? [...] Essi non appartengono pienamente né alla loro Chiesa, dato che ricevono l’Eucaristia in un’altra Chiesa, né a quella dove ricevono a volte l’Eucaristia, perché non hanno la sua fede. Essi galleggiano in un certo senso nello spazio spirituale tra le due Chiese. Non avendo una fede comune con i membri della Chiesa dove si comunicano, e non conservando più una piena comunione nei sacramenti neanche con i membri della loro Chiesa, essi hanno soltanto una comunione imperfetta con ciascuna delle due Chiese35. 35 “În fața unei atitudini favorabile față de intercomunione e cazul să ne întrebăm: membrii unei Biserici care se împărtășesc de Tainele altei Biserici, cărei Biserici aparțin?... Ei nu mai aparțin deplin nici Bisericii lor, o dată ce primesc Euharistia de la altă Biserică, nici celei de la care pri- mesc uneori Euharistia, pentru că nu au credința ei. Ei plutesc oarecum în spațiul spiritual 24 Zoltán József Bara Per quanto riguarda l’intercomunione tra la Chiesa Ortodossa e la Chiesa Roma- no-Cattolica, Stăniloae sostiene che questa non può essere proposta, finché non si arriva all’unità della fede ortodossa, fondamento della piena comunione eucaristica. In questo senso, egli esprime la sua disapprovazione nei confronti degli esponenti della Chiesa di Costantinopoli, che inclinano ad accettare la piena comunione eucaristica con la Chiesa Romano-Cattolica, argomentando che, dopo la sospensione reciproca della scomunica pronunciata nel 1054, le due Chiese si trovano, dal punto di vista dogmatico, esattamente nella situazione anteriore al 1054 e quindi non esiste più alcun ostacolo per il ristabilimento della comunione eucaristica. Di fronte a quest’affermazione, dobbiamo ricordare che allora gli anatemi sono stati indetti sulla base delle differenze apparse riguardo alla processione dello Spirito Santo e all’inizio dell’affermazione del diritto di giurisdizione del Papa sulla gerarchia orientale. E dopo questo, la divisione nella fede ha continuato con la formulazione dogmatica del primato e dell’infallibilità papale, dell’immacolata concezione, del purgatorio, ecc. 2. L’intercomunione eucaristica [...] Avendo solo pochi fedeli, gli esponenenti di quella Chiesa autocefala si affrettano a rispondere positivamente all’offerta del Papa in questo senso, in quanto non conoscono più la responsabilità verso le masse dei fedeli e non si rendono conto che per una simile intercomunione eucaristica, rendono le masse indifferenti nei confronti del rifiuto del primato papale e le portano sotto il giogo del primato papale, oppure nell’uniatismo e nella divisione36. dintre cele două Biserici. Neavând o credință comună cu membrii Bisericii celelalte cu care se împărtășesc, dar nemaipăstrând o comuniune deplină în Taine nici cu membrii Bisericii sale, ei n-au decât o comuniune nedeplină cu fiecare Biserică”. Cf. D. Stăniloae, În problema inter- comuniunii..., p. 565–566. 36 “Față de această afirmație, trebuie să amintim că anatemele s-au rostit atunci pe baza deosebirii apărute în privința purcederii Duhului Sfânt și a începutului de afirmare a dreptului de jurisdi- cție al papei asupra ierarhiei orientale. Iar de atunci încoace dezbinarea în credință a progresat, prin formularea dogmatică a primatului și a infailibilității papale, a imaculatei concepții, a pur- gatorului etc... Lipsită aproape total de credincioși, conducerea acelei Biserici Ortodoxe auto- cefale, se grăbește să răspundă pozitiv unei oferte a papei în acest sens, întrucât nu mai cuno- aște experiența răspunderii față de masele de credincioși și nu-și dă seama că printr-o astfel de intercomuniune euharistică, făcându-le indiferente față de respingerea primatului papal, le-ar arunca sub jugul primatului papal, sau în uniatism și dezbinare”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 568. Infatti, Dumitru Stăniloae si è espresso contro l’intercomunione in assenza di unità dottrinale: “Non posso capire come la comunione alla santa eucaristia possa compensare in qualche modo la non comunione nella fede”. Ha sostenuto che l’ortodossia non è risultato dell’eucaristia, ma che la vera eucaristia è risultato dell’ortodossia, cosicché la comu- nione eucaristica può aver luogo soltanto in un contesto in cui si condivide la stessa fede. Tale conclusione deriva dall’ordo della divina liturgia in cui la comunità dapprima confessa la mede- sima fede, e poi ha luogo l’epiclesi. Ma Stăniloae non vuole insinuare che la chiesa cattolica ha perduto la vera fede a tal punto che la sua eucaristia non sia valida. dintre cele două Biserici. Neavând o credință comună cu membrii Bisericii celelalte cu care se împărtășesc, dar nemaipăstrând o comuniune deplină în Taine nici cu membrii Bisericii sale, ei n-au decât o comuniune nedeplină cu fiecare Biserică”. Cf. D. Stăniloae, În problema inter- comuniunii..., p. 565–566. 2. L’intercomunione eucaristica Riconosce che la chiesa cat- tolica professa la presenza del corpo e del sangue di Cristo nell’eucaristia e, ancor più, afferma 36 “Față de această afirmație, trebuie să amintim că anatemele s-au rostit atunci pe baza deosebirii apărute în privința purcederii Duhului Sfânt și a începutului de afirmare a dreptului de jurisdi- cție al papei asupra ierarhiei orientale. Iar de atunci încoace dezbinarea în credință a progresat, prin formularea dogmatică a primatului și a infailibilității papale, a imaculatei concepții, a pur- gatorului etc... Lipsită aproape total de credincioși, conducerea acelei Biserici Ortodoxe auto- cefale, se grăbește să răspundă pozitiv unei oferte a papei în acest sens, întrucât nu mai cuno- aște experiența răspunderii față de masele de credincioși și nu-și dă seama că printr-o astfel de intercomuniune euharistică, făcându-le indiferente față de respingerea primatului papal, le-ar arunca sub jugul primatului papal, sau în uniatism și dezbinare”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 568. Infatti, Dumitru Stăniloae si è espresso contro l’intercomunione in assenza di unità dottrinale: “Non posso capire come la comunione alla santa eucaristia possa compensare in qualche modo la non comunione nella fede”. Ha sostenuto che l’ortodossia non è risultato dell’eucaristia, ma che la vera eucaristia è risultato dell’ortodossia, cosicché la comu- nione eucaristica può aver luogo soltanto in un contesto in cui si condivide la stessa fede. Tale conclusione deriva dall’ordo della divina liturgia in cui la comunità dapprima confessa la mede- sima fede, e poi ha luogo l’epiclesi. Ma Stăniloae non vuole insinuare che la chiesa cattolica ha perduto la vera fede a tal punto che la sua eucaristia non sia valida. Riconosce che la chiesa cat- tolica professa la presenza del corpo e del sangue di Cristo nell’eucaristia e, ancor più, afferma La sinodalità e l’intercomunione in Dumitru Stăniloae 25 Con la Chiesa Vecchio-Cattolica invece, pur essendo più vicina, egli considera che, anche in questo caso, la comunione eucaristica non si può realizzare finché non viene firmata una dichiarazione comune sulla piena unità nella fede. Per quanto riguarda, infine, le Chiese Antiche-Orientali, secondo lui, l’inter- comunione è ancora più facile, dato che tra queste Chiese e la Chiesa Ortodossa, come hanno mostrato le comissioni teologiche miste, non sussiste più alcuna differenza dottrinale essenziale, anzi tra esse c’è una profonda identità relativa alla spiritualità e alla concezione sacramentale ed ecclesiologica. 37 “Tocmai pentru a păstra neslăbită conștiința supremei iubiri a lui Hristos față de lume nși putința de a trăi această iubire pentru toți creștinii și pentru toți oamenii, tocmai pentru a nu goli Euha- ristia de eficacitatea acestei supreme iubiri, Ortodoxia nu se învoiește cu tratarea Euharistiei ca o ceremonie mai mult simbolică, ca o prezență echivocă și insuficient de eficace a trupului lui Hristos, ca o ceremonie cu o semnificație mai mult teoretică și sentimentală, despărțită mai mult sau mai puțin de prezența reală a trupului lui Hristos și a energiilor lui dumnezeiești; toc- mai de aceea ea nu se învoiește să considere împărtășania comună ca pe un act lipsit de supor- tul unei credințe comune, lipsit de calitatea de bază a unei înaintări moralascetice în sfințenie, che la chiesa cattolica, così come la chiesa ortodossa, ha conservato la fede nella Trinità e in Cri- sto, anche se ha aggiunto all’antica fede della chiesa il dogma del primato e dell’infallibilità del papa, il Filioque e il purgatorio. Tra tutti questi dogmi, quello del primato del papa e della sua infallibilità sono ostacoli alla comunione, e dividono la chiesa. Cf. T. Ware, Church and Eucha- rist..., p. 558; D. Stăniloae, Spiritualitate şi comunione în Liturghia Orthodoxă, Craiova 1986, p. 389–399 e 401–402. che la chiesa cattolica, così come la chiesa ortodossa, ha conservato la fede nella Trinità e in Cri- sto, anche se ha aggiunto all’antica fede della chiesa il dogma del primato e dell’infallibilità del papa, il Filioque e il purgatorio. Tra tutti questi dogmi, quello del primato del papa e della sua infallibilità sono ostacoli alla comunione, e dividono la chiesa. Cf. T. Ware, Church and Eucha- rist..., p. 558; D. Stăniloae, Spiritualitate şi comunione în Liturghia Orthodoxă, Craiova 1986, p. 389–399 e 401–402. ca pe un act lipsit de puterea care unește într-o Biserică pe cei ce îl practică”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 582. 38 “Membrii Bisericii Ortodoxe sunt chemați să se comporte ca mâinile lucrătoare ale lui Hristos, ca ochii înțelegători ai lui Hristos, ca urechile atente ale lui Hristos, ca inima iubitoare a lui Hris­tos față de toți, față de toate trebuințele și chemările altora, dovedind cu fapta că sunt măd- ularele lui Hristos”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 582. 2. L’intercomunione eucaristica Nonostante questo, egli afferma che, prima di una dichiarazione ufficiale, firmata dai sinodi locali delle due Chiese riguardo all’unità della fede, non si può ammettere che una comunione sacramentale parziale. Il rifiuto netto dell’intercomunione con le altre Chiese cristiane, non significa però una mancanza d’amore della Chiesa Ortodossa nei confronti degli altri cristiani? Secondo Stăniloae, questo rifiuto della Chiesa Ortodossa non dev’essere inteso come mancanza d’amore, ma come testimonianza a favore del vero amore di Cristo: Proprio per conservare forte la coscienza dell’amore supremo di Cristo per l’umanità, come pure la possibilità di vivere questo amore per tutti i cristiani e per tutti gli uomini, proprio per non svuotare l’Eucaristia dall’efficacia di quest’amore, l’Ortodossia non accetta di trattare l’Eucaristia come una cerimonia simbolica, come una presenza equivoca ed insufficientemente efficace del Corpo di Cristo, come una cerimonia con un significato teoretico e sentimentale, separata più o meno dalla presenza reale del Corpo di Cristo e dalle sue energie divine; proprio per questo essa non accetta di considerare la comunione come un atto privo del supporto delle fede comune, privo della qualità fondamentale per un progresso morale e ascetico nella santità, come un atto privo della forza che unisce in una Chiesa tutti coloro che lo praticano37. 26 Zoltán József Bara Secondo Stăniloae, dunque, la Chiesa Ortodossa respinge l’intercomunione eucaristica con le altre Chiese cristiane, perché vuole conservare l’Eucaristia come il nucleo supremo e inesauribile, da dove irradia l’amore di Cristo. Ciò non signi- fica una chiusura in se stessa, oppure una diminuzione della sua tensione verso l’universalità; ma l’amore che unisce i suoi membri nel Corpo mistico di Cristo, essendo alimentato dall’Eucaristia, si irradia da questi verso tutti i cristiani. Diventati membra del Corpo di Cristo, i cristiani ortodossi possono perciò esprimere in tante altre forme il loro amore verso gli altri cristiani: I membri della Chiesa Ortodossa sono chiamati a comportarsi come le mani operose di Cristo, come gli occhi comprensivi di Cristo, come gli orecchi attenti di Cristo, come il cuore amorevole di Cristo verso tutti e verso tutte le necessità e le vocazioni degli altri, manifestando praticamente che sono membra di Cristo38. ca pe un act lipsit de puterea care unește într-o Biserică pe cei ce îl practică”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 582. Conclusione Stăniloae ha definito le coordinate dell’ecumenismo dal punto di vista ortodosso. La prima coordinata, quale non abbiamo discusso in questo brano si riferisce all’unione di tutte le Chiese nell’integrità della fede apostolica, fede vissuta dalla Chiesa indivisa del primo millennio cristiano. La fede apostolica include gli scritti, la tipologia e la tradizione apostolica. Custode della fede apostolica è la Chiesa, per cui alla fede apostolica appartiene anche la dimensione istituzionale della Chiesa, cioè la vita di comunione con Dio mediante i sacramenti celebrati dai ministri ordinati. La piena valorizzazione della fede apostolica si realizza però mediante l’unità cattolico-sinodale della Chiesa. L’unità cattolico-sinodale della Chiesa è una unità dogmatica, che concilia in una misteriosa e indissolubile tensione l’unità con la diver- sità. Perciò, la seconda coordinata dell’ecumenismo riguarda l’unità dogmatica nella diversità e nell’indipendenza delle Chiese locali. In questo senso, l’unità delle Chiese si realizza attraverso la loro unione nella verità rivelata, e non attraverso una unione di tipo politico o amministrativo. Infine, la terza coordinata consiste nell’aper- tura delle Chiese al mondo. Il motivo di quest’apertura deriva dal fatto che Dio agisce anche al di là delle frontiere visibili della Chiesa. Riconoscendo che Dio agisce non solo per mezzo della Chiesa sul mondo, ma anche per mezzo del mondo sulla Chiesa, le Chiese cristiane comprendono in una nuova luce non solo la Rivelazione, La sinodalità e l’intercomunione in Dumitru Stăniloae 27 ma anche la creazione, l’Incarnazione del Figlio di Dio, la dimensione cosmica della salvezza, la comunità umana e la cattolicità-sinodale della Chiesa. In questo modo si realizza anche un’avvicinamento dottrinale tra esse. Alla luce di questi principi ecumenici, Stăniloae ha pronosticato una sinodalità o cattolicità aperta, quale apertura dogmatica e spirituale delle Chiese cristiane per ritrovare la loro unità. Alla base di questa cattolicità aperta si trova il fatto che nella Sacra Scrittura c’è una diversità di tradizioni e significati, diversità che proviene dalla diversità delle azioni di Dio nella storia di salvezza. Se l’approccio unilaterale ai sensi della Scrittura è stato nel passato causa di divisione tra i cristiani, l’approccio cattolico-sinodale a tutti i sensi della Scrittura può diventare il punto d’incontro tra tutti i cristiani. Per arrivare a questo punto d’incontro, le Chiese cristiane devono quindi evitare di attaccarsi ad uno o più di questi sensi, perché essi rappresentano solo la realtà penultima. 39 R. Bordeianu, Dumitru Staniloae: An Ecumenical Ecclesiology, Edinburgh 2013, p. 29. 40 D. Stăniloae, Coordonatele ecumenismului din punct de vedere ortodox, in “Ortodoxia” 19 (1967) 4, p. 494–540. D. Stăniloae, Coordonatele ecumenismului din punct de vedere ortodox, in “Ortodoxia” 19 (1967) 4, p. 494–540. 39 R. Bordeianu, Dumitru Staniloae: An Ecumenical Ecclesiology, Edinburgh 2013, p. 29. Conclusione Quello che le Chiese cristiane devono cercare, è invece la realtà ultima, cioè Dio infinitamente ricco, che si manifesta in diversi modi, ma che prosegue un unico scopo: la ricapitolazione di tutte le cose in Cristo. Di grande aiuto per la realizzazione dell’approccio cattolico-sinodale sono le critiche reciproche delle Chiese, come anche la promozione di un sano pluralismo teologico.39 Nel desiderio di superare le differenze dogmatiche che separano le Chiese e per arrivare alla piena comunione eucaristica, molti – secondo Stăniloae – sono caduti in un entusiasmo facile, pensando che le differenze possano essere superate con il calore dell’amore, oppure in uno spirito diplomatico, realizzando un compromesso tra le posizioni divergenti. Il risultato di queste due tendenze, che hanno alla base un diversa concezione ecclesiologica, è conosciuto con il termine di intercomunione eucaristica. Secondo Stăniloae, l’intercomunione eucaristica, oltre ad essere l’espres- sione di una mentalità relativista e transazionista, comporta una contraddizione logica, perché l’intercomunione non è comunione eucaristica piena e, quindi, i gruppi che vogliono essere in intercomunione non possono essere in comunione totale, così come invece viene concepita la comunione eucaristica. La comunione con il Corpo e il Sangue di Cristo non può essere separata dall’unità nella fede e dalla convinzione che la Chiesa rappresenta una profonda unità ontologica dei fedeli in Cristo e tra essi. Questi tre elementi sono inseparabili, e non si può avere uno senza l’altro. Per questo motivo, egli non è d’accordo con quelli che propongono l’intercomunione eucaristica tra la Chiesa Ortodossa e le altre Chiese cristiane, senza arrivare prima ad un accordo dogmatico40. Il rifiuto dell’intercomunione 28 Zoltán József Bara Zoltán József Bara non dev’essere però visto come una mancanza d’amore nei confronti di queste Chiese, ma come una testimonianza a favore dell’Eucaristia, quale nucleo supremo e inesauribile da dove irradia l’amore di Cristo. In 50 anni il dialogo tra le Chiese si è approfondito e arricchito di molti argomenti. Comunque le tesi di Stăniloae sono ancora stimolanti, specialmente quella sulla sinodalità aperta. Però, se biso- gna accettare le contraddizioni, come sostiene nella sinodalità aperta, perché non accettarle anche nella intercomunione? Bibliografia Stăniloae, D., În problema intercomuniunii, “Ortodoxia” 4 (1971), p. 561–584. Stăniloae D., Sobornicitate deschisă, “Ortodoxia” 23 (1971) 2, p. 165–180. Stăniloae D., La Liturgie de la communauté el la Liturgie intèrieure dans la vision philoca- lique (Gestes et paroles dans les diverses familles liturgiques. Confèrences Saint Serge XXV-c semaine d’étude, juillet 1977), Roma 1978. Stăniloae, D., Dogmatica I, București 2003. Stăniloae, D., Dogmatica I, București 2003. Stăniloae D., Dogmatica II, București 2003. Stăniloae D., Spiritualitate şi comunione în Liturghia Orthodoxă, Craiova 1986. Theologen des 20. Jahrhunderts. Eine Einführung, a cura di P. Neuner, G. Wenz, Darmstadt 2002, p. 145–156. Turcescu L., Eucharistic Ecclesiology or Open Sobornicity?, in: Dumitru Stăniloae: Tradi- tion and Modernity in Theology, a cura di L. Turcescu, Palm Beach 2002, p. 83–103. Ware T., Church and Eucharist, Communion and Intercommunion, “Sobornost” 7 (1978), p. 550–567. Bibliografia Bara Z., Un approccio ortodosso ecclesiologico alla dimensione sacramentale della Chiesa: la Chiesa, Sacramento di Cristo, secondo Dumitru Stăniloae, in: Sakramenty nowym wyzwaniem dla Kościoła Katolickiego, a cura di J. Radkiewicz, Szczecin 2019, p. 155–180. Bara Z., La realtà mistica della Chiesa nella riflessione ortodossa di Dumitru Stăniloae, “Studia Theologia Catholica Latina” 66 (2021) 2, p. 77–107. Bordeianu R., Dumitru Staniloae: An Ecumenical Ecclesiology, Edinburgh 2013. Bordeianu R., (In)Voluntary Ecumenism: Dumitru Stăniloae’s Interaction with the West as Open Sobornicity, in: Orthodox Constructions of the West, a cura di E.G. Demaco- poulos, A. Papanikolaou, New York 2013, p. 240–253. Bordeianu R., Ospitalità eucaristica: un’analisi fenomenologica della recente teologia ortodossa, in: Il dono dell’ospitalità. Atti del XXV Convegno ecumenico internazionale di spiritualità ortodossa (Bose, 6–9 settembre 2017), a cura di L. Chremaschi, L.D’Ayala Valva, A. Mainardi, Magnano (BI) 2018, p. 263–285. Coman V., “Open Sobornicity” and “Receptive Ecumenism”: Fruitful Models of Ecumenical Interaction, in: Just Do It? Recognition and Reception in Ecumenical Relations: Procee- dings of the 19th Academic Consultation of the Societas Oecumenica, a cura di D. Heller, M. Hietamaki, Leipzig 2018, p. 241–251. Coman V., Le Saint Esprit comme liaison de l’amour éternel entre le Père et le Fils: un cas de ‘sobornicité ouverte’ dans la théologie orthodoxe, “Irénikon” 89 (2016) 1, p. 25–51. Moşoiu N., “Sobornicitatea deschisă” ca tipologie ecumenică sau De la teologia confesională la teologia ecumenică în perspectiva viziunii despre “sobornicitatea deschisă”, “Revista Teologică” 4 (1997), p. 87–117. Nouveauté dans l’oecumenisme, Taizé 1968. Nouveauté dans l’oecumenisme, Taizé 1968. Roberson G.R., Ecumenism in the Thought of Dumitru Stăniloae, in: T. Damian, G. Alexe, E. Pentiuc, R. Roberson, J. Jonita, L. Turcescu, The Theological Legacy of Fr. Dumitru Stăniloae and its Ecumenical Actuality, Symposium VI/1, New York 1999, p. 43–54. Stavrou M., Teologia cristiana ortodossa, in: Dizionario del sapere storico-religioso del Novecento, a cura di A. Melloni, Bologna 2010. Stăniloae D., Sinteză eclesiologică, “Studii Teologice” 7 (1955) 5–6, p. 267–284. La sinodalità e l’intercomunione in Dumitru Stăniloae 29 Stăniloae D., Coordonatele ecumenismului din punct de vedere ortodox, „Ortodoxia” 19 (1967) 4, p. 494–540. Stăniloae D., Coordonatele ecumenismului din punct de vedere ortodox, „Ortodoxia” (1967) 4, p. 494–540. Stăniloae D., Iubire și adevăr: pentru o depășire a dilemei ecumenismului contemporan pe marginea întâlnirii ecumeniste de la Taizé, “Ortodoxia” 19 (1967) 2, p. 288–290. Stăniloae D., Transparenţa Bisericii în viaţa sacramentală, “Ortodoxia” 22 (1970) 4, p. 501–516. Sommario Nell’opera di Dumitru Stăniloae esiste una grande varietà di temi e problemi ecumenici, in cui egli ha assunto delle posizioni originali, a volte radicali, ma sempre favorevoli al dialogo. In questo brano vorrei presentare specialmente due temi che, in un certo senso, sintetizzano la visione ecumenico originale di Stăniloae sull’unione delle Chiese: il tema della cattolicità o sinodalità aperta, e quella dell’intercomunione eucaristica, seguendo i suoi argomenti. Stăniloae ha pronosticato una sinodalità o cattolicità aperta, quale apertura dogmatica e spirituale delle Chiese cristiane per ritrovare la loro unità. Alla base di questa cattolicità aperta si trova il fatto che nella Sacra Scrittura c’è una diversità di tradizioni e significati, diversità che provengono dalla diversità delle azioni di Dio nella storia di salvezza. L’approccio cattolico-sinodale a tutti i sensi della Scrittura può diventare il punto d’incontro tra tutti i cristiani. Dall’altra parte, nella visione di Stăniloae, l’intercomunione eucaristica deve essere una comu- nione eucaristica piena. L’Ortodossia, considerando la Chiesa come un’unità profondamente 30 Zoltán József Bara e ontologicamente ancorata in Cristo, e l’Eucaristia come il mezzo culminante che sostiene quest’unità, non può far scendere l’Eucaristia al livello di un mezzo di unità lassa, psicologica e superficiale tra i cristiani. Infatti, senza l’unità della fede e senza la comunione con il Corpo e il Sangue di Cristo, non potrebbe esistere la Chiesa nel senso pieno della parola, così come senza l’unità nella fede e senza la Chiesa non si può realizzare la comunione eucaristica. È inconcepibile un’intercomunione eucaristica che separa l’unità della fede dalla Chiesa, perché porta alla dissoluzione della Chiesa, dell’unità della fede e dell’Eucaristia. La comunione con il Corpo e il Sangue di Cristo non può essere separata dall’unità nella fede e dalla convin- zione che la Chiesa rappresenta una profonda unità ontologica dei fedeli in Cristo e tra essi. Parole Chiave: Dumitru Stăniloae, ecumenismo, cattolicità aperta (sinodalità), unione delle Chiese, intercomunione eucaristica Nota autorska Zoltán József Bara – duchowny rzymskokatolicki, adiunkt na Wydziale Teologii Rzym- skokatolickiej Uniwersytetu Babeșa i Bolyaia w Cluj-Napoca w Rumunii oraz na Wydziale Rzymsko­katolickiej Teologii Pastoralnej w Alba Iulia (Seminarium Incarnatae Sapientiae); e-mail: barazoli@gmail.com; zoltan.bara@ubbcluj.ro. Bara Z.J., La sinodalità e l’intercomunione in Dumitru Stăniloae, „Colloquia Theologica Otto- niana” 38 (2022), s. 9–30. DOI: 10.18276/cto.2022.38-01. Cytowanie Bara Z.J., La sinodalità e l’intercomunione in Dumitru Stăniloae, „Colloquia Theologica Otto- niana” 38 (2022), s. 9–30. DOI: 10.18276/cto.2022.38-01.
https://openalex.org/W2462694193
https://www.scielo.br/j/man/a/k837CYKCrNWKxJGNnNPqJtm/?lang=en&format=pdf
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Transduction and BRICS
Manuscrito
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cc-by
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CDD: 337.1 CDD: 337.1 DOI: http://dx.doi.org/10.1590/0100-6045.2015.V38N3.DP DOI: http://dx.doi.org/10.1590/0100-6045.2015.V38N3.DP Abstract: BRICS has philosophical significance. It creates new pressure on cross-cultural skill. This is analysed here as requiring transduction: a variety of defeasible practical reasoning. This replaces a simplistic model of the relation between knowledge and action with a more realistic and contemporary model. The transduction format has utility in cross-cultural training. Keywords: BRICS; cross-cultural skill; context; executive function; schema theory. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Don Peterson Shantou University Department of Computer Science Da Xue Lu, 243, Guangdong, 515063, China. 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063 peterson@stu.edu.cn Shantou University Department of Computer Science Da Xue Lu, 243, Guangdong, 515063, China. 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063 peterson@stu.edu.cn Shantou University Department of Computer Science Da Xue Lu, 243, Guangdong, 515063, China. 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063 peterson@stu.edu.cn 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063 peterson@stu.edu.cn Received: 17.01.2015; Revised: 22.10.2015; Accepted: 30.12.2015 BRICS BRICS is a collaboration between Brazil, Russia, India, China, and South Africa: it was identified as a possibility by O'Neill (2001), official discussion began in New York in 2006, and the bloc was formally initiated in Yekaterinburg in 2009. Since 2010, the BRICS nations have met annually at formal summits. Bilateral relations among BRICS nations emphasise mutual 'win-win' benefit, non-interference, and equality. There are other blocs: ASEAN (Association of Southeast Asian Nations), initiated in 1967, now includes Indonesia, Malaysia, the Philippines, Singapore, Thailand, Brunei, Cambodia, Laos, Myanmar, and Vietnam. O'Neill, who first identified BRICS, has identified: MIKT (Mexico, Indonesia, South Korea, and Turkey), MINT (Mexico, Indonesia, Nigeria, and Turkey), and the Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. 16 Don Peterson Next Eleven (Bangladesh, Egypt, Indonesia, Iran, Mexico, Nigeria, Pakistan, the Philippines, Turkey, South Korea, and Vietnam). Next Eleven (Bangladesh, Egypt, Indonesia, Iran, Mexico, Nigeria, Pakistan, the Philippines, Turkey, South Korea, and Vietnam). These blocs are, of course, an issue for economics and politics (Gutemberg, 2015). They are also an issue and direction for contemporary philosophy. The world has changed, life has changed, and the challenge to our cognitive apparatus has changed. There may be 'nothing new under the sun', but the need to interact and collaborate with 'the other' is greater now than before. In the past, of course, some people needed to interact internationally, but the obligation is greater now. The modern condition in the 21st Century is one of cooperating heterogeneity, in which we keep our differences, but we have to collaborate. In the past, we had our differences (in culture, language, religion, cuisine, legal systems, business practice, art, etc.) but we could ignore each other, or not take each other seriously, even under colonialism. In the present, we have our differences but we cannot ignore each other. There has occurred, in fact, a sea change in the demands on human cognition. This may be a change in degree rather than principle, but it is a sea change. The emergence of blocs such as BRICS makes two things clear in the problematic of the philosophy of globalisation. The first is that we will not homogenise. We will not acquire one unified world system of politics, religion, culture, law, ethics, cuisine, language, art, etc. These blocs are collaborations, they are not melting pots. The second is that we have to collaborate. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS17 A key point here is that what we need to achieve is win-win collaboration. We do not have to become the other, be like the other, agree with the other, nor like everything about the other. In fact, collaboration may be best if we retain our difference. Retaining our difference, we may have more to offer each other. This casts a sceptical philosophical light on some 'dead' metaphors. We talk of 'seeing' the other's point of view, 'knowing' their culture, putting ourselves 'in their shoes', and so on. Is this helpful? Is this accurate? The actual objective is synergy. The Greek etymology of the word 'syn-ergy' is 'together-work', and this is the point. Under BRICS, under the other blocs, under cooperating heterogeneity, we need to do this. In short, the main issue is action. We need to do the right thing, and avoid doing the wrong thing, in complex situations. Of course, knowledge matters. We need to know etiquette. We need to know that the other culture likes this and does not like that. Even better, we know their history and some reasons for their preferences. Even better, we grow to appreciate some of their gems, such as Brazilian dance, Russian bass, Indian yoga, Chinese tea, or South African music. But knowledge is not enough. We need adaptive action. At home, we might do one thing. In our own culture, we might do things a certain way. But in a trans-national working situation we may not have this simple option. We may need to adjust to the additional factor of the other culture. This is inherently more complex than operating within our own culture, but it is a feature of modern life under BRICS and other blocs, where it is now clear that we will not homogenise, and we must collaborate. The issue of adaptive action is therefore prioritised in the modern age. The basic task is to retain our integrity while working with the other. As is well known, expatriate maturity involves a conjunction of these two factors. Progress in this is illustrated as a U-curve moving through initial elation, culture shock, and then differentiated adjustment (Adler and Gundersen, 2007). Of course, we acquire knowledge of the other culture as we progress. Of course this may be enlightening, and we may change as a result. But in the end intercultural business relationships are collaborative rather than synthetic. BRICS Ignoring other cultures is no longer an option. Disrespecting other cultures is no longer an option. International business and multinational companies have become a norm rather than an exception. Accordingly, a service industry in cross-cultural skill has arisen, and is growing fast. In international business, management, and human resources management, cross-cultural skill is now seen as crucial. The field is new, and several names are currently in use: 'cross-cultural' may become 'inter-cultural', 'skill' may become 'communication', 'competence', or 'capability', etc. Countries such as those in BRICS need to do business with each other on a routine basis. Collaboration is essential. Multinational teams are normal (Halverson and Tirmizi, 2008). Expatriate failure is expensive (Chew, 2004). As a live area of applied psychology, support for cross-cultural skill is now supplied worldwide in the forms of training, consultancy, and psychometric testing (Livermore, 2015; Hofstede and Minkov, 2010; Ang and Van Dyne, 2008). Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS17 Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS17 We do not really become a member of the other culture, but we create productive synergy with members of the other culture. We now introduce the concept of transduction as an analysis of adaptive action. This does not pretend to solve all the problems of the modern 18 Don Peterson TRANSDUCTION and BRICS19 Transduction is a form of adjusted action. In neuropsychology, this is the second function identified by Norman and Shallice. In logic, it is a form of defeasible practical reasoning (reasoning to a conclusion on action, which is influenced by context which can change our decision). We start with an example. [E1] You have a standard route to work. This includes walking down a particular pavement. Today, however, you see a big puddle of water covering the width of the pavement. So on this occasion, you stop, walk round on the road, and continue after the puddle. Your feet stay dry, and nothing is disturbed. (Tomorrow, you find no puddle, and you return to your usual enactment.) We see here a pattern of four components: [S] A basic schema, which generates standard or routine action. [S] A basic schema, which generates standard or routine action. [D] A default action, which would normally be performed, in the absence of anything to the contrary. [S] A basic schema, which generates standard or routine action. [D] A default action, which would normally be performed, in the absence of anything to the contrary. [D] A default action, which would normally be performed, in the absence of anything to the contrary. [D] A default action, which would normally be performed, in the absence of anything to the contrary. [C] A context, comprising circumstances relevant to the consequences of our performed action. [V] A variant action, this being a version of [D] whose details of enactment are adjusted to [C]. [V] A variant action, this being a version of [D] whose details of enactment are adjusted to [C]. This is a basic case, but it reveals a reasoning format which is often required. We now consider a cross-cultural example. [E2] A group of long-term resident expatriates in Dubai planned to visit a museum at 2.00 pm on a Wednesday. The sign on the museum wall says that it is open at this time. On arrival, they find that the door is shut, and a man comes out and tells them to go away and come back later. The expatriates become angry. They do in fact have the necessary knowledge. They know that this is the month of Ramadan, which involves extra and obligatory prayer times for Muslims, which take priority over work. Transduction We turn first to the human brain. We turn to the basic equipment which we have. Work in neuropsychology on 'executive function' has established that our performance (behaviour, action) results from two factors, not one. Our performance results from a combination of knowledge (in the form of schemas) and executive function (control over the enactment of schemas) (Elliott, 2003). Piaget (1952) defined a schema as 'a cohesive, repeatable action sequence possessing component actions that are tightly interconnected and governed by a core meaning'. Schemas are rule-of-thumb formulae for action in standard situations. They tell us what to do normally, all things being equal. The first challenge here is that we need to select which schema to activate at any given time. We cannot do everything at once, and a juggling act is needed. We need to control when we start, when we stop, and when we temporarily suspend our schemas. Norman and Shallice (1986) attribute this function to 'contention scheduling'. This capability is associated with the prefrontal cortex. This area is at the top, and at the front of the human brain. This is thought to be only an association: executive function relies also on other areas of the brain, and this area of the brain does other things too (Alverez et al, 2006). Nevertheless, it is a strong association. The second challenge is that, having selected a schema, we may need to modulate its enactment. In standard, routine situations, we may not need to do this, but in novel situations, we may need to produce adjusted action. We may need to adjust how, where, when, and with whom we do things and say things, which may be crucial to success. Norman and Shallice recognise this as a function of the 'Supervisory Attention System' (SAS). The point for BRICS and the age of globalisation, is that 'novel' situations have become normal in international business. This is not a challenge which happens once a year. It is a challenge which happens every day. We rely on standard schemas, formulae, principles, and routines: but we also need to steer their enactment. We need to steer the detail of how we do what we do, in response to the other party, the other culture. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Situation Table for example [E2]. [S] The signs say that the museum will be open at 2.00 pm on nesday but it is not open [S] The signs say that the museum will be open at 2.00 pm on ednesday, but it is not open. Wednesday, but it is not open. TRANSDUCTION and BRICS19 It takes them some time to engage this knowledge of context [C] with their default response [D] of getting angry, so as to produce a practical variant [V] in which they retain their plan to visit Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. 20 Don Peterson the museum [S] but adjust its details of enactment, so as to go away and return at 3.00 pm as suggested. (In future, when it is not Ramadan, they rely on the sign as before.) This example introduces several new factors: mental states, culture, planning, etc. Our claim, though, is that these are details, and that the basic pattern of transduction remains the same. Both of our examples can be expressed in 'situation tables', which use the pattern of four components identified above, as follows. Situation Table for example [E1]. Situation Table for example [E1]. Situation Table for example [E1]. [S] I have a standard route to work. [D] I walk straight down the pavement. [C] Today there is a big puddle across the pavement. [V] Stop, walk round the puddle, then continue. Situation Table for example [E2]. Situation Table for example [E2]. TRANSDUCTION and BRICS21 TRANSDUCTION and BRICS21 conclusion is an action). So we might speak here of 'contrapragma' ('against circumstances'), rather than 'contradiction' ('against speaking'). The affinity with Da Costa's thinking is that an opposition (whether 'contrapragma' or 'contradiction') between [C] and [D] may be a constructive synergy. We attempt here to explicate constructive synergy. What is articulated is a process of working with the other. This is not a process of identifying with the other, agreeing with the other, or becoming the other. If these things come, then well and good: but they will be additions. The basic function is collaboration. In structural terms, [D] is changed to [V] under the influence of [C]. We see a reasoning format here which is under new pressure in the 21st Century. It is normal, it is common sense, it is not new. It is basic, it does not always work, and its articulation in situation tables is useful in cross-cultural training. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Wednesday, but it is not open. Wednesday, but it is not open. [D] Get angry. [C] This is the month of Ramadan, when Muslims have obligatory extra prayers. [C] This is the month of Ramadan, when Muslims have obligatory extra prayers. [V] Go away, and come back later. [V] Go away, and come back later. [V] Go away, and come back later. This pattern of reasoning is an addition to that identified in classical deductive logic. The pattern there is a one-step, monotonic (non-defeasible) process. That is: however complex the derivation, we deal with one set of premises and one conclusion, and introduction of new premises cannot cause retraction of a conclusion. Here we deal with two steps, [S/D] and [C/V], and we derive [V] as a version of [D]: not as a simple rejection of [D] or revision of [S], as identified in classical non-monotonic logic. Da Costa (1958, 1982), makes a related and illuminating point in paraconsistent logic. Da Costa's point is that inconsistency is not always a disaster. A database, for example, can be contradiction-tolerant, as is often needed in business systems. Da Costa is concerned with propositional reasoning (in which our conclusion is a statement), and we are concerned here with practical reasoning (in which our Discussion For the cross-cultural skill industry, then, we suggest that situation tables, as above, backed up by the analytical framework of transduction, with its four basic components, are useful in training. These provide a simple, graphical way of explaining cross-cultural situations, and the ways in which we succeed or fail to adjust to their circumstances. For philosophy, in the age of BRICS, we need to abandon a model introduced by Socrates and expressed by Plato. On this model, right action (arete) is produced by right knowledge (episteme). In the early Dialogues, unsuccessful attempts are made to produce super-schemas (logoi), and in the later Dialogues, these are sent to heaven as 'Platonic Forms' (eidoi) (Plato, 429-347 BCE/1997). On this model, action is determined by a single unit of knowledge. The better this unit is, the better the probability that we produce right action. In terms of schema theory, we seek super-schemas, which guarantee right action. For cross-cultural action, we might seek super-schemas which encompass all cross-cultural situations. We might develop perfect units of knowledge, which tell us what to do internationally. We could file these in a Library of Super-Schemas, thus assisting education and international understanding. Just as the Ten Commandments tell us how to act, and the 22 Don Peterson Highway Code (DVSA, 2015) tells us how to drive, so this Library would tell us what to do in international business. Of course this is naive. We need the Ten Commandments, we need the Highway Code, we need principles and formulae: but these are subject to transduction. j Can we ever escape from transduction? Let us take a simple case: the game of 'noughts and crosses' (or 'tic-tac-toe'). As Zaslavsky (1982) explains, the game is determinate. That is: there is an algorithm for perfect play, producing a draw or win. There are 765 different positions, and 26,830 possible games. If we know this algorithm, we cannot lose. We have a super-schema. Consider, though, the situation where we are in the middle of a business deal with a person of vanity who does not like to lose any game. It might then be expedient to lose deliberately. We have a super-schema, but we modify its enactment so as to satisfy a larger purpose: getting the deal done. What this illustrates is that we cannot escape transduction, and that Socrates was wrong. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS23 TRANSDUCTION and BRICS23 Conclusion This article is intended as a contribution to the theory and practice of cross-cultural skill. In theory, it replaces an idealist model of knowledge and action with a realistic and contemporary schema-based model. In practice, it provides an analytical framework, and a format of situation tables, for use in training. Discussion However much we optimise and expand a schema, it will never be autonomously adequate to all circumstances and surprises, since these are infinite. A related point is made by Wittgenstein (1953/2009) in his 'rule-following paradox', where he recognises that, having learned a rule, the ability to 'go on' with it to new situations is something additional. We call the idealist fantasy the 'Fallacy of Completeness'. The burden of interaction entailed by globalisation may bring a welcome release from this fallacy. We propose here a contemporary model of the relation between knowledge and action. Ideally, we would employ vast lookup tables mapping situations to facts and actions, or vast algorithms which did the same. We would employ huge but perfect formulae, there would be no need for situation-checking, and we would have the security of being right all the time. Socrates, then, would be right. Realistically, though, we are simple creatures facing a complex world, which under BRICS and other blocs has become more complex. Our cognitive resources are limited, and such an ideal strategy is not actually tenable. The point about limitation is well made by Simon (1957, 1982) in his treatment of bounded rationality. We therefore use a cognitive 'hack', and this hack is transduction. The old idealist model of knowledge and action is unrealistic and out-of-date. In the contemporary condition of BRICS, transduction is a necessity. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. References ADLER, N.J., GUNDERSEN, A. International Dimensions of Organizational Behavior. 5th Edition. South-Western College Publications, 2007. ANG, S., VAN DYNE, L. (eds). Handbook of Cultural Intelligence: Theory, Measurement and Application. Routledge, 2008. CHEW, J. "Managing MNC Expatriates through Crises: A Challenge for International Human Resource Management, Research and Practice". Human Resource Management, 12 (2), pp. 1-30, 2004. DA COSTA, N.C.A. "Nota sobre o conceito de contradição". Anuário da Sociadade Paranaense de Matemática, 1, pp. 6-8, 1958. ––––––. "The philosophical import of paraconsistent logic". The Journal of Non-Classical Logic, 1 (1), pp. 1-19, 1982. DRIVER AND VEHICLE STANDARD AGENCY (DVSA). "The Official Highway Code". UK Stationery Office, 2015. GUTEMBERG, P.L. "The Sino-Brazilian Principles in a Latin American and BRICS Context: The Case for Comparative Public Budgeting Legal Research". Wisconsin International Law Journal, Volume 33, No. 1, 2015. HALVERSON, C. B., AQEEL TIRMINIZ, S. (eds). Effective Multicultural Teams: Theory and Practice. Advances in Group Decision and Negotiation (Book 3). Springer, 2008. HOFSTEDE, G.J., MINKOV, M. Cultures and Organizations: Software of the Mind, Third Edition. McGraw-Hill Education, 2010. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. 24 Don Peterson LIVERMORE, D. Leading with Cultural Intelligence: The Real Secret to Success, 2nd Edition. AMACOM, 2015. NORMAN, D. A., SHALLICE, T. "Attention to action: Willed and automatic control of behaviour". In Davidson, R. J., G. E. Schwartz, and D. Shapiro, (eds). Consciousness and Self-Regulation: Advances in Research and Theory. Plenum Press, 1986. O'NEILL, J. "Building Better Global Economic BRICs". Global Economics Paper No: 66. Goldman Sachs, 2001. PIAGET, J. "Autobiography". In E. Boring (ed), History of Psychology in Autobiography. Clark University Press, 1952. PLATO, A. Complete Works. c. 429-347 BCE. J. M. Cooper (ed). Hackett, 1997. SIMON, H.A. Models of Man. Wiley & Sons, 1957. SIMON, H.A. Models of Man. Wiley & Sons, 1957. ––––––. Models of Bounded Rationality, Volumes 1 and 2. MIT Press, 1982. WITTGENSTEIN, L. "Philosophische Untersuchungen". Translated by G.E.M. Anscombe as Philosophical Investigations. Blackwell, 1953. Fourth edition, P. M. S. Hacker, J. Schulte (eds), Blackwell, 2009. ZASLAVSKY, C. Tic Tac Toe: And Other Three-In-A Row Games from Ancient Egypt to the Modern Computer. Crowell, 1982.
https://openalex.org/W2347204468
https://link.springer.com/content/pdf/10.1007/JHEP08(2016)122.pdf
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Search for supersymmetry in pp collisions at s = 13 $$ \sqrt{s}=13 $$ TeV in the single-lepton final state using the sum of masses of large-radius jets
˜The œJournal of high energy physics/˜The œjournal of high energy physics
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Published for SISSA by Springer Received: May 15, 2016 Accepted: July 20, 2016 Published: August 22, 2016 Search for supersymmetry in pp collisions at √s = 13 TeV in the single-lepton final state using the sum of masses of large-radius jets The CMS collaboration E-mail: cms-publication-committee-chair@cern.ch Published for SISSA by Springer Received: May 15, 2016 Accepted: July 20, 2016 Published: August 22, 2016 Search for supersymmetry in pp collisions at √s = 13 TeV in the single-lepton final state using the sum of masses of large-radius jets The CMS collaboration E-mail: cms-publication-committee-chair@cern.ch Published for SISSA by Springer Received: May 15, 2016 Accepted: July 20, 2016 Published: August 22, 2016 Received: May 15, 2016 Accepted: July 20, 2016 Published: August 22, 2016 Received: May 15, 2016 Accepted: July 20, 2016 Published: August 22, 2016 Search for supersymmetry in pp collisions at √s = 13 TeV in the single-lepton final state using the sum of masses of large-radius jets JHEP08(2016)122 The CMS collaboration , py g , for the benefit of the CMS Collaboration. The CMS collaboration Contents 1 Introduction 1 2 Detector 4 3 Simulated event samples 4 4 Event reconstruction 5 5 Trigger and event selection 8 6 Background estimation 9 6.1 Method 9 6.2 Implementation 13 6.3 Systematic uncertainties 15 7 Results and interpretation 17 8 Summary 21 The CMS collaboration 30 Contents 1 Introduction 1 2 Detector 4 3 Simulated event samples 4 4 Event reconstruction 5 5 Trigger and event selection 8 6 Background estimation 9 6.1 Method 9 6.2 Implementation 13 6.3 Systematic uncertainties 15 7 Results and interpretation 17 8 Summary 21 The CMS collaboration 30 Contents 1 Introduction 1 2 Detector 4 3 Simulated event samples 4 4 Event reconstruction 5 5 Trigger and event selection 8 6 Background estimation 9 6.1 Method 9 6.2 Implementation 13 6.3 Systematic uncertainties 15 7 Results and interpretation 17 8 Summary 21 The CMS collaboration 30 JHEP08(2016)122 The CMS collaboration The CMS collaboration E-mail: cms-publication-committee-chair@cern.ch Abstract: Results are reported from a search for supersymmetric particles in proton- proton collisions in the final state with a single, high transverse momentum lepton; multiple jets, including at least one b-tagged jet; and large missing transverse momentum. The data sample corresponds to an integrated luminosity of 2.3 fb−1 at √s = 13 TeV, recorded by the CMS experiment at the LHC. The search focuses on processes leading to high jet multiplicities, such as gluino pair production with eg →t¯t eχ0 1. The quantity MJ, defined as the sum of the masses of the large-radius jets in the event, is used in conjunction with other kinematic variables to provide discrimination between signal and background and as a key part of the background estimation method. The observed event yields in the signal regions in data are consistent with those expected for standard model backgrounds, estimated from control regions in data. Exclusion limits are obtained for a simplified model corresponding to gluino pair production with three-body decays into top quarks and neutralinos. Gluinos with a mass below 1600 GeV are excluded at a 95% confidence level for scenarios with low eχ0 1 mass, and neutralinos with a mass below 800 GeV are excluded for a gluino mass of about 1300 GeV. For models with two-body gluino decays producing on-shell top squarks, the excluded region is only weakly sensitive to the top squark mass. Keywords: Hadron-Hadron scattering (experiments), Supersymmetry ArXiv ePrint: 1605.04608 doi:10.1007/JHEP08(2016)122 doi:10.1007/JHEP08(2016)122 Open Access, Copyright CERN, for the benefit of the CMS Collaboration. Article funded by SCOAP3. 1 Introduction Supersymmetry (SUSY) [1–8] is an extension of the standard model (SM) of particle physics that is motivated by several considerations, including the gauge hierarchy problem [9–14], the existence of astrophysical dark matter [15–17], and the possibility of gauge coupling constant unification at high energy [18–22]. In SUSY models, each SM particle has a corresponding supersymmetric partner (or partners) whose spin differs by one-half, such that fermions are mapped to bosons and vice versa. Gauge quantum numbers are preserved by this symmetry, and to preserve degrees of freedom, a SM spin-1/2 Dirac particle, such as the top quark, has two spin-0 partners, the top squarks. The SUSY partner of the (spin-1) gluon, the massless mediator of the strong interactions in the SM, is the spin-1/2 gluino. In R-parity–conserving models [23, 24], SUSY particles are produced in pairs, and the lightest supersymmetric particle (LSP) is stable. If the LSP is the lightest neutralino (eχ0 1), an electrically neutral mixture of the SUSY partners of the neutral electroweak gauge and Higgs bosons, then it has weak interactions only and can in principle account for some or all of the dark matter. The gauge hierarchy problem has become more urgent with the discovery of the Higgs boson [25–30]. Although the SM is conceptually complete, the Higgs boson mass, together – 1 – with the electroweak scale, is unstable against enormous corrections from loop processes, which pull the Higgs mass to the cutoffscale of the theory, for example, the Planck scale. This outcome can be avoided within the framework of the SM only with extreme fine tuning of the bare Higgs mass parameter, a situation that is regarded as unnatural, although not excluded. This problem suggests that additional symmetries and associated degrees of freedom may be present that ameliorate these effects. So-called natural SUSY models [31– 34], in which sufficiently light SUSY partners are present, are a major focus of current new physics searches at the CERN LHC. In natural models, several of the SUSY partners are constrained to be light [33]: both top squarks, etL and etR, which have the same electroweak couplings as the left- (L) and right- (R) handed top quarks, respectively; the bottom squark with L-handed couplings (ebL); the gluino (eg); and the Higgsinos (eh). 1 Introduction While the gluino mass is not constrained by naturalness considerations as strongly as that of the lighter top squark mass eigenstate, et1, the cross section for gluino pair production is substantially larger than that for top squark pair production, for a given mass. As a consequence, the two types of searches can have comparable sensitivity to these models. Both types of searches are currently of intense interest, and CMS and ATLAS data taken at √s = 8 TeV have provided significant constraints [35] on natural SUSY scenarios. JHEP08(2016)122 This study uses the first LHC proton-proton collision data taken by the CMS experi- ment at √s = 13 TeV to search for gluino pair production. Searches targeting this process in the single-lepton final state using 8 TeV data have been performed by both ATLAS [36, 37] and CMS [38]. For meg = 1.5 TeV, somewhat above the highest gluino masses excluded at √s = 8 TeV, the cross section for gluino pair production increases dramatically with center-of-mass energy, from about 0.4 fb at √s = 8 TeV to about 14 fb at √s = 13 TeV [39]. In contrast, the cross section for the dominant background, tt production, increases much more slowly, from about 248 pb at √s = 8 TeV to 816 pb at √s = 13 TeV [40]. As a consequence, the sensitivity of this search can be significantly extended with respect to searches performed at √s = 8 TeV, even though the 13 TeV data sample has an integrated luminosity of only 2.3 fb−1, roughly one-tenth of that acquired at 8 TeV. The search targets gluino pair production with eg →tteχ0 1, which arises from eg → et1t, where the top squark is produced either on or offmass shell. The off-mass-shell scenario is shown in figure 1 (left) and is often designated T1tttt [41] in simplified model scenarios [42–44]. Results are also obtained for scenarios with on-shell top squark masses. This scenario is shown in figure 1 (right) and will be denoted by T5tttt. (For this scenario, the small contribution from the direct production of top squark pairs is also taken into account.) Regardless of whether the top squark is produced on or offmass shell, the final state is characterized by a large number of jets, four of which are b jets from top quark decays. 1 Introduction Depending on the decay modes of the accompanying W bosons, a range of lepton multiplicities is possible; we focus here on the single-lepton final state, where the lepton is either an electron or a muon. Because the two neutralinos (eχ0 1) are undetected, their production in SUSY events typically gives rise to a large amount of missing (unobserved) momentum, whose value in the direction transverse to the beam axis can be inferred from the momenta of the observed particles. The missing transverse momentum, ⃗p miss T , is a key element of searches for R-parity-conserving SUSY, and its magnitude is denoted by Emiss T . – 2 – P1 P2 ˜g ˜g ¯t t eχ0 1 eχ0 1 ¯t t P1 P2 eg eg et1 et1 ¯t t eχ0 1 eχ0 1 ¯t t Figure 1. Gluino pair production and decay for the simplified models T1tttt (left) and T5tttt (right). In T1tttt, the gluino undergoes three-body decay eg →tteχ0 1 via a virtual intermediate top squark. In T5tttt, the gluino decays via the sequential two-body process eg →et1t, et1 →teχ0 1. Because gluinos are Majorana particles, each one can decay to et1t and to the charge conjugate final state et1t. P1 P2 eg eg et1 et1 ¯t t eχ0 1 eχ0 1 ¯t t P1 P2 ˜g ˜g ¯t t eχ0 1 eχ0 1 ¯t t ¯t JHEP08(2016)122 Figure 1. Gluino pair production and decay for the simplified models T1tttt (left) and T5tttt (right). In T1tttt, the gluino undergoes three-body decay eg →tteχ0 1 via a virtual intermediate top squark. In T5tttt, the gluino decays via the sequential two-body process eg →et1t, et1 →teχ0 1. Because gluinos are Majorana particles, each one can decay to et1t and to the charge conjugate final state et1t. A challenge in performing searches for SUSY particles is obtaining sufficient sensitivity to the signal, while at the same time understanding the background contribution from SM processes in a robust manner. This analysis is designed such that the background in the signal regions arises largely from a single process, dilepton tt production, in which both W bosons from t →bW+ decay leptonically, but only one lepton satisfies the criteria associated with identification, the minimum transverse momentum (pT) requirement, and isolation from other energy in the event. 2 Detector The central feature of the CMS detector is a superconducting solenoid of 6 m internal di- ameter, providing a magnetic field of 3.8 T. Within the solenoid volume are the tracking and calorimeter systems. The tracking system, composed of silicon-pixel and silicon-strip detectors, measures charged particle trajectories within the pseudorapidity range |η| < 2.5, where η ≡−ln[tan(θ/2)] and θ is the polar angle of the trajectory of the particle with respect to the counterclockwise proton beam direction. A lead tungstate crystal electro- magnetic calorimeter (ECAL), and a brass and scintillator hadron calorimeter (HCAL), each composed of a barrel and two endcap sections, provide energy measurements up to |η| = 3. Forward calorimeters extend the pseudorapidity coverage provided by the barrel and endcap detectors up to |η| = 5. Muons are identified and measured within the range |η| < 2.4 by gas-ionization detectors embedded in the steel magnetic flux-return yoke out- side the solenoid. The detector is nearly hermetic, permitting the accurate measurement of ⃗p miss T . A more detailed description of the CMS detector, together with a definition of the coordinate system used and the relevant kinematic variables, is given in ref. [51]. JHEP08(2016)122 1 Introduction The search signature is characterized not only by the presence of high-pT jets and b-tagged jets, an isolated high-pT lepton, and large Emiss T , but also by additional kinematic variables. Apart from resolution effects, the transverse mass of the lepton + ⃗p miss T system, mT, is bounded above by mW for events with a single leptonically decaying W, and this variable is very effective in suppressing the otherwise dominant single-lepton tt background. The quantity MJ, the scalar sum of the masses of large-radius jets, is used both to characterize the mass and energy scale of the event, pro- viding discrimination between signal and background, and as a key part of the background estimation. A property of MJ exploited in this analysis is that, for the dominant back- ground, this variable is nearly uncorrelated with mT. Because of the absence of correlation between MJ and mT, the background shape at high mT, including the signal region, can be measured to a very good approximation using a low-mT control sample. The quantity MJ was first discussed in phenomenological studies, for example, in refs. [45–47]. Similar variables have been used by ATLAS for SUSY searches in all-hadronic final states using 8 TeV data [48, 49]. We have presented studies of basic MJ properties and performance using early 13 TeV data [50]. This paper is organized as follows. Section 2 gives a brief overview of the CMS de- tector. Section 3 discusses the simulated event samples used in the analysis. The event reconstruction is discussed in section 4, while section 5 describes the trigger and event selection. Section 6 presents the methodology used to predict the SM background from the event yields in control regions in data. The associated systematic uncertainties are also discussed. The event yields observed in the signal regions are presented in section 7. These – 3 – yields are compared with background predictions and used to obtain exclusion regions for the gluino pair production models shown in figure 1. Finally, section 8 presents a summary of the methodology and the results. 3 Simulated event samples The analysis makes use of several simulated event samples for modeling the SM background and signal processes. While the background estimation in the analysis is performed largely from control samples in the data, simulated event samples provide correction factors, typ- ically near unity. The equivalent integrated luminosity of the simulated event samples is at least six times that of the data, and at least 100 times that of the data in the case of tt and signal processes. The production of tt+jets, W+jets, Z+jets, and QCD multijet events is simulated with the Monte Carlo (MC) generator MadGraph5 aMC@NLO 2.2.2 [52] in leading- order (LO) mode. Single top quark events are modeled at next-to-leading order (NLO) with MadGraph5 aMC@NLO for the s-channel and powheg v2 [53, 54] for the t-channel and W-associated production. Additional small backgrounds, such as tt production in asso- ciation with bosons, diboson processes, and t¯tt¯t are similarly produced at NLO with either MadGraph5 aMC@NLO or powheg. All events are generated using the NNPDF 3.0 [55] set of parton distribution functions (PDF). Parton showering and fragmentation are per- formed with the pythia 8.205 [56] generator with the underlying event model based on the CUETP8M1 tune detailed in ref. [57]. The detector simulation is performed with Geant4 [58]. The cross sections used to scale simulated event yields are based on the highest order calculation available. For tt, in addition to using the next-to-next-to-leading order + next-to-next-to-leading logarithmic cross section calculation [40], the modeling of – 4 – the event kinematics is improved by reweighting the top quark pT spectrum to match the data [59], keeping the overall normalization fixed. Signal events for the T1tttt and T5tttt simplified models are generated in a man- ner similar to that for the SM backgrounds, with the MadGraph5 aMC@NLO 2.2.2 generator in LO mode using the NNPDF 3.0 PDF set and followed with pythia 8.205 for showering and fragmentation. The detector simulation is performed with the CMS fast simulation package [60] with scale factors applied to account for any differences with respect to the full simulation used for backgrounds. Event samples are generated for a representative set of model scenarios by scanning over the relevant mass ranges for the eg and eχ0 1, and the yields are normalized to the NLO + next-to-leading-logarithmic cross section [39, 61–64]. JHEP08(2016)122 Throughout this paper, two T1tttt benchmark models are used to illustrate typ- ical signal behavior. 3 Simulated event samples The T1tttt(1500,100) model, with masses meg = 1500 GeV and meχ0 1 = 100 GeV, corresponds to a scenario with a large mass splitting (referred to as non-compressed, or NC) between the gluino and the neutralino. This mass combination probes the sensitivity of the analysis to a low cross section (14 fb) process that has a hard Emiss T spectrum, which results in a relatively high signal efficiency. The T1tttt(1200,800) model, with masses meg = 1200 GeV and meχ0 1 = 800 GeV, corresponds to a scenario with a small mass splitting (referred to as compressed, or C) between the gluino and the neu- tralino. Here the cross section is much higher (86 fb) because the gluino mass is lower than for the T1tttt(1500,100) model, but the sensitivity suffers from a low signal efficiency due to the soft Emiss T spectrum. Finally, to model the presence of additional proton-proton collisions from the same or adjacent beam crossing as the primary hard-scattering process (“pileup” interactions), the simulated events are overlaid with multiple minimum bias events, which are also generated with the pythia 8.205 generator with the underlying event model based on the CUETP8M1 tune. The distribution of the number of overlaid minimum bias events is broad and peaks in the range 10–15. 4 Event reconstruction The reconstruction of physics objects in an event proceeds from the candidate particles identified by the particle-flow (PF) algorithm [65, 66], which uses information from the tracker, calorimeters, and muon systems to identify the candidates as charged or neutral hadrons, photons, electrons, or muons. Charged particle tracks are required to originate from the event primary vertex (PV), defined as the reconstructed vertex, located within 24 cm (2 cm) of the center of the detector in the direction along (perpendicular to) the beam axis, that has the highest value of p2 T summed over the associated charged particle tracks. The charged PF candidates associated with the PV and the neutral PF candidates are clustered into jets using the anti-kT algorithm [67] with distance parameter R = 0.4, as implemented in the fastjet package [68]. The estimated pileup contribution to the jet pT from neutral PF candidates is removed with a correction based on the area of the jet and – 5 – the average energy density of the event [69]. The jet energy is calibrated using pT- and η-dependent corrections; the resulting calibrated jet is required to satisfy pT > 30 GeV and |η| ≤2.4. Each jet must also meet loose identification requirements [70] to suppress, for example, calorimeter noise. Finally, jets that have PF constituents matched to an isolated lepton, as defined below, are removed from the jet collection. A subset of the jets are “tagged” as originating from b quarks using the combined secondary vertex (CSV) algorithm [71, 72]. For the CSV medium working point chosen for this analysis, the signal efficiency for b jets in the range pT = 30 to 50 GeV is 60–67% (51–57%) in the barrel (endcap), increasing with pT. Above pT ≈150 GeV the b tagging efficiency decreases. The probability to misidentify jets arising from c quarks is 13–15% (11–13%) in the barrel (endcap), while the misidentification probability for light-flavor quarks or gluons is 1–2%. JHEP08(2016)122 Throughout this paper, quantities related to the number of jets (Njets) or to the number of b-tagged jets (Nb) are based only on small-R jets, not on the large-R jets discussed below. Electrons are reconstructed by associating a charged particle track with an ECAL supercluster [73]. 4 Event reconstruction The resulting candidate electrons are required to have pT > 20 GeV and |η| < 2.5, and to satisfy identification criteria designed to remove light-parton jets, photon conversions, and electrons from heavy flavor hadron decays. Muons are reconstructed by associating tracks in the muon system with those found in the silicon tracker [74]. Muon candidates are required to satisfy pT > 20 GeV and |η| < 2.4. To preferentially select leptons that originate in the decay of W bosons, leptons are required to be isolated from other PF candidates. Isolation is quantified using an optimized version of the “mini-isolation” variable originally suggested in ref. [75], in which the trans- verse energy of the particles within a cone in η-φ space surrounding the lepton momentum vector is computed using a cone size that scales as 1/pℓ T, where pℓ T is the transverse momen- tum of the lepton. In this analysis, mini-isolation, Irel mini, is defined as the transverse energy of particles in a cone of radius Rmini-iso around the lepton, divided by pℓ T. The transverse energy is computed as the scalar sum of the pT values of the charged hadrons from the PV, neutral hadrons, and photons. The neutral hadron and photon contributions to this sum are corrected for pileup. The cone radius Rmini-iso varies with the pℓ T according to Rmini-iso =        0.2, pℓ T ≤50 GeV (10 GeV)/pℓ T, pℓ T ∈(50 GeV, 200 GeV) 0.05, pℓ T ≥200 GeV. (4.1) Rmini-iso =        0.2, pℓ T ≤50 GeV (10 GeV)/pℓ T, pℓ T ∈(50 GeV, 200 GeV) 0.05, pℓ T ≥200 GeV. (4.1) (4.1) The 1/pℓ T dependence is motivated by considering a two-body decay of a massive parent particle with mass M and large pT, for which the angular separation of the daughter particles is roughly ∆Rdaughters ≈2M/pT. The pT-dependent cone size reduces the rate of accidental overlaps between the lepton and jets in high-multiplicity or highly Lorentz- boosted events, particularly overlaps between b jets and leptons originating from a boosted top quark. The cone remains large enough to contain b-hadron decay products for non- prompt leptons across a range of pℓ T values. Muons (electrons) must satisfy Irel mini < 0.2 (0.1). 4 Event reconstruction The combined efficiency for the electron reconstruction and isolation requirements is about – 6 – [GeV] J M 0 200 400 600 800 1000 % events/(40 GeV) 1 − 10 1 10 2 10 Simulation CMS 13 TeV Simulation CMS 13 TeV (1500,100) 1 0 χ∼ tt → g~ , g~ g~ , 1 true lepton tt , 2 true leptons tt < 10 GeV T ISR p [GeV] J M 0 200 400 600 800 1000 % events/(40 GeV) 1 − 10 1 10 2 10 Simulation CMS 13 TeV Simulation CMS 13 TeV (1500,100) 1 0 χ∼ tt → g~ , g~ g~ , 1 true lepton tt , 2 true leptons tt > 100 GeV T ISR p Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples with a small ISR contribution (left) and a significant ISR contribution (right). These components are defined according to whether the pT of the tt system (or, in the case of signal events, that of the egeg system) is <10 GeV or >100 GeV, respectively. The T1tttt(NC) signal model (dashed red line), is described in section 3; the first model parameter in parentheses corresponds to meg and the second to meχ0 1, both in units of GeV. The events satisfy the requirements Emiss T > 200 GeV and HT > 500 GeV and have at least one reconstructed lepton. [GeV] J M 0 200 400 600 800 1000 % events/(40 GeV) 1 − 10 1 10 2 10 Simulation CMS 13 TeV Simulation CMS 13 TeV (1500,100) 1 0 χ∼ tt → g~ , g~ g~ , 1 true lepton tt , 2 true leptons tt < 10 GeV T ISR p [GeV] J M 0 200 400 600 800 1000 % events/(40 GeV) 1 − 10 1 10 2 10 Simulation CMS 13 TeV Simulation CMS 13 TeV (1500,100) 1 0 χ∼ tt → g~ , g~ g~ , 1 true lepton tt , 2 true leptons tt > 100 GeV T ISR p % events/(40 GeV) JHEP08(2016)122 Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples wi Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples with a small ISR contribution (left) and a significant ISR contribution (right). 4 Event reconstruction The variable MJ is defined as the sum of all large-R jet masses: 50% at a pℓ T of 20 GeV, increasing to 65% at 50 GeV and reaching a plateau of 80% above 200 GeV. The combined reconstruction and isolation efficiencies for muons are about 70% at a pℓ T of 20 GeV, increasing to 80% at 50 GeV and reaching a plateau of 95% at 200 GeV. We cluster R = 0.4 (“small-R”) jets and the isolated leptons into R = 1.2 (“large-R”) jets using the anti-kT algorithm. The mass of the large-R jets retains angular information about the clustered objects, as well as their pT and multiplicity. Clustering small-R jets instead of PF candidates incorporates the jet pileup corrections, thereby reducing the dependence of the mass on pileup. The variable MJ is defined as the sum of all large-R jet masses: We cluster R = 0.4 (“small-R”) jets and the isolated leptons into R = 1.2 (“large-R”) jets using the anti-kT algorithm. The mass of the large-R jets retains angular information about the clustered objects, as well as their pT and multiplicity. Clustering small-R jets instead of PF candidates incorporates the jet pileup corrections, thereby reducing the dependence of the mass on pileup. The variable MJ is defined as the sum of all large-R jet masses: MJ = X Ji=large-R jets m(Ji). (4.2) (4.2) The technique of clustering small-R jets into large-R jets has been used previously by ATLAS in, for example, ref. [76]. Leptons are included in the large-R jets to include the full kinematics of the event, and the choice R = 1.2 optimizes the background rejection power of MJ while retaining signal efficiency. Larger distance parameters were found to offer no significant additional discriminating power, while smaller parameters decrease the background rejection up to a factor of two for models with small mass splittings between the gluino and neutralino. For tt events with a small contribution from initial-state radiation (ISR), the MJ distribution has an approximate cutoffat twice the mass of the top quark, as shown in figure 2 (left). In contrast, the MJ distribution for signal events extends to larger values. The presence of a significant amount of ISR generates a high-MJ tail in the tt background, as shown in figure 2 (right). 4 Event reconstruction These components are defined according to whether the pT of the tt system (or, in the case of signal events, that of the egeg system) is <10 GeV or >100 GeV, respectively. The T1tttt(NC) signal model (dashed red line), is described in section 3; the first model parameter in parentheses corresponds to meg and the second to meχ0 1, both in units of GeV. The events satisfy the requirements Emiss T > 200 GeV and HT > 500 GeV and have at least one reconstructed lepton. Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples with a small ISR contribution (left) and a significant ISR contribution (right). These components are defined according to whether the pT of the tt system (or, in the case of signal events, that of the egeg system) is <10 GeV or >100 GeV, respectively. The T1tttt(NC) signal model (dashed red line), is described in section 3; the first model parameter in parentheses corresponds to meg and the second to meχ0 1, both in units of GeV. The events satisfy the requirements Emiss T > 200 GeV and HT > 500 GeV and have at least one reconstructed lepton. 50% at a pℓ T of 20 GeV, increasing to 65% at 50 GeV and reaching a plateau of 80% above 200 GeV. The combined reconstruction and isolation efficiencies for muons are about 70% at a pℓ T of 20 GeV, increasing to 80% at 50 GeV and reaching a plateau of 95% at 200 GeV. 50% at a pℓ T of 20 GeV, increasing to 65% at 50 GeV and reaching a plateau of 80% above 200 GeV. The combined reconstruction and isolation efficiencies for muons are about 70% at a pℓ T of 20 GeV, increasing to 80% at 50 GeV and reaching a plateau of 95% at 200 GeV. We cluster R = 0.4 (“small-R”) jets and the isolated leptons into R = 1.2 (“large-R”) jets using the anti-kT algorithm. The mass of the large-R jets retains angular information about the clustered objects, as well as their pT and multiplicity. Clustering small-R jets instead of PF candidates incorporates the jet pileup corrections, thereby reducing the dependence of the mass on pileup. 5 Trigger and event selection The data sample used in this analysis was obtained with triggers that require HT > 350 GeV and at least one electron or muon with pT > 15 GeV, where these variables are computed with online (trigger-level) quantities and typically have somewhat poorer resolution than the corresponding offline variables. To ensure high trigger efficiency with respect to the offline definition of lepton isolation described in the previous section (mini-isolation), we designed these triggers with very loose lepton isolation requirements and fixed the isolation cone size to R = 0.2. For events passing the offline selection, the total trigger efficiencies, measured in data control samples that are independently triggered, are found to be (95.1± 1.1)% for the muon channel and (94.1±1.2)% for the electron channel and are independent of the analysis variables within the uncertainties. These efficiencies are applied to the simulation as a correction. The offline event selection is summarized in table 1, which lists the event yields ex- pected from simulation for both SM background processes and for the two benchmark T1tttt signal models. We select events with exactly one isolated charged lepton (an elec- tron or a muon), HT > 500 GeV, Emiss T > 200 GeV, and at least six jets, at least one of which is b-tagged. After this set of requirements, referred in the following as the baseline selection, more than 80% of the remaining SM background arises from tt production. The contributions from events with a single top quark or a W boson in association with jets are each about 6–7%. The background from QCD multijet events after the baseline selection is negligible due to the combination of leptonic, Emiss T , and Njets requirements. After the baseline selection requirements are applied, events are binned in several other kinematic variables, both to increase the signal sensitivity and to define control regions, as described in section 6.1. To illustrate the effect of additional requirements, table 1 lists the expected yields for examples of event selection requirements on MJ, mT, Njets, and Nb. The events satisfying the baseline selection are divided in the MJ–mT plane into a signal region, defined by the additional requirements MJ > 400 GeV and mT > 140 GeV, and three control samples, bounded by MJ > 250 GeV, that are used in the background estimation. Approximately 37% of signal T1tttt events are selected with the single-lepton requirement only. 4 Event reconstruction The missing transverse energy, Emiss T , is given by the magnitude of ⃗p miss T , the negative vector sum of the transverse momenta of all PF candidates [65, 66]. Correspondence to the – 7 – true undetectable energy in the event is improved by replacing the contribution of the PF candidates associated with a jet by the calibrated four-momentum of that jet. To separate backgrounds characterized by the presence of a single W boson decaying leptonically but without any other source of missing energy, the lepton and the Emiss T are combined to obtain the transverse mass, mT, defined as: mT = q 2pℓ TEmiss T [1 −cos(∆φℓ,⃗p miss T )], (4.3) (4.3) where ∆φℓ,⃗p miss T is the difference between the azimuthal angles of the lepton momentum vector and the missing momentum vector, ⃗p miss T . Finally, we define the quantity HT as the scalar sum of the transverse momenta of all the small-R jets passing the selection. JHEP08(2016)122 5 Trigger and event selection The benchmark signal models, T1tttt(NC) and T1tttt(C), are described in section 3. The event selection requirements listed above the horizontal line in the middle of the table are defined as the baseline selection. The background estimates before the HT requirement are not specified because some of the simulated event samples do not extend to the low HT region. Given the size of the MC samples described in section 3, rows with zero yield have statistical uncertainties of at most 0.16 events, and below 0.05 events in most cases. than meχ0 1, more than half of the events passing the lepton requirement lie in the signal region. For compressed spectrum models, where meχ0 1 ≈meg −2mt, the MJ, HT, and Emiss T spectra become much softer and, as a result, only 5–10% of the single-lepton signal events are selected. As shown in figure 3, backgrounds with a single W boson decaying leptonically are strongly suppressed after the mT > 140 GeV requirement, so the total SM background in the signal region is dominated by dilepton tt events. This dilepton background falls into two categories, which make roughly equal contributions. The first involves an identified electron or muon and a hadronically decaying τ from W decay. The second source involves two leptons, each of which is an electron or a muon. One of the leptons fails to satisfy the lepton selection criteria, which include the pT and isolation requirements. This missed lepton can be produced either directly or indirectly in W decay, where in the indirect case the lepton is the daughter of a τ. 5 Trigger and event selection In non-compressed spectrum models, for which meg is significantly larger – 8 – L = 2.3 fb−1 Other QCD ttV Single t W+jets tt (1ℓ) tt (2ℓ) SM bkg. T1tttt(NC) T1tttt(C) No selection — — — — — — — — 31.3 190.0 1ℓ, pT > 20 GeV — — — — — — — — 11.9 68.7 HT > 500 GeV 4131.9 31831.5 721.9 2926.6 31885.1 27628.7 3357.8 102483.4 11.9 44.9 Emiss T > 200 GeV 310.6 154.7 89.1 457.2 4343.1 2183.6 584.0 8122.3 10.5 21.5 Njets ≥6, pT > 30 GeV 27.3 8.0 36.8 82.8 278.7 792.3 171.4 1397.4 9.6 20.4 Nb ≥1 9.4 2.7 29.6 63.9 66.3 632.2 137.4 941.4 9.1 19.1 MJ > 250 GeV 6.7 2.6 22.6 43.8 46.1 455.2 87.2 664.2 9.0 16.5 mT > 140 GeV 0.7 1.4 3.0 3.5 1.2 5.5 32.5 47.9 7.0 9.2 MJ > 400 GeV 0.4 0.8 1.1 1.4 0.6 2.8 9.7 16.7 6.4 4.5 Nb ≥2 0.16 0.04 0.55 0.68 0.00 1.29 4.52 7.24 4.87 3.47 Emiss T > 400 GeV 0.02 0.00 0.12 0.31 0.00 0.07 0.72 1.24 3.60 1.48 Njets ≥9, pT > 30 GeV 0.01 0.00 0.03 0.00 0.00 0.01 0.11 0.16 1.64 1.00 Table 1. Event yields obtained from simulated event samples, as the event selection criteria are applied. The category Other includes Drell-Yan, ttH(→bb), tttt, WZ, and WW. The yields for tt events in fully hadronic final states are included in the QCD multijet category. The category ttV includes ttW, ttZ, and ttγ. The benchmark signal models, T1tttt(NC) and T1tttt(C), are described in section 3. The event selection requirements listed above the horizontal line in the middle of the table are defined as the baseline selection. The background estimates before the HT requirement are not specified because some of the simulated event samples do not extend to the low HT region. Given the size of the MC samples described in section 3, rows with zero yield have statistical uncertainties of at most 0.16 events, and below 0.05 events in most cases. JHEP08(2016)122 Table 1. Event yields obtained from simulated event samples, as the event selection criteria are applied. The category Other includes Drell-Yan, ttH(→bb), tttt, WZ, and WW. The yields for tt events in fully hadronic final states are included in the QCD multijet category. The category ttV includes ttW, ttZ, and ttγ. 6.1 Method The prediction of the background yields in each of the signal bins takes advantage of the fact that the MJ and mT distributions of events with a significant amount of ISR are largely uncorrelated. The correlation coefficients for the single-lepton and dilepton tt events in the MJ–mT plane after the baseline selection (as shown in figure 4) are small, in the range 0.03 to 0.05. The absence of a substantial correlation allows us to measure the MJ distribution – 9 – Events/(35 GeV) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 CMS (13 TeV) -1 2.3 fb Data (1500,100) 1 0 χ∼ tt → g~ , g~ g~ (1200,800) 1 0 χ∼ tt → g~ , g~ g~ , 1 true lepton tt , 2 true leptons tt W+jets Single t ttV Other [GeV] T m 0 100 200 300 400 Data / MC 0.5 1 1.5 Figure 3. Distribution of mT in data and simulated event samples after the baseline selection is applied. The background contributions shown here are from simulation, and their total yield is normalized to the number of events observed in data. The signal distributions are normalized to the expected cross sections. The dashed vertical line indicates the mT > 140 GeV threshold that separates the signal regions from the control samples. Events/(35 GeV) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 CMS (13 TeV) -1 2.3 fb Data (1500,100) 1 0 χ∼ tt → g~ , g~ g~ (1200,800) 1 0 χ∼ tt → g~ , g~ g~ , 1 true lepton tt , 2 true leptons tt W+jets Single t ttV Other [GeV] T m 0 100 200 300 400 Data / MC 0.5 1 1.5 JHEP08(2016)122 Figure 3. Distribution of mT in data and simulated event samples after the baseline selection is applied. The background contributions shown here are from simulation, and their total yield is normalized to the number of events observed in data. The signal distributions are normalized to the expected cross sections. The dashed vertical line indicates the mT > 140 GeV threshold that separates the signal regions from the control samples. 6.1 Method [GeV] J M 0 200 400 600 800 1000 [GeV] T m 0 50 100 150 200 250 300 350 400 =0.05 ρ (1l), tt =0.03 ρ (2l), tt (1500,100) 0 1 χ∼t t → g~ , g~ g~ R1 R2 R3 R4 CMS Simulation 13 TeV Figure 4. Distribution of simulated single-lepton tt events (dark-blue triangles), dilepton tt events (light-blue inverted triangles), and T1tttt(1500,100) events (red squares) in the MJ–mT plane after the baseline selection. Each marker represents one expected event at 2.3 fb−1. Overflow events are placed on the edge of the plot. The values of the correlation coefficients ρ for each background process are given in the legend. Region R4 is the nominal signal region, while R1, R2, and R3 serve as control regions. The small signal contributions in the control regions are taken into account in one of the global fits, as discussed in the text. [GeV] J M 0 200 400 600 800 1000 [GeV] T m 0 50 100 150 200 250 300 350 400 =0.05 ρ (1l), tt =0.03 ρ (2l), tt (1500,100) 0 1 χ∼t t → g~ , g~ g~ R1 R2 R3 R4 CMS Simulation 13 TeV Figure 4. Distribution of simulated single-lepton tt events (dark-blue triangles), dilepton tt events (light-blue inverted triangles), and T1tttt(1500,100) events (red squares) in the MJ–mT plane after the baseline selection. Each marker represents one expected event at 2.3 fb−1. Overflow events are placed on the edge of the plot. The values of the correlation coefficients ρ for each background process are given in the legend. Region R4 is the nominal signal region, while R1, R2, and R3 serve as control regions. The small signal contributions in the control regions are taken into account in one of the global fits, as discussed in the text. – 10 – of the background at low mT with good statistical precision, and extrapolate it to high mT. The underlying explanation for this behavior is not immediately obvious, given that low-mT events originate mainly from tt events where only one of the top quarks decays leptonically (1ℓtt), while the high-mT regions are dominated by dilepton tt events (2ℓtt). 6.1 Method In particular, as shown in figure 2 (left), in the absence of significant ISR, the dileptonic tt events have a softer MJ spectrum than single-lepton tt events, simply because the reconstructed mass of a leptonically decaying top quark does not include the undetected neutrino. In events with substantial ISR, however, the contributions to MJ from the accidental overlap of jets can dominate the contributions due to the intrinsic mass of the top quarks. This effect is illustrated in figure 5, which compares the Njets and MJ distributions of single-lepton and dilepton tt events at high and low mT after the baseline selection is applied. Since we require at least 6 jets, single-lepton tt events must have at least 2 ISR jets and dilepton tt events must have at least 4. In this regime, the probability of additional ISR jets is similar for events with a given number of partons of similar momenta, and, as a result, the number of objects contributing to MJ (jets plus the reconstructed lepton) is comparable in 1ℓand 2ℓtt events. When these ISR jets overlap with the top quark decay products, the masses of the resulting large-R jets are dominated by the accidental overlap and, thus, the shapes of the MJ distribution of 1ℓand 2ℓtt events become more similar. This is the case for MJ > 250 GeV, where figure 5 (right) shows that the distributions of the 1ℓand 2ℓtt backgrounds have nearly the same shape, and the low-mT to high-mT extrapolation is warranted. JHEP08(2016)122 We thus divide the MJ–mT plane into four regions, three control regions (CR) and one signal region (SR): • Region R1 (CR): mT ≤140 GeV, 250 ≤MJ ≤400 GeV • Region R2 (CR): mT ≤140 GeV, MJ > 400 GeV • Region R3 (CR): mT > 140 GeV, 250 ≤MJ ≤400 GeV • Region R4 (SR): mT > 140 GeV, MJ > 400 GeV. • Region R1 (CR): mT ≤140 GeV, 250 ≤MJ ≤400 GeV • Region R2 (CR): mT ≤140 GeV, MJ > 400 GeV • Region R3 (CR): mT > 140 GeV, 250 ≤MJ ≤400 GeV • Region R3 (CR): mT > 140 GeV, 250 ≤MJ ≤400 GeV • Region R4 (SR): mT > 140 GeV, MJ > 400 GeV. 6.1 Method These regions are further subdivided into 10 bins of Emiss T , Njets, and Nb to increase signal sensitivity: • Six bins with 200 < Emiss T ≤400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb = 2, Nb ≥3) • Six bins with 200 < Emiss T ≤400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb = 2, Nb ≥3) • Four bins with Emiss T > 400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb ≥2), • Four bins with ET > 400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb ≥2), where the multiplication indicates that the binning is two dimensional in Njets and Nb. Given that the main background processes have two or fewer b quarks, the total SM contribution to the Nb ≥3 bins is very small and is driven by the b-tag fake rate. Signal events in the T1tttt and T5tttt models are expected to populate primarily the bins with Nb ≥2, while bins with Nb = 1 mainly serve to test the method in a background dominated region. here the multiplication indicates that the binning is two dimensional in Njets and Nb. – 11 – % events 0 20 40 60 Simulation CMS 13 TeV 140 GeV ≤ T , 1 true lepton: m tt > 140 GeV T , 2 true leptons: m tt jets N 6 8 10 12 T / Low m T High m 0 0.05 0.1 0.15 % events/(50 GeV) 2 − 10 1 − 10 1 10 2 10 Simulation CMS 13 TeV 140 GeV ≤ T , 1 true lepton: m tt > 140 GeV T , 2 true leptons: m tt [GeV] J M 0 500 1000 1500 T / Low m T High m 0 0.05 0.1 0.15 Figure 5. Comparison of Njets and MJ distributions, normalized to the same area, in simulated tt events with two true leptons at high mT and one true lepton at low mT, after the baseline selection is applied. The shapes of these distributions are similar. These two contributions are the dominant backgrounds in their respective mT regions. The dashed vertical line on the right-hand plot indicates the MJ > 400 GeV threshold that separates the signal regions from the control samples. 6.1 Method The shaded region corresponding to MJ < 250 GeV is not used in the background estimation. % events/(50 GeV) 2 − 10 1 − 10 1 10 2 10 Simulation CMS 13 TeV 140 GeV ≤ T , 1 true lepton: m tt > 140 GeV T , 2 true leptons: m tt [GeV] J M 0 500 1000 1500 T / Low m T High m 0 0.05 0.1 0.15 % events 0 20 40 60 Simulation CMS 13 TeV 140 GeV ≤ T , 1 true lepton: m tt > 140 GeV T , 2 true leptons: m tt jets N 6 8 10 12 T / Low m T High m 0 0.05 0.1 0.15 % events/(50 GeV) % events JHEP08(2016)122 Figure 5. Comparison of Njets and MJ distributions, normalized to the same area, in simulated tt events with two true leptons at high mT and one true lepton at low mT, after the baseline selection is applied. The shapes of these distributions are similar. These two contributions are the dominant backgrounds in their respective mT regions. The dashed vertical line on the right-hand plot indicates the MJ > 400 GeV threshold that separates the signal regions from the control samples. The shaded region corresponding to MJ < 250 GeV is not used in the background estimation. To obtain an estimate of the background rate in each of the signal bins, a modified version of an “ABCD” method is used. Here, the symbols A, B, C, and D refer to four regions in a two-dimensional space in the data, where one of the regions is dominated by signal and the other three by backgrounds. In a standard ABCD method, the background rate in the signal region is estimated from the yields in three control regions with the expression µbkg R4 = NR2 NR3/NR1, (6.1) (6.1) where the labels on the regions correspond to those shown in figure 4. The background prediction is unbiased in the limit that the two variables that define the plane (in this case, MJ and mT) are uncorrelated. The effect of any residual correlation is corrected with factors κ that can be obtained from simulated event samples: κ = NMC,bkg R4 /N MC,bkg R3 NMC,bkg R2 /N MC,bkg R1 . (6.2) (6.2) When the two ABCD variables are uncorrelated or nearly so, the κ factors are close to unity. 6.1 Method This procedure ignores potential signal contamination in the control regions, which is accounted for by incorporating the constraints in eqs. (6.1) and (6.2) into a fit that includes both signal and background components, as described in section 6.2. In principle, the background in the 10 signal bins could be estimated by applying this procedure in 10 independent planes. However, this procedure would incur large statistical uncertainties in some bins due to low numbers of events in R3. This problem is especially – 12 – 4 5 6 7 8 9 ≥ 4 5 6 7 8 9 ≥ ) T R(m 0 0.05 0.1 0.15 0.2 0.25 Simulation CMS 13 TeV Baseline selection Baseline selection = 1 b N = 2 b N 3 ≥ b N 400 GeV ≤ J M > 400 GeV J M jets N Figure 6. The ratio R(mT) of high-mT (R3 and R4) to low-mT (R1 and R2) event yields for the simulated SM background, as a function of Njets and Nb. The baseline selection requires Njets ≥6. The uncertainties shown are statistical only. 4 5 6 7 8 9 ≥ 4 5 6 7 8 9 ≥ ) T R(m 0 0.05 0.1 0.15 0.2 0.25 Simulation CMS 13 TeV Baseline selection Baseline selection = 1 b N = 2 b N 3 ≥ b N 400 GeV ≤ J M > 400 GeV J M jets N Figure 6. The ratio R(mT) of high-mT (R3 and R4) to low-mT (R1 and R2) event yields for the simulated SM background as a function of Njets and Nb The baseline selection requires Njets ≥6 ) T R(m JHEP08(2016)122 Figure 6. The ratio R(mT) of high-mT (R3 and R4) to low-mT (R1 and R2) event yields for the simulated SM background, as a function of Njets and Nb. The baseline selection requires Njets ≥6. The uncertainties shown are statistical only. important in bins with a high number of jets, where the MJ spectrum shifts to higher values and the number of background events expected in R4 can exceed the background in R3. To alleviate this problem, we exploit the fact that, after the baseline selection, the background is dominated by just one source (tt events), and the shapes of the Njets dis- tributions are nearly identical for the single-lepton and dilepton components (due to the large amounts of ISR). 6.1 Method As a result, the mT distribution is approximately independent of Njets and Nb. We study this behavior with the ratio of the number of events at high to low mT: R(mT) ≡N(mT > 140 GeV) N(mT ≤140 GeV). (6.3) (6.3) Because, as seen in figure 6, the values of R(mT) do not vary substantially across Njets and Nb bins, the predicted value of R(mT) is not sensitive to the modeling of the distributions of those quantities. We exploit this result by integrating the yields of the low-MJ regions (R1 and R3) over the Njets and Nb bins for each Emiss T bin. This procedure increases the statistical power of the ABCD method but also introduces a correlation among the predictions (eq. (6.1)) for the Njets and Nb bins associated with a given Emiss T bin. Figure 7 shows the κ factors for the 10 signal bins after summing over Njets and Nb in R1 and R3. In all cases, their values are close to unity. 6.2 Implementation The method outlined in section 6.1 is implemented with a likelihood function that incor- porates the statistical and systematic uncertainties in κ, accounts for correlations arising from the common R1 and R3 yields, and corrects for signal contamination in the control regions. – 13 – 400 ≤ miss T E > 400 miss T E 400 ≤ miss T E > 400 miss T E κ 0 0.5 1 1.5 2 Simulation CMS 13 TeV = 1 b N = 2 b N 3 ≥ b N 2 ≥ b N 8 ≤ jets N ≤ 6 9 ≥ jets N 400 ≤ miss T E > 400 miss T E 400 ≤ miss T E > 400 miss T E κ 0 0.5 1 1.5 2 Simulation CMS 13 TeV = 1 b N = 2 b N 3 ≥ b N 2 ≥ b N 8 ≤ jets N ≤ 6 9 ≥ jets N Figure 7. Values of the double-ratio κ in each of the 10 signal bins, calculated using the simulated SM background. The κ factors are close to unity, indicating the small correlation between MJ and mT. The uncertainties shown are statistical only. JHEP08(2016)122 Figure 7. Values of the double-ratio κ in each of the 10 signal bins, calculated using the simulated SM background. The κ factors are close to unity, indicating the small correlation between MJ and mT. The uncertainties shown are statistical only. The SM background contribution for each region is described as follows. We define µbkg Ri as the estimated (Poisson) mean background in each region Ri, with i = 1, 2, 3, 4. Then, in an ABCD background calculation, these four rates can be expressed in terms of three floating fit parameters µ, R(mT), and R(MJ), and the correlation correction factor κ, as µbkg R1 = µ, µbkg R2 = µ R(MJ), µbkg R3 = µ R(mT), µbkg R4 = κ µ R(MJ) R(mT). (6.4) µbkg R2 = µ R(MJ), µbkg R4 = κ µ R(MJ) R(mT). (6.4) µbkg R1 = µ, µbkg R2 = µ R(MJ), µbkg R3 = µ R(mT), µbkg R4 = κ µ R(MJ) R(mT). (6.4) (6.4) Here, µ is the background rate fit parameter for R1, R(MJ) is the ratio of the R2 to R1 rates, and R(mT) is the ratio of the R3 to R1 rates. The quantity κ is given by eq. 6.2 Implementation (6.2) after replacing the yields NMC,bkg Ri by the background rate fit parameters µMC,bkg Ri . Here, µ is the background rate fit parameter for R1, R(MJ) is the ratio of the R2 to R1 rates, and R(mT) is the ratio of the R3 to R1 rates. The quantity κ is given by eq. (6.2) after replacing the yields NMC,bkg Ri by the background rate fit parameters µMC,bkg Ri . Similarly, we define Ndata Ri as the observed data yield in each region, µMC,sig Ri as the expected signal rate in each region, and r as the parameter quantifying the signal strength relative to the expected yield across all analysis regions. We can then write the likelihood function as L = Ldata ABCD LMC κ LMC sig , (6.5) L = Ldata ABCD LMC κ LMC sig , (6.5) Ldata ABCD = 4 Y i=1 Nbins(Ri) Y k=1 Poisson(Ndata Ri,k |µbkg Ri,k + r µMC,sig Ri,k ), (6.6) LMC κ = 4 Y i=1 Nbins(Ri) Y k=1 Poisson(NMC,bkg Ri,k |µMC,bkg Ri,k ), (6.7) LMC sig = 4 Y i=1 Nbins(Ri) Y k=1 Poisson(NMC,sig Ri,k |µMC,sig Ri,k ). (6.8) (6.5) Ldata ABCD = 4 Y i=1 Nbins(Ri) Y k=1 Poisson(Ndata Ri,k |µbkg Ri,k + r µMC,sig Ri,k ), (6.6) (6.6) LMC κ = 4 Y i=1 Nbins(Ri) Y k=1 Poisson(NMC,bkg Ri,k |µMC,bkg Ri,k ), (6.7) 4 Nbins(Ri) (6.7) (6.8) – 14 – The indices k run over each of the Emiss T , Njets, and Nb bins defined in the previous section; these indices were suppressed in eq. (6.4) for simplicity. Given the integration over Njets and Nb at low MJ, Nbins(R1) = Nbins(R3) = 2, while Nbins(R2) = Nbins(R4) = 10. In eq. (6.5), Ldata ABCD accounts for the statistical uncertainty in the observed data yield in the four ABCD regions, and LMC κ and LMC sig account for the uncertainty in the computation of the κ correction factor and signal shape, respectively, due to the finite size of the MC samples. The systematic uncertainties in κ and the signal efficiency are described in the following sections. These effects are incorporated in the likelihood function as log-normal constraints with a nuisance parameter for each uncorrelated source of uncertainty. These terms are not explicitly shown in the likelihood function above for simplicity. JHEP08(2016)122 The likelihood function defined in eqs. 6.2 Implementation (6.5)–(6.8) is employed in two separate types of fits that provide complementary but compatible background estimates based on an ABCD model. The first type of fit, which we call the predictive fit, allows us to more easily establish the agreement of the background predictions and the observations in the null (i.e., the background-only) hypothesis. We do this by excluding the observations in the signal regions in the likelihood (that is, by truncating the first product in eq. (6.6) at i = 3) and fixing the signal strength r to 0. This procedure leaves as many unknowns as constraints: three data floating parameters (µ, R(MJ), and R(mT)) and three observations (Ndata Ri,k with i = 1, 2, 3) for each ABCD plane. In the likelihood function there are additional floating parameters associated with MC quantities, which have small uncertainties. As a result, the estimated background rates in regions R1, R2, and R3 converge to the observed values in those bins, and we obtain predictions for the signal regions that do not depend on the observed Ndata R4 . The predictive fit thus converges to the standard ABCD method, and the likelihood machinery becomes just a convenient way to solve the system of equations and propagate the various uncertainties. Additionally, we implement a global fit which, by making use of the observations in the signal regions, can provide an estimate of the signal strength r, while allowing for signal events to populate the control regions. This is achieved by including all four observations, Ndata Ri,k with i = 1, 2, 3, 4, in the likelihood function. Since there are four observations and three floating background parameters in each ABCD plane, there are enough constraints for the signal strength also to be determined in the fit. 6.3 Systematic uncertainties This section describes the systematic uncertainties in the background prediction, which are incorporated into the analysis as an uncertainty in the κ correction. Because the dominant background arises from 2ℓtt events, we use a control sample with two reconstructed leptons to validate our background estimation procedure and to quantify the associated uncertainty. The resulting uncertainty is augmented with simulation-based studies of effects that are not covered by this dilepton test. Table 2 summarizes all of the uncertainties in the background prediction. The ability of the ABCD method to predict the 2ℓtt background is studied using a modified ABCD plane, in which the high-mT regions, R3 and R4, are replaced with regions – 15 – Source Fractional uncertainty [%] Data sample size 28–118 Dilepton control sample test 37–88 Simulation sample size 5–17 Jet energy resolution 2–10 Jet energy corrections 1–5 ISR pT 1–5 Top pT 1–4 Non-tt background 2–11 JHEP08(2016)122 Table 2. Summary of uncertainties in the background predictions. All entries in the table except for data sample size correspond to a relative uncertainty on κ. The ranges indicate the spread of each uncertainty across the signal bins. Uncertainties from a particular source are treated as fully correlated across bins, while uncertainties from different sources are treated as uncorrelated. D3 and D4, which have two reconstructed leptons. These regions have low and high MJ, respectively, just as R3 and R4. The events in D3 and D4 pass the same selection as those in R3 and R4, except for the following changes: Njets bin boundaries are lowered by one to keep the number of large-R jet constituents the same as in the single-lepton samples; the mT requirement is not applied; and events with Nb = 0 are included to increase the size of the event sample, while events with Nb ≥3 are excluded to avoid signal contamination. We perform this test only for low Emiss T to further avoid the potentially large signal contribution in the high-Emiss T region. The low-MJ regions (R1 and D3) are integrated over Njets, while the high-MJ regions (R2 and D4) are binned in low and high Njets. The predictive fit is then used to predict the D4 event yields for both Njets bins. We predict 11.0 ± 2.3 (1.5 ± 0.5) events for the low (high) Njets bin, and we observe 12 (2) events. 6.3 Systematic uncertainties Given the good agreement between prediction and observation, the statistical precision of the test is taken as a systematic uncertainty in κ. These uncertainties are 37% and 88% for the low- and high-Njets regions, respectively. Since the event composition of regions D3 and D4 is not fully representative of that in R3 and R4, we perform studies on potential additional sources of systematic uncertainty in the simulation. We find that the main source of 1ℓtt events in the high-mT region is jet energy mismeasurement. We study the impact of mismodeling the size of this contribution by smearing the jet energies by an additional 50% with respect to the jet energy resolution measured in data [70] and calculating the corresponding shift in κ. To ensure that there are no further significant differences between the MJ shapes of events reconstructed with one or two leptons, we also calculate the shift in κ due to jet energy corrections, potential ISR pT and top quark pT mismodeling, as well as the amount of non-tt background. Even though each of these can alone have a significant effect on the MJ shape, the κ factor, as a double ratio, remains largely unaffected (table 2). Including these uncertainties in the likelihood fit produces a negligible contribution to the total uncertainty. – 16 – ) 2 Simulated events/(800 GeV 2 − 10 1 − 10 1 [GeV] J M 0 200 400 600 800 1000 1200 [GeV] T m 0 100 200 300 400 500 600 Data (1500,100) 0 1 χ∼ t t → g~ , g~ g~ CMS (13 TeV) -1 2.3 fb Figure 8. Two-dimensional distributions for data and simulated event samples in the variables mT and MJ in the Nb ≥2 region after the baseline selection. The distributions integrate over the Njets and Emiss T bins. The black dots are the data; the colored histogram is the total simulated background, normalized to the data; and the red dots are a particular signal sam- ple drawn from the expected distribution for gluino pair production in the T1tttt model with meg = 1500 GeV and meχ0 1 = 100 GeV for 2.3 fb−1. Overflow events are shown on the edges of the plot. The definitions of the signal and control regions are the same as those shown in figure 4. 6.3 Systematic uncertainties ) 2 Simulated events/(800 GeV 2 − 10 1 − 10 1 [GeV] J M 0 200 400 600 800 1000 1200 [GeV] T m 0 100 200 300 400 500 600 Data (1500,100) 0 1 χ∼ t t → g~ , g~ g~ CMS (13 TeV) -1 2.3 fb JHEP08(2016)122 Figure 8. Two-dimensional distributions for data and simulated event samples in the variables mT and MJ in the Nb ≥2 region after the baseline selection. The distributions integrate over the Njets and Emiss T bins. The black dots are the data; the colored histogram is the total simulated background, normalized to the data; and the red dots are a particular signal sam- ple drawn from the expected distribution for gluino pair production in the T1tttt model with meg = 1500 GeV and meχ0 1 = 100 GeV for 2.3 fb−1. Overflow events are shown on the edges of the plot. The definitions of the signal and control regions are the same as those shown in figure 4. 7 Results and interpretation Figure 8 shows the two-dimensional distribution of the data in the mT–MJ plane after the baseline selection, but with the additional requirement Nb ≥2. The baseline requirements include Emiss T > 200 GeV and Njets ≥6, but no further event selection is applied. For comparison, the plot also shows the expected total SM background based on simulation, as well as a particular sample of the expected signal distribution. The overall distribution of events in data is consistent with the background expectation, where the majority of events are concentrated at low mT and MJ. In R4, the nominal signal region, we observe only two events in data, while, as shown in table 3, the predicted SM background is about 5 events. The T1tttt(1500,100) (NC) model would be expected to contribute 5 additional events to R4. The validity of the central assumption of the background estimation method can be checked in the nearly signal-free Nb = 1 region by comparing the MJ shapes observed in the high- and low-mT regions in data. Figure 9 (left) shows the MJ shapes in the Nb = 1 sample, integrating over the Njets and Emiss T bins. The low mT data have been normalized to the overall yields in the corresponding high-mT data. The shapes of the MJ distributions for the high- and low-mT regions are consistent. Figure 9 (right) shows that the corresponding distributions in the Nb ≥2 sample are also consistent, as expected in the absence of signal. – 17 – [GeV] J M 200 400 600 Events/(75 GeV) 0 5 10 15 20 CMS (13 TeV) -1 2.3 fb > 140 GeV T Data, m 140 GeV ≤ T Data, m (1500,100) 1 0 χ∼ tt → g~ , g~ g~ (1200,800) 1 0 χ∼ tt → g~ , g~ g~ = 1 b N [GeV] J M 200 400 600 Events/(75 GeV) 0 5 10 15 CMS (13 TeV) -1 2.3 fb > 140 GeV T Data, m 140 GeV ≤ T Data, m (1500,100) 1 0 χ∼ tt → g~ , g~ g~ (1200,800) 1 0 χ∼ tt → g~ , g~ g~ 2 ≥ b N Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) and Nb ≥2 (right) after the baseline selection. The expected MJ distributions of the two benchmark T1tttt scenarios for mT > 140 GeV are overlaid. 7 Results and interpretation The distributions integrate over the Njets and Emiss T bins. The low-mT distribution is normalized to the number of events in the high-mT region. The dashed vertical lines indicate the MJ > 400 GeV threshold that separates the signal regions from the control samples. [GeV] J M 200 400 600 Events/(75 GeV) 0 5 10 15 CMS (13 TeV) -1 2.3 fb > 140 GeV T Data, m 140 GeV ≤ T Data, m (1500,100) 1 0 χ∼ tt → g~ , g~ g~ (1200,800) 1 0 χ∼ tt → g~ , g~ g~ 2 ≥ b N [GeV] J M 200 400 600 Events/(75 GeV) 0 5 10 15 20 CMS (13 TeV) -1 2.3 fb > 140 GeV T Data, m 140 GeV ≤ T Data, m (1500,100) 1 0 χ∼ tt → g~ , g~ g~ (1200,800) 1 0 χ∼ tt → g~ , g~ g~ = 1 b N JHEP08(2016)122 Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) an Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) and Nb ≥2 (right) after the baseline selection. The expected MJ distributions of the two benchmark T1tttt scenarios for mT > 140 GeV are overlaid. The distributions integrate over the Njets and Emiss T bins. The low-mT distribution is normalized to the number of events in the high-mT region. The dashed vertical lines indicate the MJ > 400 GeV threshold that separates the signal regions from the control samples. Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) and Nb ≥2 (right) after the baseline selection. The expected MJ distributions of the two benchmark T1tttt scenarios for mT > 140 GeV are overlaid. The distributions integrate over the Njets and Emiss T bins. The low-mT distribution is normalized to the number of events in the high-mT region. The dashed vertical lines indicate the MJ > 400 GeV threshold that separates the signal regions from the control samples. Table 3 summarizes the observed event yields, the fitted backgrounds, and the expected signal yields for the two T1tttt benchmark model points. 7 Results and interpretation Two background estimates are given: the predictive fit (PF), which uses only the yields in regions R1, R2, and R3, and the global fit (GF), which also incorporates region R4, as described in section 6. In both versions of the fit, the signal strength r is fixed to zero, giving results that are model independent. (When setting limits on individual models, we allow r to float, as discussed below.) The rows labeled R4 give the results for each of the ten signal regions, as well as the corresponding κ factors. In the absence of signal, the predictive fit and the version of the global fit performed under the null hypothesis, r = 0, should be consistent with each other. However, because the global fit incorporates more information, specifically the yields in R4, this fit has a smaller uncertainty. The regions with Nb = 1 have small expected contributions from signal. Summing over all four such signal regions (R4), the number of estimated background events from the PF and GF are 6.1±2.2 and 5.5±1.3, respectively, compared with 8 events observed in data. The consistency between the two predictions and between the predicted and observed yields in the R4 regions with Nb = 1, where the signal contribution is expected to be small, serves as a further check on the background estimation method. Summing the yields over the six signal bins with Nb ≥2, the number of estimated background events from PF and GF is 5.6±1.6 and 4.9±1.0, respectively. In data, we observe 2 events, lower than, but consistent with the background-only hypothesis. Given the absence of any significant excess, the results are interpreted first as exclusion limits on the production cross section for T1tttt model points as a function of meg and meχ0 1. Table 4 shows the ranges for the systematic uncertainties associated with predictions for the expected signal yields, including those on the signal efficiency. The largest uncertainties arise from the jet energy corrections and from the modeling of ISR. These uncertainties are generally in the range 10–20% but can increase to ∼30% as the mass splitting between – 18 – Region: bin κ T1tttt(NC) T1tttt(C) Fitted µbkg (PF) Fitted µbkg (GF) Obs. 7 Results and interpretation 200 < Emiss T ≤400 GeV R1: all Njets, Nb — 0.1 3.2 336.0 ± 18.3 335.3 ± 18.2 336 R2: 6 ≤Njets ≤8, Nb = 1 — 0.1 0.2 47.1 ± 6.9 49.5 ± 6.9 47 R2: Njets ≥9, Nb = 1 — 0.1 0.3 7.0 ± 2.6 7.5 ± 2.7 7 R2: 6 ≤Njets ≤8, Nb = 2 — 0.1 0.3 42.0 ± 6.5 41.1 ± 6.2 42 R2: Njets ≥9, Nb = 2 — 0.1 0.5 7.0 ± 2.6 6.6 ± 2.5 7 R2: 6 ≤Njets ≤8, Nb ≥3 — 0.1 0.2 12.0 ± 3.5 11.1 ± 3.2 12 R2: Njets ≥9, Nb ≥3 — 0.2 0.6 1.0 ± 1.0 0.9 ± 0.9 1 R3: all Njets, Nb — 0.2 3.8 21.0 ± 4.6 21.6 ± 4.2 21 R4: 6 ≤Njets ≤8, Nb = 1 1.12 ± 0.09 ± 0.43 0.2 0.2 3.3 ± 1.4 3.6 ± 1.0 6 R4: Njets ≥9, Nb = 1 0.91 ± 0.06 ± 0.81 0.2 0.4 0.4 ± 0.3 0.4 ± 0.2 1 R4: 6 ≤Njets ≤8, Nb = 2 1.11 ± 0.06 ± 0.42 0.3 0.4 2.9 ± 1.2 2.9 ± 0.8 2 R4: Njets ≥9, Nb = 2 1.05 ± 0.11 ± 0.94 0.3 0.6 0.5 ± 0.3 0.4 ± 0.2 0 R4: 6 ≤Njets ≤8, Nb ≥3 1.25 ± 0.11 ± 0.47 0.3 0.3 0.9 ± 0.4 0.9 ± 0.3 0 R4: Njets ≥9, Nb ≥3 1.05 ± 0.10 ± 0.93 0.3 0.7 0.1 ± 0.1 0.1 ± 0.1 0 Emiss T > 400 GeV R1: all Njets, Nb — 0.1 0.5 16.0 ± 4.0 17.1 ± 4.0 16 R2: 6 ≤Njets ≤8, Nb = 1 — 0.2 0.1 8.0 ± 2.8 6.8 ± 2.5 8 R2: Njets ≥9, Nb = 1 — 0.1 0.2 1.0 ± 1.0 1.7 ± 1.2 1 R2: 6 ≤Njets ≤8, Nb ≥2 — 0.5 0.3 3.0 ± 1.7 2.5 ± 1.4 3 R2: Njets ≥9, Nb ≥2 — 0.4 0.6 1.0 ± 1.0 0.9 ± 0.9 1 R3: all Njets, Nb — 0.4 0.9 4.0 ± 2.0 2.9 ± 1.4 4 R4: 6 ≤Njets ≤8, Nb = 1 1.09 ± 0.16 ± 0.42 0.7 0.2 2.2 ± 1.7 1.2 ± 0.7 0 R4: Njets ≥9, Nb = 1 0.98 ± 0.16 ± 0.87 0.4 0.3 0.2 ± 0.3 0.3 ± 0.2 1 R4: 6 ≤Njets ≤8, Nb ≥2 1.29 ± 0.22 ± 0.50 1.9 0.5 1.0 ± 0.8 0.5 ± 0.4 0 R4: Njets ≥9, Nb ≥2 0.90 ± 0.14 ± 0.80 1.6 1.0 0.2 ± 0.3 0.1 ± 0.1 0 Table 3. 7 Results and interpretation Observed and predicted event yields for the signal regions (R4) and background regions (R1–R3) in data (2.3 fb−1). Expected yields for the two SUSY T1tttt benchmark scenarios are also given. The results from two types of fits are reported: the predictive fit (PF) and the version of the global fit (GF) performed under the assumption of the null hypothesis (r = 0). The predictive fit uses the observed yields in regions R1, R2, and R3 only and is effectively just a propagation of uncertainties. The global fit uses all four regions. The values of κ obtained from the simulation fit are also listed. The first uncertainty in κ is statistical, while the second corresponds to the total systematic uncertainty. The benchmark signal models, T1tttt(NC) and T1tttt(C), are described in section 3. JHEP08(2016)122 Table 3. Observed and predicted event yields for the signal regions (R4) and background regions (R1–R3) in data (2.3 fb−1). Expected yields for the two SUSY T1tttt benchmark scenarios are also given. The results from two types of fits are reported: the predictive fit (PF) and the version of the global fit (GF) performed under the assumption of the null hypothesis (r = 0). The predictive fit uses the observed yields in regions R1, R2, and R3 only and is effectively just a propagation of uncertainties. The global fit uses all four regions. The values of κ obtained from the simulation fit are also listed. The first uncertainty in κ is statistical, while the second corresponds to the total systematic uncertainty. The benchmark signal models, T1tttt(NC) and T1tttt(C), are described in section 3. the gluino and LSP decreases [77]. The uncertainty associated with the renormalization and factorization scales is determined by varying the scales independently up and down by a factor of two; these are applied only as an uncertainty in the signal shape, i.e., the cross section is held constant. The uncertainty associated with the b tagging efficiency is in the range 1–15%. Uncertainties due to pileup, luminosity [78], lepton selection, and trigger efficiency are found to be ≤5%. Uncertainties for each particular source are treated as fully correlated across bins. 7 Results and interpretation A 95% confidence level (CL) upper limit on the production cross section is estimated using the modified frequentist CLS method [79–81], with a one-sided profile likelihood – 19 – Source Fractional uncertainty [%] Lepton efficiency 1–5 Trigger efficiency 1 b tagging efficiency 1–15 Jet energy corrections 1–30 Renormalization and factorization scales 1–5 Initial state radiation 1–35 Pileup 5 Integrated luminosity 3 Table 4. Typical values of the signal-related systematic uncertainties. Uncertainties due to a particular source are treated as fully correlated between bins, while uncertainties due to different sources are treated as uncorrelated. JHEP08(2016)122 Table 4. Typical values of the signal-related systematic uncertainties. Uncertainties due to a particular source are treated as fully correlated between bins, while uncertainties due to different sources are treated as uncorrelated. Table 4. Typical values of the signal-related systematic uncertainties. Uncertainties due to a particular source are treated as fully correlated between bins, while uncertainties due to different sources are treated as uncorrelated. ratio test statistic. For this test, we perform the global fit under the background-only and background-plus-signal (r floating) hypotheses. The statistical uncertainties from data counts in the control regions are modeled by the Poisson terms in eq. (6.6). All systematic uncertainties are multiplicative and are treated as log-normal distributions. Exclusion limits are also estimated for ±1σ variations on the production cross section based on the NLO+NLL calculation [39]. Figure 10 shows the corresponding excluded region at a 95% CL for the T1tttt model in the meg–meχ0 1 plane. At low eχ0 1 mass we exclude gluinos with masses of up to 1600 GeV. The highest limit on the eχ0 1 mass is 800 GeV, attained for meg of approximately 1300 GeV. The observed limits are within the 1σ uncertainty in the expected limits. The central value is slightly higher because the observed event yield is less than the SM background prediction, as shown in table 3. In the context of natural SUSY models, it is important to extend the interpretation to scenarios in which the top squark is lighter than the gluino. Rather than considering a large set of models with independently varying top squark masses, we consider the extreme case in which the top squark has approximately the smallest mass consistent with two-body decay, met1 ≈mt + meχ0 1, for a range of gluino and neutralino masses. 7 Results and interpretation The decay kinematics for such extreme, compressed mass spectrum models correspond to the lowest signal efficiency for given values of meg and meχ0 1, because the top quark and the eχ0 1 are produced at rest in the top squark frame. As a consequence, the excluded signal cross section for fixed values of meg and meχ0 1 and with meg > met1 ≥mt + meχ0 1 is minimized around this extreme model point. For physical consistency, the signal model used in this study, both in the fit procedure and in the theoretical cross section used to obtain mass limits, includes not only gluino-pair production, but also direct et1et1 production. However, the effect of the direct top squark contribution on the results is small, ≲2% for meχ0 1 > 400 GeV and up to 20% for low values of meχ0 1. χ1 Figure 11 shows the excluded region in the meg–meχ0 1 plane for this combined model with both gluino-mediated top squark production and direct top squark pair production. – 20 – [GeV] g~ m 800 1000 1200 1400 1600 1800 [GeV] 1 0 χ∼ m 0 200 400 600 800 1000 1200 1400 1600 1800 3 − 10 2 − 10 1 − 10 1 (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼ t t → g~ , g~ g~ → pp theory σ 1 ± Observed experiment σ 1 ± Expected 95% CL upper limit on cross section [pb] Figure 10. Interpretation of results in the T1tttt model. The colored regions show the upper limits (95% CL) on the production cross section for pp →egeg,eg →tteχ0 1 in the meg–meχ0 1 plane. The curves show the expected and observed limits on the corresponding SUSY particle masses obtained by comparing the excluded cross section with theoretical cross sections. [GeV] g~ m 800 1000 1200 1400 1600 1800 [GeV] 1 0 χ∼ m 0 200 400 600 800 1000 1200 1400 1600 1800 3 − 10 2 − 10 1 − 10 1 (13 TeV) -1 2.3 fb CMS NLO+NLL exclusion 1 0 χ∼ t t → g~ , g~ g~ → pp theory σ 1 ± Observed experiment σ 1 ± Expected 95% CL upper limit on cross section [pb] JHEP08(2016)122 Figure 10. Interpretation of results in the T1tttt model. 7 Results and interpretation In this kinematic region, the sensitivity to the signal is dominated by the events that have at least two leptonic W boson decays, which produce additional Emiss T , as well as a tail in the mT distribution. Although such dilepton events are nominally excluded in the analysis, a significant number of these signal events escape the dilepton veto. These events include both W decays to τ leptons that decay hadronically, and W decays to electrons or muons that are below kinematic thresholds or are outside of the detector acceptance. 7 Results and interpretation The colored regions show the upper limits (95% CL) on the production cross section for pp →egeg,eg →tteχ0 1 in the meg–meχ0 1 plane. The curves show the expected and observed limits on the corresponding SUSY particle masses obtained by comparing the excluded cross section with theoretical cross sections. The top squark mass is assumed to be 175 GeV above that of the neutralino. For most of the excluded region, the boundary is close to that obtained for the T1tttt model, showing that there is only a weak sensitivity to the value of the top squark mass. The uncertainty on the boundary of the excluded region for the T5tttt model is similar to that shown for the T1tttt model in figure 10. For meχ0 1 > 150 GeV, the excluded value of meg is typically within 60 GeV of that excluded for T1tttt. Models that have low values of meχ0 1 show a reduced sensitivity because the neutralino carries very little momentum, reducing the value of mT. In this kinematic region, the sensitivity to the signal is dominated by the events that have at least two leptonic W boson decays, which produce additional Emiss T , as well as a tail in the mT distribution. Although such dilepton events are nominally excluded in the analysis, a significant number of these signal events escape the dilepton veto. These events include both W decays to τ leptons that decay hadronically, and W decays to electrons or muons that are below kinematic thresholds or are outside of the detector acceptance. The top squark mass is assumed to be 175 GeV above that of the neutralino. For most of the excluded region, the boundary is close to that obtained for the T1tttt model, showing that there is only a weak sensitivity to the value of the top squark mass. The uncertainty on the boundary of the excluded region for the T5tttt model is similar to that shown for the T1tttt model in figure 10. For meχ0 1 > 150 GeV, the excluded value of meg is typically within 60 GeV of that excluded for T1tttt. Models that have low values of meχ0 1 show a reduced sensitivity because the neutralino carries very little momentum, reducing the value of mT. 8 Summary Using a sample of proton-proton collisions at √s = 13 TeV with an integrated luminosity of 2.3 fb−1, a search for supersymmetry is performed in the final state with a single lepton, – 21 – [GeV] g~ m 600 800 1000 1200 1400 1600 [GeV] 1 0 χ∼ m 0 200 400 600 800 1000 1200 CMS (13 TeV) -1 2.3 fb 1 0 χ∼ t t → g~ , g~ g~ → pp = 175 GeV) 1 0 χ∼ - m 1t~ (m 1 0 χ∼ t → 1t~ t, 1t~ → g~ , 1t~ 1t~ + g~ g~ → pp Expected Observed Figure 11. Excluded region (95% CL), shown in blue, in the meg–meχ0 1 plane for a model combining T5tttt, gluino pair production, followed by gluino decay to an on-shell top squark, together with a model for direct top squark pair production. The top squarks decay via the two-body process et →teχ0 1. The neutralino and top squark masses are related by the constraint met1 = meχ0 1 +175 GeV. For comparison, the excluded region (95% CL) from figure 10 for the T1tttt model, which has three- body gluino decay, is shown in red. The small difference between the two boundary curves shows that the limits for the scenarios with two-body gluino decay have only a weak dependence on the top squark mass. [GeV] g~ m 600 800 1000 1200 1400 1600 [GeV] 1 0 χ∼ m 0 200 400 600 800 1000 1200 CMS (13 TeV) -1 2.3 fb 1 0 χ∼ t t → g~ , g~ g~ → pp = 175 GeV) 1 0 χ∼ - m 1t~ (m 1 0 χ∼ t → 1t~ t, 1t~ → g~ , 1t~ 1t~ + g~ g~ → pp Expected Observed JHEP08(2016)122 Figure 11. Excluded region (95% CL), shown in blue, in the meg–meχ0 1 plane for a model combining T5tttt, gluino pair production, followed by gluino decay to an on-shell top squark, together with a model for direct top squark pair production. The top squarks decay via the two-body process et →teχ0 1. The neutralino and top squark masses are related by the constraint met1 = meχ0 1 +175 GeV. For comparison, the excluded region (95% CL) from figure 10 for the T1tttt model, which has three- body gluino decay, is shown in red. 8 Summary The small difference between the two boundary curves shows that the limits for the scenarios with two-body gluino decay have only a weak dependence on the top squark mass. b-tagged jets, and large missing transverse momentum. The search focuses on final states resulting from the pair production of gluinos, which subsequently decay via eg →tteχ0 1, leading to high jet multiplicities. A key feature of the analysis is the use of the variable MJ, the sum of the masses of large-R jets, which are formed by clustering anti-kT R = 0.4 jets and leptons. Used in conjunction with the variable mT, the transverse mass of the system consisting of the lepton and the missing transverse momentum vector, MJ provides a powerful background estimation method that is well suited to this high jet multiplicity search. After the baseline selection is applied, signal (R4) and control regions (R1, R2, and R3) are defined in the MJ–mT plane, which are further divided into bins of Emiss T , Njets, and Nb to provide additional sensitivity. In regions R3 and R4, the requirement mT > 140 GeV provides strong suppression of the single-lepton tt background, so that dilepton tt events dominate over all other background sources. For these dilepton events to enter a signal – 22 – region, however, they must contain a substantial amount of initial-state radiation (ISR). For this extreme range of ISR jet momentum and multiplicity, the single-lepton and dilepton tt events have very similar kinematic properties. The variables MJ and mT are nearly uncorrelated, even though different processes dominate the low- and high-mT regions. As a consequence, the low-mT regions (R1 and R2) can be used to measure the background shape for the MJ distribution at high mT. A correction factor, near unity, is taken from simulation and is used to account for a possible correlation between MJ and mT. The observed event yields in the signal regions are consistent with the predictions for the SM background contributions, and exclusion limits are set on the gluino pair production cross sections in the meg–meχ0 1 plane, as described by the simplified models T1tttt and T5tttt, where the latter is augmented with a model of direct top squark pair production for consistency. In the T1tttt model, gluinos decay via the three-body process eg →tteχ0 1, which proceeds via a virtual top squark in the intermediate state. 8 Summary Under the assumption of a 100% branching fraction to this final state, the cross section limit for each model point is compared with the theoretical cross section to determine the excluded particle masses. Gluinos with a mass below 1600 GeV are excluded at a 95% CL for scenarios with low eχ0 1 mass, and neutralinos with a mass below 800 GeV are excluded for a gluino mass of about 1300 GeV. In the T5tttt model, the top squark is lighter than the gluino, which therefore decays via a two-body process. The boundary of the excluded region in the meg–meχ0 1 plane for T5tttt is found to be only weakly sensitive to the top squark mass. These results significantly extend the sensitivity of single-lepton searches based on data at √s = 8 TeV. JHEP08(2016)122 Acknowledgments Forschungszentren, Germany; the General Secretariat for Research and Technology, Greece; the National Scientific Research Foundation, and National Innovation Office, Hungary; the Department of Atomic Energy and the Department of Science and Technology, India; the Institute for Studies in Theoretical Physics and Mathematics, Iran; the Science Founda- tion, Ireland; the Istituto Nazionale di Fisica Nucleare, Italy; the Ministry of Science, ICT and Future Planning, and National Research Foundation (NRF), Republic of Korea; the Lithuanian Academy of Sciences; the Ministry of Education, and University of Malaya (Malaysia); the Mexican Funding Agencies (BUAP, CINVESTAV, CONACYT, LNS, SEP, and UASLP-FAI); the Ministry of Business, Innovation and Employment, New Zealand; the Pakistan Atomic Energy Commission; the Ministry of Science and Higher Education and the National Science Centre, Poland; the Funda¸c˜ao para a Ciˆencia e a Tecnologia, Portugal; JINR, Dubna; the Ministry of Education and Science of the Russian Federation, the Federal Agency of Atomic Energy of the Russian Federation, Russian Academy of Sciences, and the Russian Foundation for Basic Research; the Ministry of Education, Sci- ence and Technological Development of Serbia; the Secretar´ıa de Estado de Investigaci´on, Desarrollo e Innovaci´on and Programa Consolider-Ingenio 2010, Spain; the Swiss Funding Agencies (ETH Board, ETH Zurich, PSI, SNF, UniZH, Canton Zurich, and SER); the Ministry of Science and Technology, Taipei; the Thailand Center of Excellence in Physics, the Institute for the Promotion of Teaching Science and Technology of Thailand, Special Task Force for Activating Research and the National Science and Technology Development Agency of Thailand; the Scientific and Technical Research Council of Turkey, and Turkish Atomic Energy Authority; the National Academy of Sciences of Ukraine, and State Fund for Fundamental Researches, Ukraine; the Science and Technology Facilities Council, U.K.; the US Department of Energy, and the US National Science Foundation. JHEP08(2016)122 Individuals have received support from the Marie-Curie program and the European Re- search Council and EPLANET (European Union); the Leventis Foundation; the A.P. Acknowledgments Finally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the following funding agencies: the Austrian Federal Ministry of Science, Research and Economy and the Austrian Science Fund; the Belgian Fonds de la Recherche Scientifique, and Fonds voor Wetenschappelijk Onderzoek; the Brazilian Funding Agencies (CNPq, CAPES, FAPERJ, and FAPESP); the Bulgarian Ministry of Education and Science; CERN; the Chinese Academy of Sciences, Ministry of Science and Technology, and National Natural Science Foundation of China; the Colombian Funding Agency (COLCIENCIAS); the Croatian Ministry of Science, Education and Sport, and the Croatian Science Foundation; the Research Promotion Foundation, Cyprus; the Ministry of Education and Research, Estonian Research Council via IUT23-4 and IUT23- 6 and European Regional Development Fund, Estonia; the Academy of Finland, Finnish Ministry of Education and Culture, and Helsinki Institute of Physics; the Institut National de Physique Nucl´eaire et de Physique des Particules/CNRS, and Commissariat `a l’´Energie Atomique et aux ´Energies Alternatives/CEA, France; the Bundesministerium f¨ur Bildung und Forschung, Deutsche Forschungsgemeinschaft, and Helmholtz-Gemeinschaft Deutscher – 23 – Forschungszentren, Germany; the General Secretariat for Research and Technology, Greece; the National Scientific Research Foundation, and National Innovation Office, Hungary; the Department of Atomic Energy and the Department of Science and Technology, India; the Institute for Studies in Theoretical Physics and Mathematics, Iran; the Science Founda- tion, Ireland; the Istituto Nazionale di Fisica Nucleare, Italy; the Ministry of Science, ICT and Future Planning, and National Research Foundation (NRF), Republic of Korea; the Lithuanian Academy of Sciences; the Ministry of Education, and University of Malaya (Malaysia); the Mexican Funding Agencies (BUAP, CINVESTAV, CONACYT, LNS, SEP, and UASLP-FAI); the Ministry of Business, Innovation and Employment, New Zealand; the Pakistan Atomic Energy Commission; the Ministry of Science and Higher Education and the National Science Centre, Poland; the Funda¸c˜ao para a Ciˆencia e a Tecnologia, Portugal; JINR, Dubna; the Ministry of Education and Science of the Russian Federation, the Federal Agency of Atomic Energy of the Russian Federation, Russian Academy of Sciences, and the Russian Foundation for Basic Research; the Ministry of Education, Sci- ence and Technological Development of Serbia; the Secretar´ıa de Estado de Investigaci´on, Desarrollo e Innovaci´on and Programa Consolider-Ingenio 2010, Spain; the Swiss Funding Agencies (ETH Board, ETH Zurich, PSI, SNF, UniZH, Canton Zurich, and SER); the Ministry of Science and Technology, Taipei; the Thailand Center of Excellence in Physics, the Institute for the Promotion of Teaching Science and Technology of Thailand, Special Task Force for Activating Research and the National Science and Technology Development Agency of Thailand; the Scientific and Technical Research Council of Turkey, and Turkish Atomic Energy Authority; the National Academy of Sciences of Ukraine, and State Fund for Fundamental Researches, Ukraine; the Science and Technology Facilities Council, U.K.; the US Department of Energy, and the US National Science Foundation. 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Acknowledgments Sloan Foundation; the Alexander von Humboldt Foundation; the Belgian Federal Science Policy Office; the Fonds pour la Formation `a la Recherche dans l’Industrie et dans l’Agriculture (FRIA-Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT- Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Science and Industrial Research, India; the HOMING PLUS program of the Foundation for Polish Science, cofinanced from European Union, Regional Develop- ment Fund; the Mobility Plus program of the Ministry of Science and Higher Educa- tion (Poland); the OPUS program of the National Science Center (Poland); the Thalis and Aristeia programs cofinanced by EU-ESF and the Greek NSRF; the National Priori- ties Research Program by Qatar National Research Fund; the Programa Clar´ın-COFUND del Principado de Asturias; the Rachadapisek Sompot Fund for Postdoctoral Fellowship, Chulalongkorn University (Thailand); the Chulalongkorn Academic into Its 2nd Century Project Advancement Project (Thailand); and the Welch Foundation, contract C-1845. 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Gascon, – 32 – M. Gouzevitch, G. Grenier, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, S. Perries, A. Popov16, D. Sabes, V. Sordini, M. Vander Donckt, P. Verdier, S. Viret Georgian Technical University, Tbilisi, Georgia A. Khvedelidze8 Tbilisi State University, Tbilisi, Georgia Tbilisi State University, Tbilisi, Georgia Z. Tsamalaidze8 Z. Tsamalaidze8 Z. Tsamalaidze8 RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, S. Beranek, L. Feld, A. Heister, M.K. Kiesel, K. Klein, M. Lipinski, A. Ostapchuk, M. Preuten, F. Raupach, S. Schael, C. Schomakers, J.F. Schulte, J. Schulz, T. Verlage, H. Weber, V. Zhukov16 JHEP08(2016)122 RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany M. Brodski, E. Dietz-Laursonn, D. Duchardt, M. Endres, M. Erdmann, S. Erdweg, T. Esch, R. Fischer, A. G¨uth, T. Hebbeker, C. Heidemann, K. Hoepfner, S. Knutzen, M. Merschmeyer, A. Meyer, P. Millet, S. Mukherjee, M. Olschewski, K. Padeken, P. Papacz, T. Pook, M. Radziej, H. Reithler, M. Rieger, F. Scheuch, L. Sonnenschein, D. Teyssier, S. Th¨uer RWTH Aachen University, III. Physikalisches Institut B, Aachen, Germany V. Cherepanov, Y. Erdogan, G. Fl¨ugge, F. Hoehle, B. Kargoll, T. Kress, A. K¨unsken, J. Lingemann, A. Nehrkorn, A. Nowack, I.M. Nugent, C. Pistone, O. Pooth, A. Stahl15 V. Cherepanov, Y. Erdogan, G. Fl¨ugge, F. Hoehle, B. Kargoll, T. Kress, A. K¨unsken, J. Lingemann, A. Nehrkorn, A. Nowack, I.M. Nugent, C. Pistone, O. Pooth, A. Stahl15 National and Kapodistrian University of Athens, Athens, Greece National and Kapodistrian University of Athens, Athens, Greece A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi University of Io´annina, Io´annina, Greece I. Evangelou, G. Flouris, C. Foudas, P. Kokkas, N. Loukas, N. Manthos, I. Papadopoulos, E. Paradas MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University N. Filipovic Wigner Research Centre for Physics, Budapest, Hungary G. Bencze, C. Hajdu, P. Hidas, D. Horvath20, F. Sikler, V. Veszpremi, G. Vesztergombi21, A.J. Zsigmond Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi22, A. Makovec, J. Molnar, Z. Szillasi University of Debrecen, Debrecen, Hungary M. Bart´ok21, P. Raics, Z.L. Trocsanyi, B. Ujvari National Institute of Science Education and Research, Bhubaneswar, India S. Bahinipati, S. Choudhury23, P. Mal, K. Mandal, A. Nayak24, D.K. Sahoo, N. Sahoo, S.K. Swain Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, R. Gupta, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, A. Mehta, M. Mittal, J.B. Singh, G. Walia University of Delhi, Delhi, India Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, S. Keshri, A. Kumar, S. Mal- hotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma University of Io´annina, Io´annina, Greece I. Evangelou, G. Flouris, C. Foudas, P. Kokkas, N. Loukas, N. Manthos, I. Papadopoulos, E. Paradas Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi22, A. Makovec, J. Molnar, Z. Szillasi University of Debrecen, Debrecen, Hungary M. Bart´ok21, P. Raics, Z.L. Trocsanyi, B. Ujvari Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi22, A. Makovec, J. Molnar, Z. Szillasi National Institute of Science Education and Research, Bhubaneswar, India S. Bahinipati, S. Choudhury23, P. Mal, K. Mandal, A. Nayak24, D.K. Sahoo, N. Sahoo, S.K. Swain S. Bahinipati, S. Choudhury23, P. Mal, K. Mandal, A. Nayak24, D.K. Sahoo, N. Saho S.K. Swain University of Hamburg, Hamburg, Germany V. Blobel, M. Centis Vignali, A.R. Draeger, T. Dreyer, E. Garutti, K. Goebel, D. Gonzalez, J. Haller, M. Hoffmann, A. Junkes, R. Klanner, R. Kogler, N. Kovalchuk, T. Lapsien, T. Lenz, I. Marchesini, D. Marconi, M. Meyer, M. Niedziela, D. Nowatschin, J. Ott, F. Pantaleo15, T. Peiffer, A. Perieanu, J. Poehlsen, C. Sander, C. Scharf, P. Schleper, A. Schmidt, S. Schumann, J. Schwandt, H. Stadie, G. Steinbr¨uck, F.M. Stober, M. St¨over, H. Tholen, D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald – 33 – Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, R. Gupta, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, A. Mehta, M. Mittal, J.B. Singh, G. Walia Institut f¨ur Experimentelle Kernphysik, Karlsruhe, Germany C. Barth, C. Baus, J. Berger, E. Butz, T. Chwalek, F. Colombo, W. De Boer, A. D C. Barth, C. Baus, J. Berger, E. Butz, T. Chwalek, F. Colombo, W. De Boer, A. Dierlamm, S. Fink, R. Friese, M. Giffels, A. Gilbert, D. Haitz, F. Hartmann15, S.M. Heindl, U. Husemann, I. Katkov16, P. Lobelle Pardo, B. Maier, H. Mildner, M.U. Mozer, T. M¨uller, Th. M¨uller, M. Plagge, G. Quast, K. Rabbertz, S. R¨ocker, F. Roscher, M. Schr¨oder, G. Sieber, H.J. Simonis, R. Ulrich, J. Wagner-Kuhr, S. Wayand, M. Weber, T. Weiler, S. Williamson, C. W¨ohrmann, R. Wolf Institute of Nuclear and Particle Physics (INPP), NCSR Demokritos, Aghia Paraskevi, Greece JHEP08(2016)122 G. Anagnostou, G. Daskalakis, T. Geralis, V.A. Giakoumopoulou, A. Kyriakis, D. Loukas, I. Topsis-Giotis Saha Institute of Nuclear Physics, Kolkata, India Saha Institute of Nuclear Physics, Kolkata, India R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh, N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur Saha Institute of Nuclear Physics, Kolkata, India R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghos R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh, N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur Indian Institute of Technology Madras, Madras, India P.K. Behera Bhabha Atomic Research Centre, Mumbai, India Bhabha Atomic Research Centre, Mumbai, India R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty15, P.K. Netrakanti, L.M. Pant, P. Shukla, A. Topkar Bhabha Atomic Research Centre, Mumbai, India R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty15, P.K. Netrakanti, L.M. Pant, P. Shukla, A. Topkar JHEP08(2016)122 Tata Institute of Fundamental Research-A, Mumbai, India Tata Institute of Fundamental Research-A, Mumbai, India T. Aziz, S. Dugad, G. Kole, B. Mahakud, S. Mitra, G.B. Mohanty, N. Sur, B. Sutar T. Aziz, S. Dugad, G. Kole, B. Mahakud, S. Mitra, G.B. Mohanty, N. Sur, B. Sutar Tata Institute of Fundamental Research-B, Mumbai, India Tata Institute of Fundamental Research-B, Mumbai, India S. Banerjee, S. Bhowmik25, R.K. Dewanjee, S. Ganguly, M. Guchait, Sa. Jain, S. Kumar, M. Maity25, G. Majumder, K. Mazumdar, B. Parida, T. Sarkar25, N. Wickramage26 S. Banerjee, S. Bhowmik25, R.K. Dewanjee, S. Ganguly, M. Guchait, Sa. Jain, S. Kuma M. Maity25, G. Majumder, K. Mazumdar, B. Parida, T. Sarkar25, N. Wickramage26 Indian Institute of Science Education and Research (IISER), Pune, India S. Chauhan, S. Dube, A. Kapoor, K. Kothekar, A. Rane, S. Sharma Institute for Research in Fundamental Sciences (IPM), Tehran, Iran Institute for Research in Fundamental Sciences (IPM), Tehran, Iran H. Bakhshiansohi, H. Behnamian, S. Chenarani27, E. Eskandari Tadavani, S.M. Etesami27, A. Fahim28, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi, F. Rezaei Hosseinabadi, B. Safarzadeh29, M. Zeinali University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald INFN Sezione di Baria, Universit`a di Barib, Politecnico di Baric, Bari, Italy M. Abbresciaa,b, C. Calabriaa,b, C. Caputoa,b, A. Colaleoa, D. Creanzaa,c, L. Cristellaa,b, N. De Filippisa,c, M. De Palmaa,b, L. Fiorea, G. Iasellia,c, G. Maggia,c, M. Maggia, G. Minielloa,b, S. Mya,b, S. Nuzzoa,b, A. Pompilia,b, G. Pugliesea,c, R. Radognaa,b, A. Ranieria, G. Selvaggia,b, L. Silvestrisa,15, R. Vendittia,b, P. Verwilligena University of Delhi, Delhi, India Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, S. Keshri, A. Kumar, S. Mal- hotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma – 34 – INFN Sezione di Bolognaa, Universit`a di Bolognab, Bologna, Italy INFN Sezione di Bolognaa, Universit`a di Bolognab, Bologna, Italy INFN Sezione di Bologna , Universita di Bologna , Bologna, Italy G. Abbiendia, C. Battilana, D. Bonacorsia,b, S. Braibant-Giacomellia,b, L. Brigliadoria,b, R. Campaninia,b, P. Capiluppia,b, A. Castroa,b, F.R. Cavalloa, S.S. Chhibraa,b, G. Codispotia,b, M. Cuffiania,b, G.M. Dallavallea, F. Fabbria, A. Fanfania,b, D. Fasanellaa,b, P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria, F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,15 F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,15 NFN Sezione di Firenzea, Universit`a di Firenzeb, Firenze, Italy INFN Sezione di Firenzea, Universit`a di Firenzeb, Firenze, Italy G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, V. Goria,b, P. Lenzia,b, M. Meschinia, S. Paolettia, G. Sguazzonia, L. Viliania,b,15 G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, V. Goria P. Lenzia,b, M. Meschinia, S. Paolettia, G. Sguazzonia, L. Viliania,b,15 INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera15 INFN Sezione di Genovaa, Universit`a di Genovab, Genova, Italy V. Calvellia,b, F. Ferroa, M. Lo Veterea,b, M.R. Mongea,b, E. Robuttia, S. Tosia,b INFN Sezione di Milano-Bicoccaa, Universit`a di Milano-Bicoccab, Milano, Italy L. Brianza, M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, A. Ghezzia,b, P. Govonia,b, S. Malvezzia, R.A. Manzonia,b,15, B. Marzocchia,b, D. Menascea, L. Moronia, M. Paganonia,b, D. Pedrinia, S. Pigazzini, S. Ragazzia,b, T. Tabarelli de Fatisa,b JHEP08(2016)122 INFN Sezione di Napolia, Universit`a di Napoli ’Federico II’b, Napoli, Italy, Universit`a della Basilicatac, Potenza, Italy, Universit`a G. Marconid, Roma, Italy S. Buontempoa, N. Cavalloa,c, G. De Nardo, S. Di Guidaa,d,15, M. Espositoa S. Buontempoa, N. Cavalloa,c, G. De Nardo, S. Di Guidaa,d,15, M. Espositoa,b, F Fabozzia,c A O M Iorioa,b G Lanzaa L Listaa S Meolaa,d,15 M Merolaa S. Buontempoa, N. Cavalloa,c, G. De Nardo, S. Di Guidaa,d,15, M. Espositoa,b, F. Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,15, M. Merolaa, P P l a 15 C S a b F Th S. Buontempoa, N. Cavalloa,c, G. De Nardo, S. Di Guidaa,d,15, M. Espositoa,b, F. Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,15, M. Merolaa, P. Paoluccia,15, C. Sciaccaa,b, F. Thyssen F. Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,15, M. Merolaa, P. Paoluccia,15, C. Sciaccaa,b, F. Thyssen INFN Sezione di Padovaa, Universit`a di Padovab, Padova, Italy, Universit`a di Trentoc, Trento, Italy P. Azzia,15, L. Benatoa,b, D. Biselloa,b, A. Bolettia,b, R. Carlina,b, A. Carvalho Antunes De Oliveiraa,b, P. Checchiaa, M. Dall’Ossoa,b, P. De Castro Manzanoa, , , , , , Antunes De Oliveiraa,b, P. Checchiaa, M. Dall’Ossoa,b, P. De Castro Manzanoa, T. Dorigoa, U. Dossellia, F. Gasparinia,b, F. Gonellaa, S. Lacapraraa, M. Margonia,b, A T Meneguzzoa,b J Pazzinia,b,15 M Pegoraroa N Pozzobona,b P Ronchesea,b T. Dorigoa, U. Dossellia, F. Gasparinia,b, F. Gonellaa, S. Lacapraraa, M. Margonia,b, A.T. Meneguzzoa,b, J. Pazzinia,b,15, M. Pegoraroa, N. Pozzobona,b, P. Ronchesea,b, M. Sgaravattoa, F. Simonettoa,b, E. Torassaa, M. Zanetti, P. Zottoa,b, A. Zucchettaa,b, G. Zumerlea,b INFN Sezione di Paviaa, Universit`a di Paviab, Pavia, Italy INFN Sezione di Cataniaa, Universit`a di Cataniab, Catania, Ita INFN Sezione di Cataniaa, Universit`a di Cataniab, Catania, Italy S. Albergoa,b, M. Chiorbolia,b, S. Costaa,b, A. Di Mattiaa, F. Giordanoa,b, R. Potenzaa,b, A. Tricomia,b, C. Tuvea,b A. Tricomia,b, C. Tuvea,b – 35 – INFN Sezione di Firenzea, Universit`a di Firenzeb, Firenze, Italy INFN Sezione di Paviaa, Universit`a di Paviab, Pavia, Italy A. Braghieria, A. Magnania,b, P. Montagnaa,b, S.P. Rattia,b, V. Rea, C. Riccardia,b, P. Salvinia, I. Vaia,b, P. Vituloa,b INFN Sezione di Perugiaa, Universit`a di Perugiab, Perugia, Italy L. Alunni Solestizia,b, G.M. Bileia, D. Ciangottinia,b, L. Fan`oa,b, P. Laricciaa,b, R. Leonardia,b, G. Mantovania,b, M. Menichellia, A. Sahaa, A. Santocchiaa,b INFN Sezione di Pisaa, Universit`a di Pisab, Scuola Normale Superiore di Pisac, Pisa, Italy K. Androsova,30, P. Azzurria,15, G. Bagliesia, J. Bernardinia, T. Boccalia, R. Castaldia, M.A. Cioccia,30, R. Dell’Orsoa, S. Donatoa,c, G. Fedi, A. Giassia, M.T. Grippoa,30, F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy- Navarroa,31, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy- Navarroa,31, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia – 36 – INFN Sezione di Romaa, Universit`a di Romab, Roma, Italy L. Baronea,b, F. Cavallaria, M. Cipriania,b, G. D’imperioa,b,15, D. Del Rea,b,15, M. Diemoz S. Gellia,b, C. Jordaa, E. Longoa,b, F. Margarolia,b, P. Meridiania, G. Organtinia,b, R. Paramattia, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b INFN Sezione di Torinoa, Universit`a di Torinob, Torino, Italy, Universit`a del Piemonte Orientalec, Novara, Italy N. Amapanea,b, R. Arcidiaconoa,c,15, S. Argiroa,b, M. Arneodoa,c, N. Bartosik N. Amapanea,b, R. Arcidiaconoa,c,15, S. Argiroa,b, M. Arneodoa,c, N. Bartosika, R. Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, R. Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, L. Fincoa,b, B. Kiania,b, C. Mariottia, S. Maselli A. Deganoa,b, N. Demariaa, L. Fincoa,b, B. Kiania,b, C. Mariottia, S. Masellia, E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M.M. Obertinoa,b, L. Pachera,b, N. Pastronea, JHEP08(2016)122 E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M.M. Obertinoa,b, L. Pachera,b, N. Pastrone M. Pelliccionia, G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa,b, M. Ruspaa,c, R. Sacchia,b, K. Shchelinaa,b, V. Solaa, A. Solanoa,b, A. Staianoa, P. Traczyka,b INFN Sezione di Triestea, Universit`a di Triesteb, Trieste, Italy S. Belfortea, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, C. La Licataa,b, A. Schizzia,b, A. Zanettia Kyungpook National University, Daegu, Korea Warsaw, Poland K. Bunkowski, A. Byszuk35, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Misiura, M. Olszewski, M. Walczak Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal Kyungpook National University, Daegu, Korea D.H. Kim, G.N. Kim, M.S. Kim, S. Lee, S.W. Lee, Y.D. Oh, S. Sekmen, D.C. Son, Y.C. Yang Chonbuk National University, Jeonju, Korea H. Kim, A. Lee Chonbuk National University, Jeonju, Korea Chonbuk National University, Jeonju, Korea H. Kim, A. Lee y, j , H. Kim, A. Lee Hanyang University, Seoul, Korea J.A. Brochero Cifuentes, T.J. Kim Korea University, Seoul, Korea S. Cho, S. Choi, Y. Go, D. Gyun, S. Ha, B. Hong, Y. Jo, Y. Kim, B. Lee, K. Lee, K.S. Lee, S. Lee, J. Lim, S.K. Park, Y. Roh Seoul National University, Seoul, Korea J. Almond, J. Kim, S.B. Oh, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu y, , J. Almond, J. Kim, S.B. Oh, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu University of Seoul, Seoul, Korea M. Choi, H. Kim, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu M. Choi, H. Kim, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu Sungkyunkwan University, Suwon, Korea Sungkyunkwan University, Suwon, Korea Y. Choi, J. Goh, C. Hwang, D. Kim, J. Lee, I. Yu Y. Choi, J. Goh, C. Hwang, D. Kim, J. Lee, I. Yu Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus V. Dudenas, A. Juodagalvis, J. Vaitkus National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia I. Ahmed, Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali32, F. Mohamad Idris33, W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli – 37 – Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada, R. Lopez-Fernandez, J. Mejia Guisao, A. Sanchez-Hernandez g y , y, H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada, R. Lopez-Fernandez, J. Mejia Guisao, A. Sanchez-Hernandez Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, C. Oropeza Barrera, F. Vazquez Valencia Benemerita Universidad Autonoma de Puebla, Puebla, Mexico S. Carpinteyro, I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico A. Morelos Pineda JHEP08(2016)122 University of Auckland, Auckland, New Zealand D. Krofcheck University of Canterbury, Christchurch, New Zealand P.H. Butler National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, M.A. Shah, M. Shoaib, M. Waqas National Centre for Nuclear Research, Swierk, Poland National Centre for Nuclear Research, Swierk, Poland National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrock K. Romanowska-Rybinska, M. Szleper, P. Zalewski Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland Moscow, Russia A. Baskakov, A. Belyaev, E. Boos, M. Dubinin41, L. Dudko, A. Ershov, A. Gribushin, V. Klyukhin, O. Kodolova, I. Lokhtin, I. Miagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev Institute for Theoretical and Experimental Physics, Moscow, Russia Institute for Theoretical and Experimental Physics, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, V. Popov, I. Pozdnyakov, G. Safronov, A. Spiridonov, M. Toms, E. Vlasov, A. Zhokin National Research Nuclear University ‘Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia M. Chadeeva40, M. Danilov40, O. Markin P.N. Lebedev Physical Institute, Moscow, Russia V. Andreev, M. Azarkin37, I. Dremin37, M. Kirakosyan, A. Leonidov37, S.V. Rusakov, A. Terkulov JHEP08(2016)122 Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho, M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Rodrigues Antunes, J. Seixas, O. Toldaiev, D. Vadruccio, J. Varela, P. Vischia Joint Institute for Nuclear Research, Dubna, Russia S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Kar- javin, A. Lanev, A. Malakhov, V. Matveev36,37, P. Moisenz, V. Palichik, V. Perelygin, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, N. Voytishin, A. Zarubin Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia L. Chtchipounov, V. Golovtsov, Y. Ivanov, V. Kim38, E. Kuznetsova39, V. Murzin, V. Oreshkin, V. Sulimov, A. Vorobyev Institute for Nuclear Research, Moscow, Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov, N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin Institute for Nuclear Research, Moscow, Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsano N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin – 38 – State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, D. Elumakhov, V. Kachanov, A. Kalinin, D. Kon- stantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P. Adzic42, P. Cirkovic, D. Devetak, J. Milosevic, V. Rekovic Centro de Investigaciones Energ´eticas Medioambientales y Tecnol´ogicas (CIEMAT), Madrid, Spain (CIEMAT), Madrid, Spain , , , , , , A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quin- tario Olmeda, I. Redondo, L. Romero, M.S. Soares Universidad Aut´onoma de Madrid, Madrid, Spain J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad Aut´onoma de Madrid, Madrid, Spain J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad de Oviedo, Oviedo, Spain J. Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, E. Palencia Cortezon, S. Sanchez Cruz, J.M. Vizan Garcia Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain I.J. Cabrillo, A. Calderon, J.R. Casti˜neiras De Saa, E. Curras, M. Fernandez, J. Garcia- Ferrero, G. Gomez, A. Lopez Virto, J. Marco, C. Martinez Rivero, F. Matorras, J. Piedra – 39 – Gomez, T. Rodrigo, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte Gomez, T. Rodrigo, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte CERN, European Organization for Nuclear Research, Geneva, Switzerland D. Abbaneo, E. Auffray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Barney, P. Bloch, A. Bocci, A. Bonato, C. Botta, T. Camporesi, R. Castello, M. Cepeda, G. Cermi- nara, M. D’Alfonso, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola, F. De Guio, A. De Roeck, E. Di Marco43, M. Dobson, M. Dordevic, B. Dorney, T. du Pree, D. Duggan, M. D¨unser, N. Dupont, A. Elliott-Peisert, S. Fartoukh, G. Franzoni, J. Fulcher, W. Funk, D. 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Xu Purdue University Calumet, Hammond, U.S.A. N. Parashar, J. Stupak Rice University, Houston, U.S.A. Rice University, Houston, U.S.A. A. Adair, B. Akgun, Z. Chen, K.M. Ecklund, F.J.M. Geurts, M. Guilbaud, W. Li, B. Michlin, M. Northup, B.P. Padley, R. Redjimi, J. Roberts, J. Rorie, Z. Tu, J. Zabel University of Rochester, Rochester, U.S.A. University of Rochester, Rochester, U.S.A. B. Betchart, A. Bodek, P. de Barbaro, R. Demina, Y.t. Duh, T. Ferbel, M. Galanti, A. Garcia-Bellido, J. Han, O. Hindrichs, A. Khukhunaishvili, K.H. Lo, P. Tan, M. Verzetti Rutgers, The State University of New Jersey, Piscataway, U.S.A. J.P. Chou, E. Contreras-Campana, Y. Gershtein, T.A. G´omez Espinosa, E. Halkiadakis, M. Heindl, D. Hidas, E. Hughes, S. Kaplan, R. Kunnawalkam Elayavalli, S. Kyriacou, J.P. Chou, E. Contreras-Campana, Y. Gershtein, T.A. Gomez Espinosa, E. Halkiadak M. Heindl, D. Hidas, E. Hughes, S. Kaplan, R. Kunnawalkam Elayavalli, S. Kyriaco – 45 – A. Lath, K. Nash, H. Saka, S. Salur, S. Schnetzer, D. Sheffield, S. Somalwar, R. Stone, S. Thomas, P. Thomassen, M. Walker University of Tennessee, Knoxville, U.S.A. M. Foerster, J. Heideman, G. Riley, K. Rose, S. Spanier, K. Thapa Texas A&M University, College Station, U.S.A. Woods †: Deceased 1: Also at Vienna University of Technology, Vienna, Austria 2: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China 3: Also at Institut Pluridisciplinaire Hubert Curien, Universit´e de Strasbourg, Universit´e de Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France 3: Also at Institut Pluridisciplinaire Hubert Curien, Universit´e de Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 5: Also at Centre National de la Recherche Scientifique (CNRS) — IN2P3, Paris, France 5: Also at Centre National de la Recherche Scientifique (CNRS) — IN2P3, Paris, France 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 8: Also at Joint Institute for Nuclear Researc 9: Also at Helwan University, Cairo, Egypt 9: Also at Helwan University, Cairo, Egypt 10: Now at Zewail City of Science and Technology, Zewail, Egypt 10: Now at Zewail City of Science and Technology, Zewail, Egypt 11: Also at Ain Shams University, Cairo, Egypt 11: Also at Ain Shams University, Cairo, Egypt 12: Also at Fayoum University, El-Fayoum, Egypt 12: Also at Fayoum University, El-Fayoum, Egypt 13: Now at British University in Egypt, Cairo, Egypt 13: Now at British University in Egypt, Cairo, Egypt 14: Also at Universit´e de Haute Alsace, Mulhouse, France 14: Also at Universit´e de Haute Alsace, Mulhouse, France – 46 – 15: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 16: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 17: Also at RWTH Aachen University, III. Texas A&M University, College Station, U.S.A. Physikalisches Institut A, Aachen, Germany 18: Also at University of Hamburg, Hamburg, Germany 19: Also at Brandenburg University of Technology, Cottbus, Germany 20: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 21: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University, Budapest, Hungary 22: Also at University of Debrecen, Debrecen, Hungary 23: Also at Indian Institute of Science Education and Research, Bhopal, India JHEP08(2016)122 24: Also at Institute of Physics, Bhubaneswar, India 25: Also at University of Visva-Bharati, Santiniketan, India 26: Also at University of Ruhuna, Matara, Sri Lanka 27: Also at Isfahan University of Technology, Isfahan, Iran 28: Also at University of Tehran, Department of Engineering Science, Tehran, Iran 28: Also at University of Tehran, Department of Engineering 29: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Aza University, Tehran, Iran 30: Also at Universit`a degli Studi di Siena, Siena, Italy 31: Also at Purdue University, West Lafayette, U.S.A. 31: Also at Purdue University, West Lafayette, U.S.A. 32: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 32: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 33: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 34: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 35: Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Polan 36: Also at Institute for Nuclear Research, Moscow, Russia 36: Also at Institute for Nuclear Research, Moscow, Russia t Institute for Nuclear Research, Moscow, Russia 37: Now at National Research Nuclear University ’Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia ( ) 38: Also at St. Petersburg State Polytechnical University, St.˜Petersburg, Russia 38: Also at St. Petersburg State Polytechnical University, St.˜Petersburg, Russia 39: Also at University of Florida, Gainesville, U.S.A. 40: Also at P.N. Lebedev Physical Institute, Moscow, Russia 41: Also at California Institute of Technology, Pasadena, U.S.A. Texas A&M University, College Station, U.S.A. 42: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 42: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 43: Also at INFN Sezione di Roma, Universit`a di Roma, Roma, Italy at National Technical University of Athens, Athe 44: Also at National Technical University of Athens, Athens, Greece 45: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy 46: Also at National and Kapodistrian University of Athens, Athens, Greece 47: Also at Riga Technical University, Riga, Latvia 48: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 49: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 49: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 50: Also at Adiyaman University, Adiyaman, Turkey 51: Also at Mersin University, Mersin, Turkey 52: Also at Cag University, Mersin, Turkey 53: Also at Piri Reis University, Istanbul, Turkey 53: Also at Piri Reis University, Istanbul, Turkey 54: Also at Gaziosmanpasa University, Tokat, Turkey 54: Also at Gaziosmanpasa University, Tokat, Turkey 55: Also at Ozyegin University, Istanbul, Turkey 55: Also at Ozyegin University, Istanbul, Turkey 56: Also at Izmir Institute of Technology, Izmir, Turkey 56: Also at Izmir Institute of Technology, Izmir, Turkey 57: Also at Marmara University, Istanbul, Turkey 57: Also at Marmara University, Istanbul, Turkey 58: Also at Kafkas University, Kars, Turkey 59: Also at Istanbul Bilgi University, Istanbul, Turkey 59: Also at Istanbul Bilgi University, Istanbul, Turkey – 47 – 60: Also at Yildiz Technical University, Istanbul, Turkey 61: Also at Hacettepe University, Ankara, Turkey 62: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 63: Also at School of Physics and Astronomy, University of Southampton, Southampton, Unit Kingdom 64: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 65: Also at Utah Valley University, Orem, U.S.A. 66: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia 67: Also at Facolt`a Ingegneria, Universit`a di Roma, Roma, Ital Also at Argonne National Laboratory, Argonne JHEP08(2016)122 69: Also at Erzincan University, Erzincan, Turkey 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 71: Also at Texas A&M University at Qatar, Doha, Qatar 72: Also at Kyungpook National University, Daegu, Korea – 48 –
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Coupling Mechanism and Coupling Degree Measurement Model of Shipbuilding Industry Cluster
Polish Maritime Research
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POLISH MARITIME RESEARCH Special Issue 2016 S1 (91) 2016 Vol. 23; pp. 78-85 10.1515/pomr-2016-0049 POLISH MARITIME RESEARCH Special Issue 2016 S1 (91) 2016 Vol. 23; pp. 78-85 10.1515/pomr-2016-0049 POLISH MARITIME RESEARCH Special Issue 2016 S1 (91) 2016 Vol. 23; pp. 78-85 10.1515/pomr-2016-0049 COUPLING MECHANISM AND COUPLING DEGREE MEASUREMENT MODEL OF SHIPBUILDING INDUSTRY CLUSTER AN Xiao li a) School of Economics and Management, Harbin Engineering University,P.R.China,150001 b) School of Economics and Management, Tsinghua University,P.R.China,10010 ABSTRACT This article was aimed to analyse the working condition of the coupling mechanism in Chinese shipbuilding industry cluster. Literature review was conducted to unveil the coupling mechanism of shipbuilding industry cluster, providing analysis of the present development of shipbuilding industry. A theoretical model was proposed by using the efficacy function and the capacity of the coupling coefficient while measuring the coupling of the shipbuilding industry cluster. Four coupling characteristics were found including relevant, systemic, diversity, and collaborative in the shipbuilding industry cluster. The shipbuilding industry cluster has two levels of coupling mechanism including the core layer coupling mechanism and the support layer coupling mechanism respectively. The improvement through coupling the core layer, support layer, and the coupling between two levels, is an effective way to achieve long-term stable development in Chinese shipbuilding industry cluster. Keywords: coupling mechanism, coupling degree measurement model, shipbuilding industry cluster INTRODUCTION greater contribution to military and civilian. Shipbuilding industry cluster is an effective way for the development of shipbuilding industry as a strategic tool (Huang, 2006)[1]. Scholars focus on the studies on development elements (Cao and Liu, 2007)[2], development models (Tao and Yang, 2007; Li, 2008; Hu and Yu, 2013)[3-5], the formation mechanism of shipbuilding industrial cluster (Tao, 2005; Zhao, 2007; Wang, 2010)[6-8], and the life cycle of South Korea’s shipbuilding industry cluster (Shin et al., 2011)[9], shipbuilding industry cluster development theoretical model (Zong, 2013)[10], development opportunities and challenges of shipbuilding industry cluster (Zhang and Zeng, 2014)[11], etc. However, the literatures rarely involve the study of the complex mechanism of the shipbuilding industry cluster. The coupling theory provides a new theoretical support for this field of research. Chinese Shipbuilding Industry is the supporting industry of Chinese maritime transportation, ocean development and national defense construction, and strategic industry of combination of military and civilian. It has important significance to the national defense capability and the national economic growth. The construction of shipbuilding industrial cluster is an effective way to enhance the competitiveness of shipbuilding industry, and the appropriate coupling mechanism can make positive synergy effect on shipbuilding industry cluster. The industrial clusters of shipbuilding industry can take appropriate measures to improve the coupling degree to make the cluster maintain high coupling operation in a state of highly efficient operation, and make POLISH MARITIME RESEARCH, No S1/2016 78 The coupling theory is applied to the study of the complex relationships within the systems, which is mainly used in manufacturing industry and other related fields. Li and Li (2005) presented that the equipment manufacturing industry cluster coupling contains three layers coupling mechanisms, namely the manufacturing enterprise’s coupling mechanism, coupling mechanism of industry cluster and regional social network coupling mechanism, proposed the coupling degree model of equipment manufacturing industry cluster, analyzed the influence factors of equipment manufacturing industry cluster coupling degree and coupling mechanism, and finally made empirical analysis on Shenyang equipment manufacturing industry cluster[12]. Ruan and Zuan (2010) put forward the coupling degree model of manufacturing industry cluster, and made empirical analysis of the coupling degree on Qingdao manufacturing industry cluster[13]. Yuan et al. (2012) divided the network structure of the manufacturing industry into core manufacturing layer, industry matching layer, service support layer and analyzed coupling mechanism among layers[14]. Fu et al. INTRODUCTION (2012) made the research on the coupling of multiple wastewater using network design to achieve agile manufacturing[16]. Du et al. (2012) established the coupling coordination model about the industrial technology innovation ability. The model mainly includes three basic parts, the efficiency function, the coupling degree model and the evaluation index system. This coupling coordination model was used to evaluate whether the coal resource cities should develop some leading industries, and took Qitaihe as an example[17].Liu and Sun (2014) established the coupling degree measurement model of knowledge innovation and technology innovation in high-end equipment manufacturing industry, and proved the validity of the coupling degree measure model from the empirical point of view[15]. Zou et al. (2014) analyzed the development trend of the equipment manufacturing industry in Liaoning by using factor analysis method, and proposed the coupling development “Trilogy” of technology innovation and technological upgrading of Liaoning province equipment manufacturing industry combined with technological innovation and technological upgrading coupling improvement mechanism, respectively, exploration and development model, the leading domestic development model and international development model[18].The research of the coupling mechanism and coupling degree measurement model of shipbuilding industry cluster can provide theoretical guidance and decision-making basis for the industrial cluster of shipbuilding industry, which has important significance to both theoretical and practical development. DEVELOPMENT STATUS QUO OF CHINESE SHIPBUILDING INDUSTRY completion. The market share of China was more than 45% both in the year of 2013 and 2014, followed by South Korea and Japanese with the market share of about 30% and 20% respectively. hi The new received orders in China kept the world’s first in 2013 and 2014. From the perspective of market share, China, South Korea and Japan are the world shipbuilding countries led by China. China’s shipbuilding industry development is quite superior and stability. Fig.1. Market share of major shipbuilding countries in the world in 2013 Fig.1. Market share of major shipbuilding countries in the world in 2013 Fig.1. Market share of major shipbuilding countries in the world in 2013 Fig.2. Market share of major shipbuilding countries in the world in 2014 Fig.2. Market share of major shipbuilding countries in the world in 2014 DEVELOPMENT STATUS QUO OF CHINESE SHIPBUILDING INDUSTRY From 2001 to 2014, China’s annual shipbuilding output , the new received orders, the overall holding orders had showed a rising trend, and reached the highest value in 2012, 2007 and 2008, respectively. China’s annual shipbuilding output had been declining in the last three years, but the degree of declining has slowed. The number of new received orders had increased significantly, and then fell slightly. China’s annual shipbuilding output, the new received orders, and the overall holding orders had been showing a fluctuation states, however the general trends are still upward. INDUSTRIAL CONCENTRATION HAS BEEN INCREASED, AND THE COMPREHENSIVE STRENGTH HAS BEEN GREATLY IMPROVED On one hand, from the ecological point of view, the shipbuilding industry cluster is a industry species ecological system; on the other hand, from the innovation economics point of view, shipbuilding industry cluster is a special kind of regional innovation system[19].This paper argues that the shipbuilding industry cluster refers to taking ship manufacturing enterprises as the center, and united the related enterprises or organizations of ship industry, such as: supporting enterprises, universities, research institutions, vocational training institutions and shipbuilding industry association, gathering together to form an organic system in a particular geographic location [8][20]. The industry cluster, which has complete industrial structure, robust support system, can be formed through specialization and depth cooperation of subjects within the cluster. Initially coupling is a physical concept, which has been widely used in the field of system science and social sciences. The concept of coupling is used to explore or describe the relationship, mutual influence and common cooperation and evolution process of various systems. In physics, coupling refers to the phenomenon that two or more than two of the circuit elements or the input and output of the electrical network are closely matched with each other, and the transmission of energy from one side to the other side through the interaction. In this article, the coupling is defined as a dynamic relationship formed by positive interaction, matching and coordination among system elements or subsystems to achieve the coordinated development. The coupling of Chinese shipbuilding industry cluster is a kind of dynamic relationship and synergistic action process among the related enterprises and organizations of shipbuilding industry. The coupling of shipbuilding industry cluster is not only coordination among the agents, but is also on collaborative development, so as to achieve the purposes, such as complementary advantages, mutual promotion and coordinated development. In the year of 2014, there were more than 90 percent orders has been completed in the three major shipbuilding bases, which were in the place of the Bohai Bay region, Yangtze River Delta and Pearl River Delta. The concentration degree of top 10 enterprises, top 20 enterprises and top 30 enterprises was up to 50.6%, 71.6% , 83.8%, respectively, which increased 3.2%,5.9% and 7.4% respectively comparing to the year of 2013. This means the degree of industrial concentration has been significantly improved. INDUSTRIAL CONCENTRATION HAS BEEN INCREASED, AND THE COMPREHENSIVE STRENGTH HAS BEEN GREATLY IMPROVED There has been formed a group of key enterprises in China, and four of them are the world’s top ten enterprises, in terms of new orders. We can obtain that Chinese shipbuilding enterprises’ market competitiveness and the international status had been improved. CHINA REMAINED THE WORLD’S LARGEST SHIPBUILDING COUNTRY The data of Figure 1 and 2 are obtained from China Association of the National Shipbuilding Industry Website. The new orders of China, South Korea, and Japan already occupied more than 90% of the world shipbuilding total 79 POLISH MARITIME RESEARCH, No S1/2016 MARKET DEMAND IS NOT BALANCED, AND THE DEVELOPMENT POTENTIAL IS SLIGHTLY INADEQUATE In the year of 2014, China’s new receive orders quantity maintained a high level, which was about 60 million deadweight tons. The quantity of new receive orders was showing a quarterly decline trend, which was 25 million deadweight tons, 14 million deadweight tons, 11 million deadweight tons and 7 million deadweight tons, from first quarter to the fourth quarter, respectively. Except the first quarter, the situation of shipbuilding enterprises had deteriorated significantly in the year of 2014. The main reason is as following: first of all, shipping market supply and demand contradictions problems have not been fundamentally resolved, and the order quantity is gradually reduced. Secondly, as a significant reduction in international oil prices, which dampened the enthusiasm of the enterprise to manufacture energy-saving ships, and postponed dismantling of old ships, the market demand for new ships continued to decrease. In recent years, the Chinese shipbuilding outputhas reached 20 million tons. Facing the depresseding market, China’s shipbuilding industry capacity utilization efficiency is not high, and does not reach a reasonable level of the industry. THE COUPLING CHARACTERISTICS OF SHIPBUILDING INDUSTRY CLUSTER Shipbuilding industry cluster has the characteristics of relevance, systematic, diversity, collaborative etc. Firstly, correlation. The coupling agents including core ship manufacturing enterprises, the ship supporting enterprises, the ship parts production enterprises, and the service organizations are interrelated and interact with each other. Shipbuilding industry cluster is not a closed static system. Obviously, Chinese shipbuilding industry cluster has the prerequisites for application of coupling theory. Secondly, systematic. The main subjects or elements in the coupling system form a new system through cooperating with each other according to certain rules. The main bodies of Chinese shipbuilding industry cluster form the new system of shipbuilding industry cluster through cooperating with each other according to the rules and regulations, such as cooperation contract, contract etc. Thirdly, diversity. Each INDUSTRIAL CONCENTRATION HAS BEEN INCREASED, AND THE COMPREHENSIVE STRENGTH HAS BEEN GREATLY IMPROVED Unique symbiotic cluster of shipbuilding industry enterprises was formed through the exchange of knowledge, information, human resources, equipment and capital of each subject in the cluster. On one hand, from the ecological point of view, the shipbuilding industry cluster is a industry species ecological system; on the other hand, from the innovation economics point of view, shipbuilding industry cluster is a special kind of regional innovation system[19].This paper argues that the shipbuilding industry cluster refers to taking ship manufacturing enterprises as the center, and united the related enterprises or organizations of ship industry, such as: supporting enterprises, universities, research institutions, vocational training institutions and shipbuilding industry association, gathering together to form an organic system in a particular geographic location [8][20]. The industry cluster, which has complete industrial structure, robust support system, can be formed through specialization and depth cooperation of subjects within the cluster. Initially coupling is a physical concept, which has been widely used in the field of system science and social sciences. The concept of coupling is used to explore or describe the relationship, mutual influence and common cooperation and evolution process of various systems. In physics, coupling refers to the phenomenon that two or more than two of the circuit elements or the input and output of the electrical network are closely matched with each other, and the transmission of energy from one side to the other side through the interaction. In this article, the coupling is defined as a dynamic relationship formed by positive interaction, matching and coordination among system elements or subsystems to achieve the coordinated development. The coupling of Chinese shipbuilding industry cluster is a kind of dynamic relationship and synergistic action process among the related enterprises and organizations of shipbuilding industry. The coupling of shipbuilding industry cluster is not only coordination among the agents, but is also on collaborative development, so as to achieve the purposes, such as complementary advantages, mutual promotion and coordinated development. Unique symbiotic cluster of shipbuilding industry enterprises was formed through the exchange of knowledge, information, human resources, equipment and capital of each subject in the cluster. THE COUPLING CONNOTATION OF SHIPBUILDING INDUSTRY CLUSTER As a typical large-scale equipment manufacturing industry, shipbuilding industry enterpriseshave many characteristics such as long industrial chain involved and high requirements for supporting industry [4].A mature shipbuilding industry cluster should include the shipping core manufacturing enterprises, the ship supporting enterprises, the ship parts production enterprises, and the service oriented institutions. POLISH MARITIME RESEARCH, No S1/2016 80 enterprises, the ship matching enterprises, and the ship core manufacturing enterprises are closely linked to form a vertical industrial chain. coupled subsystem has its own organizational characteristics. The links between the elements of the subsystems are complicated, which constitute a variety of coupling modes. Each subject of shipbuilding industry cluster has its own organization and management mode and characteristics, so the links of enterprises and support service agencies are very complex, and the relationship between them is dynamic. Finally, collaborative. The coupled subsystems in shipbuilding industry cluster interact with each other through the way of complementary advantages to achieve the collaborative development of the entire system [21]. Fig.3. The coupling structure of shipbuilding industry cluster core layer THE COUPLING MECHANISM OF SHIPBUILDING INDUSTRY CLUSTER Large ship manufacturing enterprises are the core of agglomeration of Chinese shipbuilding industry cluster, and a series of related businesses(ship matching enterprises, ship parts production enterprises and shipbuilding supporting enterprise) and service organizations (such as scientific research institutions, intermediary institutions, government agencies, educational institutions, and shipbuilding industry association) are derived from the large ship manufacturing enterprise. These service institutions provide the related services, such as technical, information, policy guidance and coordination for the cluster. Chinese Shipbuilding Industry Cluster coupling structure contains two levels of coupling mechanisms: the core layer coupling mechanism, and the support layer coupling mechanism[12]. The coupling of the shipbuilding industry cluster is the complex combination of the two levels of coupling mechanisms rather than a simple superposition. The two coupling layers are closely linked to each other based on knowledge sharing and economic transactions, but independent of each other. The coupling dynamics of one level will have an effect on the other coupling layer, thus affecting the overall function of the cluster. A positive effect is produced by the good coupling of two levels, to promote the formation of a high coupling degree of the entire cluster, so as to maintain sustainable competitiveness. Fig.3. The coupling structure of shipbuilding industry cluster core layer Fig.3. The coupling structure of shipbuilding industry cluster core layer The main contents of the core layer coupling mechanism are ship manufacturing enterprises, which are the core of the shipbuilding industry cluster and play the role of the components supplied by the supporting enterprises to be assembled into the ship. The matching enterprises and the ship spare parts manufacturing enterprises provide the ship core manufacturing enterprises with spare parts and necessary components of the ship. Of course, the ship spare parts production enterprises also provide spare parts for the ship matching enterprises. Shipbuilding supply enterprises provide the cores of ship manufacturing enterprises, the ship matching enterprises and ship spare parts production enterprises with all kinds of raw materials for shipbuilding. These adaptation stages are the initial stage of coupling, and the closely and efficiently cooperation and interaction among the enterprises are the key points. Each enterprise needs follow-up and support of other links as key link in the vertical industry chain, and the benign interaction among enterprises to produce the adhesive effect, so as to achieve the coordinated COUPLING MECHANISM OF SUPPORT LAYER Fig.4 .The support coupling layer structure of shipbuilding industrial cluster CONSTRUCTION OF COUPLING DEGREE MEASUREMENT MODEL OF SHIPBUILDING INDUSTRY CLUSTER Previous research illustrates the coupling mechanism of shipbuilding industry cluster. In reality, it is necessary to measure the coupling degree of shipbuilding industry cluster scientifically in order to clear the coupling condition of shipbuilding industrial cluster. Then relevant institutions can take pertinent measures to improve cluster coupling degree. Construction process of coupling degree measurement model of shipbuilding industry cluster is as follows. Fig.4 .The support coupling layer structure of shipbuilding industrial cluster Fig.4 .The support coupling layer structure of shipbuilding industrial cluster Support coupling mechanism is also the outer coupling mechanism, mainly provides technology information, resource, policy guidance and other services for shipbuilding industry cluster. The coupling layer mainly includes the relation and coupling between some support service institutions and the inner cluster. The inner cluster includes the shipping core manufacturing enterprises, the ship matching enterprises, the ship spare parts production enterprises and the shipbuilding supply enterprises. This coupling layer is important, but can easily be neglected. Support service institutions, which are parts of support coupling layer, include government agencies, intermediary institutions, scientific research institutions, education institutions, financial institutions and shipbuilding industry association, etc. In general, these support service institutions support cluster inner layer. Support coupling layer structure is as follows.h COUPLING MECHANISM OF CORE LAYER The core layer coupling mechanism is mainly the coupling of the core ship manufacturing enterprises, the ship matching enterprises, the ship spare parts manufacturing enterprises and the shipbuilding supply enterprises. In this coupling layer, the cooperation of the enterprises is not only between supply and demand, but the matching of the upstream and downstream enterprises in the product, service and manpower, etc, based on the complete vertical industrial chain. Industrial connection is a prerequisite for the coupling of industry cluster, and the information flow in unobstructed and timely manner are the conditions of realizing the benign coupling. Shipbuilding industry cluster is a typical vertical industry chain agglomeration model, which is longer, involving a wide range of services, with high requirements for matching services. The ship spare parts manufacturing POLISH MARITIME RESEARCH, No S1/2016 81 services for the cluster, playing the role of middle bridge and avoiding low coupling degree of cluster caused by the non-smooth information. Scientific research institutions include universities and research institutes, which are the most important innovation institutions of knowledge and technology. Shipbuilding industry cluster inner layer can cooperate with scientific research institutions, such as forming the Research Alliance to continue to learn new technologies, to absorb shipbuilding talents. Scientific research institutions become the birthplace of the high- tech and technical talents of the cluster inner layer in this way. Education and training institutions train managers, so cluster coupling degree is improved by the managers by making the scientific management more effectively in the cluster inner layer. Financial institutions provide funding for enterprises. Shipbuilding industry associations offer a variety of social services for shipbuilding industry cluster, and maintain the legitimate rights of interests of the member units and the industry. The overall effect is much greater than their respective effect, resulting in efficiency overflow proliferation and positive effect on the whole cluster, thus forming a virtuous circle, expanding the overall positive effect of the cluster, so as to achieve benign coupling by support service institutions coordinating with cluster inner layer, and producing synergistic effect in dynamic interaction. development and the coupling. Good coupling mechanism can promote the formation of positive coupling effect bundle. The positive effect of the coupling effect bundle is amplified to achieve positive cycle. COUPLING MECHANISM OF SUPPORT LAYER EFFICIENCY FUNCTION OF SHIPBUILDING INDUSTRY CLUSTER Set variable (i=1,2,...,n) is the order parameter of the shipbuilding industry cluster. The core ship manufacturing enterprise, the ship matching enterprise, the ship spare parts manufacturing enterprise, the shipbuilding supply enterprise, and support service institution are the five order parameters. uij is the j-index of i-parameter, the value is Xij (i=1,2,..., n). Aij, Bij are the upper and lower limits of order parameter of shipbuilding industry cluster steady state. And efficiency function of the core ship manufacturing enterprise, the ship matching enterprise, the ship spare parts manufacturing enterprise, the shipbuilding supply enterprise, and support service institution orderly to shipbuilding industrial cluster system can be expressed by: The main contents of support layer coupling mechanism are government agencies involving in shipbuilding industrial cluster coupled by providing policy support, macro-environment creation, etc. for cluster inner layer. Government agencies play a guiding role on the behavior of all the subjects in the cluster inner layer by industrial preferential policies, tax policies to bring the cluster to benign coupling. Intermediary agencies provide information uij has a negative effect uij has a positive effect (1) (1) POLISH MARITIME RESEARCH, No S1/2016 82 The uij is the efficiency contribution size of variable Xij to the system. uij represents the satisfaction degree of each index reaching the goal, uij is between 0 and 1, reaching to 0 for the most unsatisfied, closing to 1 for the most satisfactory. The core ship manufacturing enterprise, the ship matching enterprise, the ship spare parts manufacturing enterprise, the shipbuilding supply enterprise and support service institution are mutually connected and influenced subsystems, the total contribution of each index to subsystems is achieved by integrated method, and the linear weighted method is generally used[22]. same. Therefore, it is obviously not a good reflection of the shipbuilding industry cluster synergy and the overall effect only using the coupling degree to evaluate the development of the cluster. In this case, the significance of conclusion by using the coupling degree to the development of cluster is not obvious. The modified coupling degree measurement model of shipbuilding industry cluster is[25-27]: (4) (4) (2) (2) where, C is coupling degree; C’ is modified coupling degree; T is subsystem comprehensive harmonic index of shipbuilding industrial cluster, which reflects the overall synergistic effect of shipbuilding industrial cluster; a, b, c, etc. are undetermined coefficients, which can determined by absorbing the experts’ advice. The modified coupling is divided into four levels. CONCLUSIONS Through the review of the prior literature on the Chinese shipbuilding industrial cluster and manufacturing, this paper focuses on the current development of the Chinese shipbuilding industry and related concepts. The shipbuilding industrial cluster includes two basic coupling mechanism layers: the core layer and support layer on top of the prior papers, this paper developed a new coupling degree measurement model In order to achieve the long-term stable development of the shipbuilding industry cluster, the following aspects were proposed. (3) (3) According to the above equation, the core layer consists of four sub-systems, namely the core ship manufacturing enterprise, the ship matching enterprise, the ship spare parts manufacturing enterprise, the shipbuilding supply enterprise. Support layer consists of five subsystems, namely the core ship manufacturing enterprise, the ship matching enterprise, the ship spare parts manufacturing enterprise, the shipbuilding supply enterprise and support service institution. The core layer coupling degree measurement model and the support layer coupling degree measurement model are constructed with subsystem number instead of n of the formula. The value of C is between 0 and 1, 1 represents subsystem coupled with maximum degree and the coupling condition of subsystems benign. 0 represents subsystems coupled with minimum degree, and the subsystems are independent. First of all, the ship matching enterprises play an important role in the shipbuilding industry cluster. Accelerated development of matching industries is useful for improving the coupling degree of the core layer, promoting the development of the shipbuilding industry cluster. When the matching capacity cannot catch up with the development of the shipbuilding industry, the ship manufacturing enterprises will not carry out effective production activities, in other words, the core layer coupling degree of shipbuilding industry cluster is delayed by the supporting layer. The development of matching industries can be accelerated by implementing professional operation, e.g. establishing the main auxiliary instrument, machinery and other professional company; concentrating financial resources, material resources, manpower to engage in specialized production[6].Specialized production can be applied to improve the matching capacity of shipbuilding industry cluster, to promote the establishment of a good coupling mechanism. Coupling degree C is an important parameter to judge the coupling state of the subsystems, but it is difficult to reflect the overall effect of the cluster coupling in some cases, especially in the cluster contrast study. Due to the development strategy, the characteristics of each shipbuilding industry cluster are different. EFFICIENCY FUNCTION OF SHIPBUILDING INDUSTRY CLUSTER Coupling layer of shipbuilding industrial cluster is in the low coordination coupling stage, if 0 < C’ ≤ 0.4. Coupling layer of shipbuilding industrial cluster is in the moderate coordination coupling stage, if 0.4 < C’ ≤ 0.6. Coupling layer of shipbuilding industrial cluster is in the high coordination coupling stage, if 0.6 < C’ ≤ 0.8. Coupling layer of shipbuilding industrial cluster is in the extreme coordination coupling stage, if 0.8 < C’ ≤ 1. (2) ui is the contribution of subsystem to total system order degree. m is the number indicators of subsystem, λij is index weights. The weights of each index can be determined by analytic hierarchy process and entropy weight method[22] [23]. COUPLING DEGREE FUNCTION OF SHIPBUILDING INDUSTRY CLUSTER The calculation of the coupling degree of shipbuilding industry cluster draw lessons from the concept of capacity coupling and capacity coupling coefficient model in physics[24]. In this paper, it is extended to the coupling degree measurement model of multiple systems. REFERENCE Secondly, the support layer coupling can be strengthened by enhancing the flow of knowledge and technological innovation of the shipbuilding industry cluster. As the current spillover of the Chinese shipbuilding industry is still at a lower level, the coupling degree of the support layer is restricted at relative lower level. It is obviously that the improvement of knowledge spillover of shipbuilding industry cluster can optimize the coupling mechanism of the cluster, and promote the development of coupling of shipbuilding industry. Technology innovation can save the cost of shipbuilding for the cluster, but also can make up the in shortage of raw materials in shipbuilding, so that the cluster is more competitive. The knowledge creation and flow can be strengthened through enhancing cooperation of the industry and universities, increasing the flow of talents and enhancing the sharing degree of infrastructure facilities[3].h 1. Huang Jin. Chinese shipbuilding industry cluster development stratify[D].Hunan University.2006.(in Chinese) 2. Cao Yousheng,Liu Xisong. Research on elemental factors and countermeasures in promoting cluster of China shipbuilding industry[J].Ship Building of China,2007(3):91- 95. (in Chinese) 3. Tao Yonghong,Yang Haisong. On development mode and strategy of shipbuilding industrial cluster in Yangtze River Delta[J].Journal of Jiangsu University of Science and Technology(Social Science Edition), 2007(6):33-38. (in Chinese) g g g Thirdly, the two level coupling state of cluster can be promoted by taking measures together and developing producer service industry. Chinese shipbuilding industry can better achieve structural adjustment, and the cluster can better maintain a good coupling state by speeding up the development of producer service industry. Efficient logistic services, perfect marketing and after-sale service network systems are good signs of the core layer coupling status, sound financial services system is a good performance of the support layer coupling status. Accordingly, the coupling degree of the two layers can be improved by the following ways, so that the cluster can maintain a high degree of coupling status. First, the cluster can establish information and intelligent logistic service systems by making full use of modern information technology. Second, it can build brands, and improve market share by establishing and improving the global after-sale services and marketing network systems. Third, Cluster can improve the construction of the ship financial services system to provide better financial support for the development of the shipbuilding industry cluster by strengthening cooperation with domestic and foreign financial institutions, developing multi-channel financing, and improving financing means. 4. Li Hao. CONCLUSIONS For example, development stages of cluster A and cluster B are different, but the coupling degree is the POLISH MARITIME RESEARCH, No S1/2016 83 REFERENCE Research on the Yangtze River Delta ship industry cluster development[D].Lanzhou University.2009. (in Chinese) 5. Hu Wei,Yu Yingying. Research on the development model and strategic of Wuhu ship industrial cluster[J].Machine Design and Manufacturing Engineering,2013(3):22-25. (in Chinese) 6. Tao Yonghong. 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This work was supported by National Natural Science Foundation of China(71403066), Doctoral Fund of Ministry of Education of China(20122304120021), China Postdoctoral Science Foundation(2013T60351), Heilongjiang Province Postdoctoral Start Foundation (LBH-Q13050), the Special Foundation of Central Universities Basic Research Fee (HEUCF140907). 12. Li Kai,Li Shijie. Coupling structure of equipment manufacture industrial clusters: a new view on the industrial clusters[J].China Industrial Economy, 2005(2):51- 57. (in Chinese) POLISH MARITIME RESEARCH, No S1/2016 84 13. Ruan Yanyan,Qi Zhenfa. The study of coupling degree of manufacturing clusters[J].Journal of Shandong University of Technology(Natural Science Edition),2010(11):107-110. (in Chinese) 25. Kowalski, Jakub; Lesniewski, Wojciech; Litwin, Wojciech. 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Direct in situ photolithography of perovskite quantum dots based on photocatalysis of lead bromide complexes
Nature communications
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cc-by
10,945
Direct in situ photolithography of perovskite quantum dots based on photocatalysis of lead bromide complexes Pingping Zhang1, Gaoling Yang 2 , Fei Li3, Jianbing Shi1 & Haizheng Zhong 1 Photolithography has shown great potential in patterning solution-processed nanomaterials for integration into advanced optoelectronic devices. However, photolithography of perovskite quantum dots (PQDs) has so far been hindered by the incompatibility of perovskite with traditional optical lithography pro- cesses where lots of solvents and high-energy ultraviolet (UV) light exposure are required. Herein, we report a direct in situ photolithography technique to pattern PQDs based on the photopolymerization catalyzed by lead bromide complexes. By combining direct photolithography with in situ fabrication of PQDs, this method allows to directly photolithograph perovskite precursors, avoiding the complicated lift-off processes and the destruction of PQDs by solvents or high-energy UV light, as PQDs are produced after lithography exposure. We further demonstrate that the thiol-ene free-radical photo- polymerization is catalyzed by lead bromide complexes in the perovskite precursor solution, while no external initiators or catalysts are needed. Using direct in situ photolithography, PQD patterns with high resolution up to 2450 pixels per inch (PPI), excellent fluorescence uniformity, and good stability, are successfully demonstrated. This work opens an avenue for non-destructive direct photolithography of high-efficiency light-emitting PQDs, and potentially expands their application in various integrated optoelectronic devices. Perovskite quantum dots (PQDs) exhibit narrow photoluminescence (PL) emission with high quantum yields which is tunable over the entire visible range, have shown great potential in a range of optoelectronic applications1–4. In particular, there is intense interest in applying PQDs in displays5–8. Despite the great progress, PQDs based light-emitting diodes (LEDs) still suffer from some issues that prevent their incor- poration into commercial display products such as cell phones or lap- tops. One of them is patterning, multicolor LEDs have been hindered by the difficulty in patterning red, green, and blue (RGB) PQD films into arrays of individual devices rather than bulk films9–12. Therefore, the capability of patterning PQDs into pixel arrays is urgently needed. Over the past few years, various patterning techniques have been developed, including inkjet printing13, nanoimprinting14, laser direct writing15, photolithography16, and so on. Among all these methods, photolithography has attracted considerable attention owing to its high resolution, wide availability, and high throughput. There are two cate- gories of this technique, conventional photoresist-assisted method, and direct photolithography. The former uses the conventional lift-off technique where the photoresist is first patterned before QDs are deposited17–20. Article https://doi.org/10.1038/s41467-022-34453-9 Description of direct in situ photolithography method Cross-section transmis- sion electron microscopy (TEM) measurement was performed to clearly demonstrate the QD structure of perovskite, and exhibit their homo- geneous dispersion in micropatterns, as shown in Fig. 1c. High crystal- linity and continuous lattice fringes can be seen clearly from further high-resolution TEM (HRTEM) image in Fig. 1d, the lattice constant of 0.301 nm is consistent with (002) interplanar spacing of MAPbBr3. The bright and uniform PQD-polymer films can be ascribed to the good protection of polymer matrix on the exposed region, which was proved by the calculation of the retention of PQDs defined as the percentage of PQDs retained in the exposed films after developing24. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) and induc- tively coupled plasma-mass spectroscopy (ICP-MS) were used for the analysis of Pb atom in the developer solvent and polymer film to determine the retention of PQDs. And the figure was recorded as up to 85%, which accounts for the good protection effect of polymer on perovskite thus obtaining high luminescent patterns. Exploration of photochemical mechanism In general, external initiators or catalysts are essential for poly- Most of these photolithography technologies are based on pho- topolymerization, which is the curing process of monomers triggered by photosensitizers or photoinitiators exposed to light. Recently, perovskite has shown great promise as an efficient catalyst31,32, and is used to directly initiate the photopolymerization process for the fab- rication of various PQD-polymer compounds, such as PQD-polymethyl methacrylate33, PQD-polystyrene34, and PQD-polyaniline35. However, these photopolymerizations can be easily inhibited in the air due to the quenching effect of oxygen on carbon radicals36. While the thiol-ene reaction is insensible to oxygen and high-efficiency, which has been applied in photolithography for microdevice fabrication37. A typical radical thiol-ene reaction often starts from thiyl radicals, then they attack double bonds to generate carbon radicals, which in turn react with thiol monomers to release thiyl radicals, resulting in the chain growth and transfer38. The insensitivity to oxygen comes from that the ready hydrogen abstraction of peroxy radicals from thiols to generate active thiyl radicals, which have the ability to continue the free-radical chain process39. Thioethers have been photosynthesized with PQDs as a photocatalyst40. However, most reactions with PQDs as a photo- catalyst normally involve blue or white light irradiation for a long time of several hours which may also potentially destroy the PQDs41 and are hard to incorporate existing photolithography process. Description of direct in situ photolithography method Description of direct in situ photolithography method Fine PQDs patterns were fabricated from the original perovskite pre- cursor solution with monomers by direct in situ photolithography method. As shown in Fig. 1a, prior to photolithography, the glass sub- strate is cleaned by sonication in various solvents, then silane coupling agents are used to functionalize the substrate with ethenyl or thiol groups exposing. Such functionalized substrate creates strong covalent bonding sites for the resultant polymer film, which is critical to the successful patterning of perovskite films. The patterning of PQDs can start from simply casting the photosensitive perovskite precursor resist (PPR) directly onto the functionalized substrate. The PPR is key to the direct in situ photolithography, which is prepared by dissolving reagent salts, multifunctional thiol and ethenyl monomers in polar aprotic sol- vents. Specifically, for green PPR, MABr (MA = methylammonium) and PbBr2 are chosen as perovskite precursor salts, which have been proved to be very effective reagents to generate perovskite. For monomers, trimethylolpropane tris (3-mercaptopropionate) (TTMP), a multi-thiol crosslinking reagent, is used as a thiol monomer, while triallyl iso- cyanurate (TAIC) provides electron-rich vinyl groups. To dissolve per- ovskite precursor and monomers better, polar aprotic solvents such as N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) are the best choice. Unlike previous methods, direct in situ photolithography approach directly patterns original perovskite precursors instead of prepared QD inks, which often need extensive cleaning steps and can be destroyed under high-energy UV light in the presence of oxygen51,52. Second, perovskite precursor film is exposed with UV light through a photomask. Upon UV irradiation, a photochemically activated reaction between TTMP and TAIC occurs, leading to the solidification of the PPR in the exposed area. Third, the unexposed PPR is removed by spin- washing with chloroform as a developer. Cured products on exposed areas can adhere to the substrate strongly during spin-washing because of the covalent interactions formed between functionalized substrates and TTMP or TAIC in PPRs, making the developing process much easier. Finally, the prepared perovskite precursor patterns are directly annealed in the ambient air to evaporate residual solvents, when the concentration reaches its critical value for in situ nucleation, lumines- cent perovskite patterns are formed. Figure 1b shows the corresponding fluorescence image of the patterned arrays of square with a size of 30 μm on a substrate, the patterned pixel arrays emit bright and uni- form green fluorescence under UV irradiation. Direct in situ photolithography of perovskite quantum dots based on photocatalysis of lead bromide complexes However, PQDs are easily dissolved in polar solvents and re-dispersed in nonpolar solvents due to their ionic nature, making them usually incompatible with traditional photolithography processes 1MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081, China. 2MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Optics and Photonics, Beijing Institute of Technology, Beijing 100081, China. 3QD LAB, Hefei Innovation Research Institute of Beihang University, Hefei, Anhui 230001, China. e-mail: glyang@bit.edu.cn 1MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081, China. 2MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Optics and Photonics, Beijing Institute of Technology, Beijing 100081, China. 3QD LAB, Hefei Innovation Research Institute of Beihang University, Hefei, Anhui 230001, China. e-mail: glyang@bit.edu.cn Nature Communications| (2022) 13:6713 Nature Communications| (2022) 13:6713 Nature Communications| (2022) 13:6713 1 Article https://doi.org/10.1038/s41467-022-34453-9 synthesis strategy, clearly provides an efficient platform for the man- ufacture of high-resolution patterned optoelectronic devices. where a lot of solvents are needed in photoresists, developers, etchants, or strippers11. Alternatively, direct photolithography is quite simple and effective, which patterns photosensitive QDs directly through the light- induced change in their solubility21. Recently, several research groups presented high-resolution QD patterns based on direct photo- lithography with different photochemistry mechanisms, such as unsa- turated double bond cross-linking22–24, azide cross-linking24–27, polarity change28–30, and so on. Description of direct in situ photolithography method Hence, PQDs may not be the best option for photocatalysis during the existing photolithography process. Additionally, in the existing direct photolithography, almost all photosensitive QDs are prepared through the hot injection approach and subsequent ligand exchange method beforehand, which usually involve high reaction temperature, harsh inert gas environment, complex synthesis steps, and extensive cleaning steps. In addition, these photosensitive QDs suffer from preserving their optical prop- erties in the lithography exposure process, especially for PQDs. In situ fabrication has been proved to be a powerful method to synthesize PQDs directly on a substrate or in a matrix42, including polymer matrix43, glass matrix44, molecular sieves45, or crystals46, due to the low formation enthalpy and high defect tolerant of ionic perovskite47,48. Recently, in situ fabrication has been utilized to provide patterned PQDs pixels with tunable luminescence for integrating into optoelec- tronic devices49,50. Thus motivated, here we propose a direct photolithography method to pattern in situ fabricated PQDs based on polymerization catalyzed by lead bromide complexes. This method produces high- resolution patterns by direct photolithography of perovskite pre- cursors using UV light to trigger the photopolymerization, PQDs are in situ fabricated in polymer by final annealing, avoiding high-energy UV light to destroy PQDs since they were produced after exposure. In particular, we demonstrate that the photopolymerization under UV irradiation is based on lead bromide complexes catalyzing the thiol- ene free-radical reaction, without any external initiators or catalysts that are harmful to the stability of patterns. Furthermore, uniform and residual-free luminescent pixel arrays are fabricated via direct photo- lithography of in situ fabricated PQDs, in which the high uniformity of emission intensities reflects the homogeneous distribution of PQDs. Lastly, we successfully fabricate red, green and blue PQD patterns, the minimum feature size of 5 μm corresponding to a resolution of up to 2450 pixels per inch (PPI), the maximal thickness of 10 μm enables to absorb blue or UV light more efficiently when used as color convention pixels. The direct photolithography method combined with in situ Exploration of photochemical mechanism p p In general, external initiators or catalysts are essential for poly- merization, but things are different in our case. It seems perovskite precursors have the ability of photocatalysis, which makes direct Nature Communications| (2022) 13:6713 2 https://doi.org/10.1038/s41467-022-34453-9 Article Casting UV exposure Developing Annealing In situ crystallization Photopolymerization MABr + PbBr2 + monomers in DMF and (or) DMSO Silane coupling agent MA + Br − Pb 2+ Monomer Polymer PQD PPR Developer d (002) = 0.301 nm 2 nm 200 nm 90 μm a b c d g. 1 | Schematic illustration of PQD patterning method. a Schematic descrip- n of the direct in situ photolithography method from the perovskite precursor ist (PPR), annealing refers to heating the samples at a specific temperature. Fluorescence image of green MAPbBr3 PQD patterns (30 μm size square) fabricated by the direct in situ photolithography method. c Cross-section TEM image of MAPbBr3 PQD patterns fabricated by the direct in situ photolithograp method. d HRTEM image of single MAPbBr3 PQD in the patterns fabricated by t direct in situ photolithography method. Casting UV exposure Developing Annealing In situ crystallization Photopolymerization MABr + PbBr2 + monomers in DMF and (or) DMSO PPR Developer a a Developer Casting Annealing Developing MABr + PbBr2 + monomers in DMF and (or) DMSO Photopolymerization In situ crystallization MABr + PbBr2 + monomers in DMF and (or) DMSO Silane coupling agent Monomer 90 μm b b fabricated by the direct in situ photolithography method. c Cross-section TEM image of MAPbBr3 PQD patterns fabricated by the direct in situ photolithography method. d HRTEM image of single MAPbBr3 PQD in the patterns fabricated by the direct in situ photolithography method. Fig. 1 | Schematic illustration of PQD patterning method. a Schematic descrip- tion of the direct in situ photolithography method from the perovskite precursor resist (PPR), annealing refers to heating the samples at a specific temperature. b Fluorescence image of green MAPbBr3 PQD patterns (30 μm size square) 100 s irradiation (Fig. 2b), confirming the thiol-ene photopolymeriza- tion system. The extent of polymerization was also determined by FT- IR spectra, where 83% and 84% were ascribed to the conversion rate of the ethenyl and thiol group, suggesting the nearly 1:1 stoichiometry rection (Supplementary Fig. 2). Furthermore, various monomers with varying quantities of functional groups (Supplementary Fig. Exploration of photochemical mechanism 3a, b) were also examined under the same reaction conditions, all the com- binations were cured within 30 min under a low power UV light (10 mW cm−2) (Supplementary Fig. 3c and Supplementary Table 2), demonstrating the universality of thiol-ene reactions photocatalyzed by perovskite precursors. in situ photolithography of PQDs possible. We seek to understand the photocatalysis mechanism by investigating the type of polymerization, photocatalytic species, and reactive site. To verify the photocatalysis of perovskite precursors, equal volumes of monomer systems with and without perovskite precursors were irradiated under 365 nm UV light for 2 min. As can be seen in Fig. 2a, with perovskite precursors, soli- dified gelatin was formed at the bottom of the bottle, while without precursors, the ink was still liquid, appreciably illustrating the photo- catalysis of perovskite precursors. To further explore whether the photopolymerization was conducted by self-polymerization31,33 or copolymerization37, monomer systems either with one single mono- mer (ethenyl or thiol) or with both of them mixing with perovskite precursor were irradiated under UV light. Evidently, the monomer system containing both ethenyl and thiol monomers was cured within 30 s (entry 1 in Supplementary Table 1 and Supplementary Fig. 1), while for a single monomer, no polymerization occurred (entries 2 and 3 in Supplementary Table 1 and Supplementary Fig. 1). This indicates that the PPR underwent the thiol-ene reaction. Additionally, this reaction system was further verified by Fourier-transform infrared (FT-IR) spectra of the PPR before and after UV irradiation, both peaks of –CH=CH2 (3080 cm−1) and –SH (2527 cm−1) reduced dramatically after in situ photolithography of PQDs possible. We seek to understand the photocatalysis mechanism by investigating the type of polymerization, photocatalytic species, and reactive site. To verify the photocatalysis of perovskite precursors, equal volumes of monomer systems with and without perovskite precursors were irradiated under 365 nm UV light for 2 min. As can be seen in Fig. 2a, with perovskite precursors, soli- dified gelatin was formed at the bottom of the bottle, while without precursors, the ink was still liquid, appreciably illustrating the photo- catalysis of perovskite precursors. To further explore whether the photopolymerization was conducted by self-polymerization31,33 or copolymerization37, monomer systems either with one single mono- mer (ethenyl or thiol) or with both of them mixing with perovskite precursor were irradiated under UV light. Exploration of photochemical mechanism Therefore, we speculated that the photopolymerization in this system may be catalyzed by the lead bromide complexes. To confirm the catalytic capacity of lead bromide complexes, PPRs with different bromide concentrations were tested. As can be seen in Fig. 3a, with the increase of [MABr], the curing time dramatically reduced, with a maximum increase in catalytic effect by a factor of ten. Since high coordinated lead bromide complexes are formed at high bromide concentration, which can be confirmed by the redshift of absorption (Supplementary Fig. 10a)56, the enhanced catalytic effect at high [MABr] can imply the stronger catalytic capacity of high coordi- nated lead bromide complexes. To further demonstrate that, per- ovskite precursors were dissolved in solvents with different coordination abilities (DMSO > DMF > GBL (γ-butyrolactone))55. As solvent molecules can compete with halides for coordination with lead ions, when the coordination ability of a solvent was reduced, the coordination of halide and Pb2+ would increase to form lead halide complexes with high coordination numbers57, which can be proved by the redshift of their absorption spectra (Supplementary Fig. 10b). As shown in Fig. 3b, when the curing yields after 2 min 30 s UV light irradiation were plotted against the decreasing coordination ability of solvents, a clear upward trend emerged (0% for DMSO, 20% for DMF, 70% for complex DMF/GLB), strongly suggesting the key photo- catalytic role of high coordinated lead bromide complexes. In per- ovskite precursor solutions, PbBr2, PbBr3 ‒, PbBr4 2‒ have been reported in the literature56, while PbBr2 has been excluded in the previous experiment, we speculated that high coordinated PbBr4 2‒ is the key photocatalyst in this reaction. To prove that, UV lights with various wavelengths were used, since lead bromide complexes with different coordination numbers show different absorption of light at different wavelengths56,58. As shown in Fig. 3c, when exposed under 311 nm UV light close to the absorption peak of PbBr3 ‒, an acceptable exposure dose for completely curing were required, combining with the abso- lutely predominated concentration of PbBr3 ‒ in the PPR, indicating a relatively weak catalytic effect of PbBr3 ‒. When replacing with 365 nm UV light close to the absorption peak of PbBr4 2‒, only half exposure dose was needed even under a relatively low concentration, implying the strong photocatalytic capacity of PbBr4 2‒. Exploration of photochemical mechanism c EPR spectra of the PPR collected after 0 min and 2 min UV irradiation. Source data are provided as a Source Data file. 3100 2600 2500 2400 Absorbance (arb. u.) Wavenumbers (cm−1) PPR Cured PPR b With precursors Without precursors 1 cm a Gelatin Liquid 3460 3480 3500 3520 3540 3560 Intensity (arb. u.) Magnetic field (G) 0 min 2 min c b c a spectra of the perovskite precursor resist (PPR) and cured PPR after 365 nm UV light (5 W) irradiation for 100 s. c EPR spectra of the PPR collected after 0 min and 2 min UV irradiation. Source data are provided as a Source Data file. Fig. 2 | Investigation of the photopolymerization type. a Photograph of the resultant inks without (left side) and with (right side) perovskite precursors in the monomer system after irradiating with 365 nm UV light (20 W) for 2 min. b FT-IR The ink with HBr had a comparable curing rate to that with MABr (Supplementary Fig. 9), along with the photocatalysis capacity of PbBr2 demonstrated before, which absolutely excluded the photocatalysis effect of PQDs in our case. These results suggest that the primary catalyst of this reaction is neither from any single reagent salts and their constituent ions in the perovskite precursor solution nor PQDs. system is not the thiol-Michael addition mechanism. When a negligible amount of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a free- radical inhibitor, no polymerization happened under the same UV light environment (entries 6 vs 4 in Supplementary Table 3 and Supple- mentary Fig. 4), confirming the inhibiting effect of the free-radical inhibitor for this reaction system. To further confirm the presence of free radicals, electron paramagnetic resonance (EPR) spectroscopy measurement was carried out. A clear EPR signal assigned to carbon radicals was detected after 2 min irradiation of PPR using a UV light source (Fig. 2c), unambiguously proving the growth and transfer of polymer chain was initiated by free radicals. Together, both the inhi- bition reaction and EPR results strongly confirm the free-radical reac- tion pathway. Perovskite precursor solutions are actually colloid54, where halides and solvent molecules coordinate with lead ions to obtain a variety of lead halide complexes PbXn 2−n (X = Cl, Br or I; n = the coor- dination number of halide), all the various complexes are in dynamic equilibrium55. Exploration of photochemical mechanism Evidently, the monomer system containing both ethenyl and thiol monomers was cured within 30 s (entry 1 in Supplementary Table 1 and Supplementary Fig. 1), while for a single monomer, no polymerization occurred (entries 2 and 3 in Supplementary Table 1 and Supplementary Fig. 1). This indicates that the PPR underwent the thiol-ene reaction. Additionally, this reaction system was further verified by Fourier-transform infrared (FT-IR) spectra of the PPR before and after UV irradiation, both peaks of –CH=CH2 (3080 cm−1) and –SH (2527 cm−1) reduced dramatically after 100 s irradiation (Fig. 2b), confirming the thiol-ene photopolymeriza- tion system. The extent of polymerization was also determined by FT- IR spectra, where 83% and 84% were ascribed to the conversion rate of the ethenyl and thiol group, suggesting the nearly 1:1 stoichiometry rection (Supplementary Fig. 2). Furthermore, various monomers with varying quantities of functional groups (Supplementary Fig. 3a, b) were also examined under the same reaction conditions, all the com- binations were cured within 30 min under a low power UV light (10 mW cm−2) (Supplementary Fig. 3c and Supplementary Table 2), demonstrating the universality of thiol-ene reactions photocatalyzed by perovskite precursors. Generally, the thiol-ene reaction has two polymerization mechanisms: thiol-Michael addition which is catalyzed mainly by bases or nucleophiles53, and thiol-ene free-radical addition which is induced by photoinitiators or high-energy UV light irradiation37. To unravel the polymerization mechanism of the PPRs, proton source and free-radical inhibitors were added, which can effectively inhibit the thiol-Michael addition and free-radical addition, respectively. When a proton source such as HCl was added in the PPR, the cured polymer was obtained after 19 s irradiation using a 365 nm UV light source (entries 5 vs 4 in Supplementary Table 3 and Supplementary Fig. 4), implying this Nature Communications| (2022) 13:6713 3 https://doi.org/10.1038/s41467-022-34453-9 Article 3100 2600 2500 2400 Absorbance (arb. u.) Wavenumbers (cm−1) PPR Cured PPR With precursors Without precursors 1 cm 3460 3480 3500 3520 3540 3560 Intensity (arb. u.) Magnetic field (G) 0 min 2 min b c a Gelatin Liquid Fig. 2 | Investigation of the photopolymerization type. a Photograph of the resultant inks without (left side) and with (right side) perovskite precursors in the monomer system after irradiating with 365 nm UV light (20 W) for 2 min. b FT-IR spectra of the perovskite precursor resist (PPR) and cured PPR after 365 nm UV light (5 W) irradiation for 100 s. Exploration of photochemical mechanism At 405 nm, the y We were intrigued by the unexpected photocatalytic ability of perovskite precursors and curious about which part plays a major role. Firstly, to explore whether the reagent salts MABr and PbBr2 were the primary catalyst, a series of control experiments were performed with various constituents. We found that nearly all the liquid was cured in 6 min when MABr and PbBr2 were used together (entry 7 in Supple- mentary Table 4 and Supplementary Fig. 5a), reaction conducted using PbBr2 also gave cured products but at a low yield after 20 min irra- diation (entry 8 in Supplementary Table 4 and Supplementary Fig. 5b), no cured product appeared when MABr was used (entry 9 in Supple- mentary Table 4 and Supplementary Fig. 5b). This result implies the fast catalytic effect of the mixture of MABr and PbBr2, the slow cata- lytic effect of PbBr2, and excludes the catalytic possibility of MABr, MA+ and Br– in this system. Obviously, PbBr2 does not play a major catalytic role in this photocatalytic reaction, but it does show some catalytic effect, so we wondered if Pb2+ might be the catalyst. Therefore, we examined its catalytic ability by changing the halides from Br to I and Cl. However, no cured product was obtained under UV light (311, 365, and 405 nm) irradiation (entries 11 and 12 vs 10 in Supplementary Table 5, Supplementary Fig. 6), excluding the catalytic effect of Pb2+ either. Since the PQDs have been reported to be used as photocatalyst for thiol-ene reactions under blue light irradiation40, it is easy to relate the photocatalytic effect to the PQDs. However, both the PL and ultraviolet-visible (UV–Vis) spectra (Supplementary Fig. 7) of the PPR exhibit distinctly different emission and large Stokes shift compared with the typical PQDs, demonstrating that there was no PQD generated from PPR no matter how long the solution was stirred. All the PPRs with different stirring time can be cured within 2 min (the left five in Sup- plementary Fig. 8), while no cured product was found in the ink using PQDs as photoinitiator (the rightest in Supplementary Fig. 8), which at least indicates that PQDs cannot photocatalyze the polymerization as efficiently as the perovskite precursors. Furthermore, MABr was sub- stituted by HBr to eliminate A site ion that is necessary for the fabri- cation of perovskite, avoiding any possibility to produce perovskite. Exploration of photochemical mechanism f PLE (solid line) and PL (dot-dash line) spectra of the perovskite precurso solution (PPS) and PPR. g Proposed photochemical mechanism of lead bromide complexes catalyzed initiation process of thiol-ene free-radical addition, L stands for a solvent molecule. Source data are provided with this paper. Electron ho Pb Br Br Br B LUMO HOMO UV light O Pb Br Br Br Br L H S Pb Br Br Br H Br S δ+ δ− Coordination g Electron hole transfer H Pb Br Br Br Br S δ+ δ− h+ e− LUMO HOMO e− UV light O2 O2·− r H Br S δ+ δ− ation Pb Br Br Br Br L H+ S· Initiation Electron hole transfer H Pb Br Br Br Br S δ+ δ− h+ e− LUMO HOMO e− UV light O2 O2·− Pb Br Br Br Br L H S Pb Br Br Br H Br S δ+ δ− Coordination Pb Br Br Br Br L H+ S· Initiation g Electron hole transfer wavelengths (line graph corresponding to the right axis). EPR spectra of the inks respectively mixed by d TAIC and e TTMP with the same perovskite precursor solution. f PLE (solid line) and PL (dot-dash line) spectra of the perovskite precursor solution (PPS) and PPR. g Proposed photochemical mechanism of lead bromide complexes catalyzed initiation process of thiol-ene free-radical addition, L stands for a solvent molecule. Source data are provided with this paper. Fig. 3 | Exploration of the photochemical mechanism. a Curing time of per- ovskite precursor resists (PPRs) with an increasing ratio of MABr to PbBr2 at a constant concentration of PbBr2. b Curing yield of PPRs with different solvents at constant concentrations of all reagent salts and monomers. c Exposure dose of UV lights with different wavelengths to cure the PPR completely (histogram corre- sponding to the left axis) and absorbance of the PPR at different UV light the perovskite precursor solution (Fig. 3e), while no carbon radical was detected when TAIC was used (Fig. 3d). The EPR measurements sug- gest that the monomers catalyzed by lead bromide complexes are thiols. Together, all these results confirm that the sulfur–lead coordi- nation bond is the reactive site. absorbance is nearly 0, indicating the low concentration of higher coordinated lead bromide complexes. Exploration of photochemical mechanism [MABr]:[PbBr2] molar radio 0:1 0.5:1 1:1 2:1 4:1 0 2 4 6 8 10 Curing time (min) DMSO DMF DMF&GBL 0.0 0.2 0.4 0.6 0.8 Solvent Curing ratio a b [MABr]:[PbBr2] molar radio 0:1 0.5:1 1:1 2:1 4:1 0 2 4 6 8 10 Curing time (min) DMSO DMF DMF&GBL 0.0 0.2 0.4 0.6 0.8 Solvent Curing ratio Ab b ( b ) a b c Exposure dose (J cm‒2) 311 nm 365 nm 405 nm 0 3 6 9 12 15 18 UV light 0 2 4 6 8 10 [MABr]:[PbBr2] molar radio 0:1 0.5:1 1:1 2:1 4:1 0 2 4 6 8 10 Curing time (min) a DMSO DMF DMF&GBL 0.0 0.2 0.4 0.6 0.8 Solvent Curing ratio b Absorbance (abs. u.) c Exposure dose (J cm‒2) 311 nm 365 nm 405 nm 0 3 6 9 12 15 18 UV light 0 2 4 6 8 10 b c a 3460 3480 3500 3520 3540 Intensity (arb. u.) Magnetic field (G) 0 min 8 min f PL intensity (arb. u.) 320 380 440 500 560 620 0 50 100 150 200 250 0 50 100 150 200 250 PPS PPS PPR PPR PLE intensity (arb. u.) Wavelength (nm) d 3460 3480 3500 3520 3540 3560 Intensity (arb. u.) Magnetic field (G) 0 min 8 min f d Intensity (arb. u.) e e PLE intensity (arb. u.) Intensity (arb. u.) Wavelength (nm) Magnetic field (G) Wavelength (nm) g Electron hole transfer H Pb Br Br Br Br S δ+ δ− h+ e− LUMO HOMO e− UV light O2 O2·− Pb Br Br Br Br L H S Pb Br Br Br H Br S δ+ δ− Coordination Pb Br Br Br Br L H+ S· Initiation g Fig. 3 | Exploration of the photochemical mechanism. a Curing time of per- ovskite precursor resists (PPRs) with an increasing ratio of MABr to PbBr2 at a constant concentration of PbBr2. b Curing yield of PPRs with different solvents at constant concentrations of all reagent salts and monomers. c Exposure dose of UV lights with different wavelengths to cure the PPR completely (histogram corre- sponding to the left axis) and absorbance of the PPR at different UV light wavelengths (line graph corresponding to the right axis). EPR spectra of the inks respectively mixed by d TAIC and e TTMP with the same perovskite precursor solution. Exploration of photochemical mechanism Nature Communications| (2022) 13:6713 4 https://doi.org/10.1038/s41467-022-34453-9 Article Electron hole transfer H Pb Br Br Br Br S δ+ δ− h+ e− LUMO HOMO e− UV light O2 O2·− Pb Br Br Br Br L H S Pb Br Br Br H Br S δ+ δ− Coordination Pb Br Br Br Br L H+ S· Initiation g [MABr]:[PbBr2] molar radio 3460 3480 3500 3520 3540 Intensity (arb. u.) Magnetic field (G) 0 min 8 min 0:1 0.5:1 1:1 2:1 4:1 0 2 4 6 8 10 Curing time (min) DMSO DMF DMF&GBL 0.0 0.2 0.4 0.6 0.8 Solvent Curing ratio Absorbance (abs. u.) e f d a b c 3460 3480 3500 3520 3540 3560 Intensity (arb. u.) Magnetic field (G) 0 min 8 min PL intensity (arb. u.) 320 380 440 500 560 620 0 50 100 150 200 250 0 50 100 150 200 250 PPS PPS PPR PPR PLE intensity (arb. u.) Wavelength (nm) Exposure dose (J cm‒2) 311 nm 365 nm 405 nm 0 3 6 9 12 15 18 UV light 0 2 4 6 8 10 ig. 3 | Exploration of the photochemical mechanism. a Curing time of per- vskite precursor resists (PPRs) with an increasing ratio of MABr to PbBr2 at a onstant concentration of PbBr2. b Curing yield of PPRs with different solvents at onstant concentrations of all reagent salts and monomers. c Exposure dose of UV ghts with different wavelengths to cure the PPR completely (histogram corre- ponding to the left axis) and absorbance of the PPR at different UV light wavelengths (line graph corresponding to the right axis). EPR spectra of the inks respectively mixed by d TAIC and e TTMP with the same perovskite precursor solution. f PLE (solid line) and PL (dot-dash line) spectra of the perovskite precursor solution (PPS) and PPR. g Proposed photochemical mechanism of lead bromide complexes catalyzed initiation process of thiol-ene free-radical addition, L stands for a solvent molecule. Source data are provided with this paper. Demonstration of PQD patterns via direct in situ photolithography p g p y Through the direct in situ lithography method, we fabricated a series of micropatterns from mesoscale to microscale. Figure 4a shows the fluorescence microscope image of 20 μm blue stripes with high con- trast between the bright blue and dark states, exhibiting clearly defined and sharp edges. The scanning electron microscope (SEM) image further demonstrates the smooth surface and high contrast between the exposed and unexposed regions in these pattern arrays (Fig. 4b). Figure 4c and Supplementary Fig. 13a show the corre- sponding cross-section SME image, the sharp and clean edges indicate that all the exposed line patterns were cured and the unexposed parts were removed completely. The thickness of patterns was adjustable by altering UV exposure time (Supplementary Fig. 13a), and a propor- tional relationship was recorded (Supplementary Fig. 13b). The inset image of the enlarged view of a single stripe (Fig. 4c) shows the thickness of patterns was more than 10 μm, which is much larger than most of the patterns within 1 μm obtained by conventional patterning processes. The absorbance spectra of the PQD-polymer films were shown in Supplementary Fig. 14, the large absorption suggests these thick films enable UV or blue light to be fully absorbed and blocked to improve energy efficiency and avoid the leakage of light60. We further demonstrated the luminescence uniformity of PQD patterns using a fluorescence microscope. Figure 4d depicts the emission intensity of a PQD array composed of 196 circle pixels with a diameter of 20 μm, desirable fluorescence homogeneity across all pixels can be seen. The corresponding statistical histogram of their fluorescence intensity is shown in Fig. 4e, about 70% of pixels were distributed in the range of 2000–2200, indicating the good luminescence uniformity of the PQD pixel arrays. Moreover, the radial and surface fluorescence intensity distribution of an individual 20 μm circle pixel were shown in Fig. 4f, illustrating the great fluorescence homogeneity of each pixel in PQD patterns. The 3D fluorescence image of 50 μm blue stripe patterns further confirmed the uniformity of each stripe while no residue can be seen at the unexposed regions (Supplementary Fig. 15), verifying the high efficiency and feasibility of the direct in situ photolithography. The higher-resolution images were created, including circle and square patterns with a size of 10 μm in Supplementary Fig. 21a and Supplementary Fig. 21b, circle patterns with a diameter of 5 μm in Fig. Demonstration of PQD patterns via direct in situ photolithography 5f, corresponding to the resolution of up to 2540 PPI, sufficiently enabling for the resolution of augmented reality (AR) and virtual reality (VR) display. This resolution was limited by the apparatus we used, whose minimum feature size is 5 μm, the method can be extended to fabricate higher resolution patterns by using advanced apparatus. We further evaluated the applicability of this method to various substrates which were functioned with VTMS or MPTS to enable ethenyl or thiol groups exposing, the PPR can react with these active groups to form covalent bonding under UV exposure. Clear patterns were formed not only on rigid substrates, such as glasses and wafers (Fig. 5h), but also on flexible substrates like polyethylene terephthalate (PET) (Fig. 5g, Supplementary Fig. 21c), implying broader integration of the direct in situ photolithography method. What’s more, owing to no external initiators involved and effective polymer encapsulation, the fabricated PQD patterns exhibited good stability, which is highly important for further applications. The films with and without 2 w% initiators (1- hydroxycyclohexyl phenyl ketone) were evaluated with respect to UV and heat. Supplementary Fig. 22a shows the sample with initiators dropped to 7% of the original PL intensity after continuous six-day irradiation, while the one without initiators remained basically unchanged despite fluctuations. Under the heat of 60 °C, the PL intensity of the sample with initiators decreased by 82% in the first 16 h and continued dropping to 6% of the origin, while the figure for that without initiators fluctuated and remained the origin intensity until the end of test for 64 h (Supplementary Fig. 22b). In addition, at ambient temperature and normal atmosphere with an average humidity of 54%, the PL intensity of the sample without initiators can maintain 85% of the origin after 30 days (Supplementary Fig. 22c). The good stability of the PQDs might benefit from the effective encapsulation of polymer to discourage PQDs from ion migration, crystal aggregation, as well as permeation of O2 and moisture65. By contrast, the films with external initiators may generate radicals upon irradiation, which quenched PQDs easily. Furthermore, harsher solvent tests were performed to confirm the good protection of the polymer. As can be seen in Fig. 5h and Supplementary Fig. Exploration of photochemical mechanism Colorful PQDs (Fig. 5e and Supplementary Fig. 19) were also fabricated on a substrate by using SiO2 as an intermediate layer between different layers to efficiently protect the preceding layer from being destroyed by DMF and DMSO. The UV-Vis absorbance, PL emission and time- resolved PL spectra of the patterned film with stripes of 100 μm period (Fig. 5d) are shown in Supplementary Fig. 20. The peak wavelengths, PL quantum yields (QYs) and average PL lifetimes are 464 nm, 17% and 36.4 ns for blue patterns, 521 nm, 87% and 40.3 ns for green patterns, 638 nm, 58% and 61.6 ns for red patterns, respectively. A mechanism of in situ PQD fabrication can account for the good performance: a polymer matrix forms before the perovskite nucleation; the developer acts as an antisolvent to increase the supersaturation instantaneously and produce plenty of smaller nuclei62; further annealing of the film facilitates the growth of nuclei into larger PQDs due to the removal of solvent63 and heat-induced diffusion of the precursor ions in the polymer matrix. The polymer matrix formed beforehand enables the controlled nucleation and growth of perovskite to facilitate a uniform PQD distribution due to the spatial confinement and the local deple- tion of precursors43,64. thiol positive radicals, which can remove the hydrogen ions immedi- ately to generate sulfur radicals with the assistance of bromides. Finally, the lead bromide complexes that have lost holes return to the ground state under the oxidation of oxygen in the solution to com- plete the catalytic regeneration cycle in the aerobic atmospheric conditions. The role of oxygen as the electron scavenger was con- firmed by the increasing amount of thiyl radicals in the mixture of the perovskite precursor solution and TTMP from N2, to air, to O2 atmo- sphere (Supplementary Fig. 11). In addition, the universe photo- catalytic effect of lead bromide complexes was also demonstrated in other radical reactions by transforming aniline into polyaniline (Sup- plementary Fig. 12). Exploration of photochemical mechanism Based on these results, it can be concluded that the highly coordinated lead bromide complex has a strong catalytic capacity, while in our case, it is PbBr4 2‒. To gain further insight into the photopolymerization mechanism, severalexperiments wereconducted to determine the reactive site. We first added different kinds of monomers into the perovskite precursor solution separately (entries 13 and 14 in Supplementary Table 6), where pH went down evidently only after TTMP was added. Meanwhile, a significant decrease in pH can be seen when PbBr2 was added to the monomer system (entry 15 in Supplementary Table 6). The increased acidity of the solutions may come from the weakening of sulfur–hydrogen bonding, indicating the effective coordination between sulfur in thiol groups and lead ions. To further verify this statement, oleic acid (OA), which can coordinate with lead ions effectively was added into the PPR59. Almost double curing time can be explained by the reduction of coordination between sulfur and lead ions, verifying the reactive site is on the lead ion (entries 16 and 17 in Supplementary Table 7). Moreover, strong sulfur radical signals were detected after 8 min irradiation when TTMP was added separately into Furthermore, fluorescence quenching studies were performed to prove the electron–hole transfer between PbBr4 2‒ and organic mono- mers. As shown in Fig. 3f, both the intensity of photoluminescence excitation (PLE) and PL spectra of the perovskite precursor solution drop more than 35% with limited blue shift after adding organic monomers, indicating that the organic monomers may act as quenchers to extract the electrons and/or holes from the excited PbBr4 2‒ 58. Based on our photopolymerization reaction results and mechanism studies, we proposed a mechanism of thiol-ene free-radi- cal addition photocatalyzed by lead bromide complexes as shown in Fig. 3g. Firstly, the sulfur atoms in thiol monomers coordinate with the lead ions, while the hydrogen atoms coordinate with bromides. Upon UV light irradiation, lead bromide complexes transition from the ground state to the excited state accompanied by the generation of electrons and holes. The holes transfer to the thiol groups to generate Nature Communications| (2022) 13:6713 5 https://doi.org/10.1038/s41467-022-34453-9 Article red perovskite due to the ascending sequence lying in the solubility of precursors61 and formation enthalpy48. Examples of the cartoon, let- ters, and the school logo composed of circle red, green, and blue pixels with a diameter and spacing of 20 μm were demonstrated in Fig. 5a–c. Demonstration of PQD patterns via direct in situ photolithography a b c 40 μm 40 μm 40 μm 10.4 μm a b 40 μm 40 μm c 40 μm 10.4 μm b c f 0 5 10 15 20 25 30 0.0 0.2 0.4 0.6 0.8 1.0 Norm. PL intensity Distance (μm) f PL intensity (abr. u.) d d e 1800 2000 2200 2400 2600 0 35 70 105 140 Average count PL intensity (arb. u.) e Average count 196 circle pixels with a diameter of 20 μm. Error bars represent standard deviation (the statistical data and errors were presented in Supplementary Table 8). f Radial emission intensity distribution of a single 20 μm circle pixel, inlet image is the surface-emission intensity of the pixel. Source data are provided with this paper. Fig. 4 | Characterization of PQD patterns prepared via direct in situ photo- lithography. a Fluorescence image, b SEM image, and c cross-section SEM image (inset image is enlarged view of a single stripe) of blue stripe patterns. d Emission intensity distribution image, e average emission intensity statistical distribution of Patterned PQDs Ethanol 50 μm 0.2 cm 100 μm 100 μm 100 μm 0.5 cm 50 μm 0.2 cm 0.2 cm 50 μm 500 μm 50 μm 0.5 cm a b c d e f g h Fig. 5 | Multicolored PQDs patterns via direct in situ photolithography. Fluor- escence images of a red cartoon, b green letters, c blue logo and corresponding pixels; d red, green and blue stripe patterns; e red and green double color pat- terned film with squares of 250 μm; f blue circle patterns with a diameter of 5 μm, the scale bar in the inset image is 10 μm. g Photograph of green cartoon on the flexible PET under UV light. h Photograph of green logo dipping into ethanol under UV light. 100 μm 100 μm 100 μm 500 μm 50 μm d e f a b Patterned PQDs Ethanol 0.5 cm h 0.5 cm g h 50 μm 0.2 cm c g c the scale bar in the inset image is 10 μm. g Photograph of green cartoon on the flexible PET under UV light. h Photograph of green logo dipping into ethanol under UV light. Fig. 5 | Multicolored PQDs patterns via direct in situ photolithography. Demonstration of PQD patterns via direct in situ photolithography 23a, the luminescent patterns still kept com- plete morphology and bright fluorescence when immersing into water and polar ethanol which can destroy PQDs easily, about 66 and 60% of the origin PL QY were retained even after 10 h (Supplementary Fig. 23b). The migration of PQDs in ethanol was determined by The direct in situ photolithography can also be extended to other color PQD patterns. To make sure blue and red PPRs can be patterned, it is critical to determine the proportion of Br‒ substituted by Cl‒ or I‒ to ensure enough lead bromide complexes exist in the PPR. That is because both the amounts of PbBr4 2– and the curing rates of corre- sponding PPRs decreased with the increasing ratio of [Cl‒] and [I‒] (Supplementary Fig. 16a), and a proportional linear relationship was recorded between the curing rate and the content of PbBr4 2‒ (Sup- plementary Fig. 16b). To balance the ratio involving Cl‒ and I‒ to satisfy the formation of blue and red PQDs and the quantity of PbBr4 2‒ required to photocatalyze the polymerization, suitable ratios were chosen for blue and red PQDs and their catalysis capacities were confirmed by the variation of PL and PLE spectra (Supplementary Fig. 17). To obtain PQDs with good performance, annealing process was optimized for different PQDs (Supplementary Fig. 18), the climb- ing annealing temperature is respectively applied from blue, green and Nature Communications| (2022) 13:6713 6 Article https://doi.org/10.1038/s41467-022-34453-9 0 5 10 15 20 25 30 0.0 0.2 0.4 0.6 0.8 1.0 Norm. PL intensity Distance (μm) a b c 40 μm 40 μm 40 μm PL intensity (abr. u.) d e f 10.4 μm 1800 2000 2200 2400 2600 0 35 70 105 140 Average count PL intensity (arb. u.) Fig. 4 | Characterization of PQD patterns prepared via direct in situ photo- lithography. a Fluorescence image, b SEM image, and c cross-section SEM image (inset image is enlarged view of a single stripe) of blue stripe patterns. d Emission intensity distribution image, e average emission intensity statistical distribution of 196 circle pixels with a diameter of 20 μm. Error bars represent standard deviation (the statistical data and errors were presented in Supplementary Table 8). f Radial emission intensity distribution of a single 20 μm circle pixel, inlet image is the surface-emission intensity of the pixel. Source data are provided with this paper. Article Article an attenuated total reflection (ATR) accessory, with zinc-selenide dia- mond coated plate. EPR spectra were measured by Bruker EMXplus-6/ 1, no chemicals were needed to detect carbon radicals, while 5,5- dimethyl-1-pyrroline-N-oxide (DMPO) was used as a radical trapping agent to detect thiyl radical. Steady-state UV–Vis absorption spectra were measured by PerkinElmer Lambda1050+, and the texting solu- tions were loaded in a standard quartz cuvette with an optical path of 1 mm. PLE and PL spectra were measured using an F-380 fluorescence spectrometer (Tianjin Gangdong Sci. & Tech. Development Co., Ltd.), the standard quartz cuvette with an optical path of 1 mm loaded with texting solutions was placed at a 45° angle to the light source. an attenuated total reflection (ATR) accessory, with zinc-selenide dia- mond coated plate. EPR spectra were measured by Bruker EMXplus-6/ 1, no chemicals were needed to detect carbon radicals, while 5,5- dimethyl-1-pyrroline-N-oxide (DMPO) was used as a radical trapping agent to detect thiyl radical. Steady-state UV–Vis absorption spectra were measured by PerkinElmer Lambda1050+, and the texting solu- tions were loaded in a standard quartz cuvette with an optical path of 1 mm. PLE and PL spectra were measured using an F-380 fluorescence spectrometer (Tianjin Gangdong Sci. & Tech. Development Co., Ltd.), the standard quartz cuvette with an optical path of 1 mm loaded with texting solutions was placed at a 45° angle to the light source. analyzing the content of Pb atoms in the film from ICP-MS and the immersed solution from ICP-OES, the proportion of 14% was assigned to the PQDs leaching from the film after 10 h immersion, illustrating the robustness of the direct in situ photolithography method. Collec- tively, these results demonstrate that the direct in situ photo- lithography technology allows for high-resolution, full-color and patterned applications. In conclusion, by combining direct optical lithography and in situ fabrication of PQDs, we propose a non-destructive lithography method that can directly lithograph perovskite precursor solutions and then grow PQDs in situ. We demonstrate the photocatalytic role of lead bromide complexes in polymerization, thus enabling fast photo- lithography without the need for additional initiators or catalysts. Different from the traditional photolithography method, PQDs are generated after the lithography process, which eliminates the complex process required to prepare the PQDs in advance, at the same time effectively avoids the degradation of PQDs by solvents and high- energy UV light in the lithography process. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Preparation and characterization of PPRs The data generated in this study are provided in the Source Data file. Source data are provided with this paper. All operations were performed under atmospheric conditions. A green PPR was prepared by dissolving 0.3 mmol MABr, 0.15 mmol PbBr2, 1 mmol TAIC, and 1 mmol TTMP into 0.8 mL DMF and 0.2 mL DMSO. A blue PPR was prepared by dissolving 0.4 mmol MACl, 0.2 mmol PbBr2, 0.1 mmol PEABr, 1 mmol TAIC, and 1 mmol TTMP into 0.8 mL DMF and 0.2 mL DMSO. A red PPR was prepared by dissolving 0.09 mmol CsI, 0.21 mmol MAI, 0.05 mmol PbI2, 0.15 mmol PbBr2, 0.12 mmol PEABr 1 mmol TAIC and 1 mmol TTMP into 0.8 mL DMF and 0.2 mL DMSO. All PPRs need to be stirred for 4 h at room temperature. References Materials All reagents were used as received without further purification: lead bromide (PbBr2, 99%), lead iodide (PbI2, 98%), cesium bromide (CsBr, 99.9%), cesium iodide (CsI, 99.9%), methylamine hydrobromide (MABr, 98%), methylamine hydroiodide (MAI, 98%), methylamine hydrochloride (MACl, 98%), isocyanuric acid tris(2-acryloyloxyethyl) ester (IATE, >80%) were purchased from Aladdin. 2-Phenylethanamine bromide, (PEABr, >99.5%) was purchased from Xi’an Polymer Light Technology Corp. Triallyl isocyanurate (TAIC, 98%), trimethylolpro- pane trimethacrylate (TMPTMA, 90%), trimethylolpropane tris(3- mercaptopropionate) (TTMP, 85%), pentaerythritol tetra(3-mercapto- proionate) (PTMP, 95%), vinyltrimethoxysilane (VTMS, 97%), 3-mercaptopropyltriethoxysilane (MPTS, 98%) were purchased from Meryer. 1,4-Butanediol bis(thioglycolate) (BBT, 98%) was purchased from Macklin. N,N-dimethylformamide (DMF, 99.5%), dimethyl sulf- oxide (DMSO, ≥99.8%), trichloromethane (CHCl3, ≥99%) were pur- chased from Beijing Tong Guang Fine Chemicals Company Co., Ltd. 5,5-Dimethyl-1pyrroline N-oxide (DMPO, 98%) was purchased from Energy-Chemical Co., Ltd. Article By controlling the poly- merization and in situ fabrication conditions, non-destructive efficient PQD pixel patterns can be obtained, with pixel resolution up to 2450 PPI, thicknesses up to 10 μm, and good stability to light, heat, and solvents. The direct in situ photolithography technology provides a useful idea for the preparation of non-destructive and high-efficiency light-emitting PQD pixels, and offers a simple technical route for the development of high-resolution patterned optoelectronic applica- tions, such as Micro-LED, laser, and AR/VR devices. In the future, lead- free perovskite, including Sn-based, Bi-based, Cu-based and double perovskite, can be explored for direct in situ photolithography to avoid the use of lead. It is of great importance to develop scale-up film coating methods with high utilization of materials, such as blade, spray coating, and roll-to-roll processes. Patterning and characterization of PQDs All operations were performed under atmospheric conditions. All substrates were cleaned in an ultrasonic bath using deionized water, ethanol, acetone, ethanol, isopropanol for 15 min each and were blown dry with a nitrogen gun. VTMS (MPTS) modifying substrates were carried out with the vapor-deposition method: a small drop of VTMS (MPTS) was dropped onto the substrates, subsequently, these sub- strates were heated to 80 (100) °C for 4 h. After casting 10 μL of PPR on the center of the substrate, the substrate was then bound between a black PMMA board and a patterned chrome mask. Then, a 20 W 365 nm handheld-LED was put to expose above the mask for 3–10 s for green patterns, 5–12 s for blue patterns, and 4–8 min for red patterns. After exposure, the substrate was placed on a spin coater. The uncured zone was rinsed out by spin-washing with clean chloroform, yielding micropatterns. The substrate was then annealed at 80 °C for 3 min for green patterns, room temperature for blue patterns, and 130 °C for 5 min for red patterns to remove residual solvents, and PQDs were in situ fabricated in the polymer matrix. For colorful patterns on the same substrate, an intermediate layer of SiO2 was deposited between each PQD layer via vacuum magnetron sputtering. Methods i l Fluorescent images were obtained using a Nikon N-SIM A1R microscope. SEM images were taken on a ZEISS GeminiSEM. The retention and the leaching rate of the PQD-polymer films were deter- mined by analyzing the content of Pb atom in the solvents and films based on the Agilent ICP-OES and ICP-MS 7800, and the testing sam- ples were digested in an acidic condition and diluted by deionized water. The PL intensity of the optical images was manipulated using the camera software or ImageJ. Optical absorption spectra of pat- terned films were collected using a UV-6100 UV–Vis spectro- photometer (Shanghai Mapada Instruments Co., Ltd.). Photoluminescence spectra were collected with an F-380 fluorescence spectrometer (Tianjin Gangdong Sci. & Tech. Development Co., Ltd.). 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Coordination chemistry dictates the structural defects in lead halide perovskites. Chemphyschem 17, 2795–2798 (2016). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-022-34453-9. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-022-34453-9. 56. Yoon, S. J., Stamplecoskie, K. G. & Kamat, P. V. How lead halide complex chemistry dictates the composition of mixed halide per- ovskites. J. Phys. Chem. Lett. 7, 1368–1373 (2016). Correspondence and requests for materials should be addressed to Gaoling Yang. 57. Hamill, J. C., Schwartz, J. & Loo, Y. L. Influence of solvent coordi- nation on hybrid organic–inorganic perovskite formation. ACS Energy Lett. 3, 92–97 (2017). Peer review information Nature Communications thanks Dezhi Tan, Katharina Ehrmann, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. 58. Oldenburg, K. & Vogler, A. Electronic spectra and photochemistry of tin(II), lead(II), antimony(III), and bismuth(III) bromide complexes in solution. Z. Naturforsch. B 48, 1519–1523 (1993). 59. Almeida, G., Infante, I. & Manna, L. Resurfacing halide perovskite nanocrystals. Science 364, 833–834 (2019). Competing interests p g The authors declare no competing interests. Acknowledgements © The Author(s) 2022 The authors would like to acknowledge the Experimental Center of Advanced Materials of Beijing Institute of Technology for the support in The authors would like to acknowledge the Experimental Center of Advanced Materials of Beijing Institute of Technology for the support in 10 Nature Communications| (2022) 13:6713
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Bacterial Toxins Fuel Disease Progression in Cutaneous T-Cell Lymphoma
Toxins
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Toxins 2013, 5, 1402-1421; doi:10.3390/toxins5081402 toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Review Bacterial Toxins Fuel Disease Progression in Cutaneous T-Cell Lymphoma Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2, Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1, Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,* 1 Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen 2200, Denmark; E-Mails: awo@sund.ku.dk (A.W.-O.); thorkr@sund.ku.dk (T.K.); cmenne@sund.ku.dk (C.M.B.); cge@sund.ku.dk (C.G.); awoetmann@sund.ku.dk (A.W.) 2 Department of Dermatology, Aarhus University Hospital, Aarhus 8000, Denmark; E-Mails: lise.lindahl@ki.au.dk (L.M.L.); lars.iversen@ki.au.dk (L.I.) 3 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; E-Mail: wasik@mail.med.upenn.edu 4 Department of Pathology, NYU Langone Medical Center, New York, NY 10016, USA; E-Mail: sergei.koralov@nyumc.org 5 Department of Biomedicine, Aarhus University, Aarhus 8000, Denmark; E-Mail: kilian@microbiology.au.dk * Author to whom correspondence should be addressed; E-Mail: ndum@sund.ku.dk; Tel.: +45-3532-7879. Received: 4 July 2013; in revised form: 2 August 2013 / Accepted: 6 August 2013 / Published: 14 August 2013 Abstract: In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency. Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus. Bacterial toxins such as h l l i ( ) h l b d b i l d i h OPEN ACCESS Toxins 2013, 5, 1402-1421; doi:10.3390/toxins5081402 toxins ISSN 2072-6651 www.mdpi.com/journal/toxins OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins OPEN ACCESS Keywords: cutaneous T-cell lymphoma; infections; Staphylococcus aureus; enterotoxins; superantigens Keywords: cutaneous T-cell lymphoma; infections; Staphylococcus aureus; enterotoxins; superantigens 1. Introduction Cutaneous T-cell lymphoma (CTCL) comprises a heterogeneous group of lymphoproliferative disorders defined by primary expansion of malignant T lymphocytes in the skin. The two most common forms, mycosis fungoides (MF) and Sézary syndrome (SS), constitute approximately 50%–70% of all de novo cases of CTCL, with MF accounting for the majority of cases. In this review, CTCL will refer exclusively to mycosis fungoides and Sézary syndrome. Early skin lesions in CTCL usually present as erythematous patches that notoriously resemble benign inflammatory skin disorders like psoriasis, chronic eczema or atopic dermatitis—collectively, making an early diagnosis very difficult [1–6] even though promising new approaches using miRNA expression profiling seem to discriminate between the benign inflammatory and malignant conditions inflammation with high accuracy [7,8]. Although patients diagnosed in the early stages of disease often experience an indolent disease course, a subgroup of patients experience an aggressive clinical course with tumor development, skin ulceration, involvement of lymph nodes, bone marrow and internal organs and gradual development of immunodeficiency at later stages of disease. Concomitant with disease progression there is a decrease in normal lymphocyte count and functionality and, consequently, advanced disease may be associated with profound immune deregulation [1,2,9,10]. The etiology of CTCL has long puzzled researchers and a wide range of risk factors has been examined in this regard. Chromosomal instability and abnormal expression of genes involved in cell cycle control and proliferation has been reported several times in CTCL studies [11–13]. However, in contrast to other hematological disorders, in CTCL well documented etiological or predisposing genetic factors remain elusive. Occupational and environmental factors have been proposed in some studies but with limited reproducibility and a lack of any evident biological causality [14–16]. Yet, a recent finding by Duvic and colleagues sheds light on a previously suspected link between drugs (thiazide used in the treatment of hypertension) and CTCL [17] indicating that environmental factors might indeed play a role in a subset of patients with chemical or biological agents acting as inciting agents in the context of this T cell lymphoma. Familial aggregation of CTCL incidences has been described [18] and a correlation between CTCL disease occurrence and certain human leukocyte antigen (HLA) alleles has also been observed [19]. Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2, Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1, Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,* Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2, Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1, Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,* Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2, Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1, Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,* 1 Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen 2200, Denmark; E-Mails: awo@sund.ku.dk (A.W.-O.); thorkr@sund.ku.dk (T.K.); cmenne@sund.ku.dk (C.M.B.); cge@sund.ku.dk (C.G.); awoetmann@sund.ku.dk (A.W.) 1 Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen 2200, Denmark; E-Mails: awo@sund.ku.dk (A.W.-O.); thorkr@sund.ku.dk (T.K.); cmenne@sund ku dk (C M B ); cge@sund ku dk (C G ); awoetmann@sund ku dk (A W ) 2 Department of Dermatology, Aarhus University Hospital, Aarhus 8000, Denmark; E-Mails: lise.lindahl@ki.au.dk (L.M.L.); lars.iversen@ki.au.dk (L.I.) 3 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; E-Mail: wasik@mail.med.upenn.edu 4 Department of Pathology, NYU Langone Medical Center, New York, NY 10016, USA; E-Mail: sergei.koralov@nyumc.org 5 Department of Biomedicine, Aarhus University, Aarhus 8000, Denmark; E-Mail: kilian@microbiology.au.dk * Author to whom correspondence should be addressed; E-Mail: ndum@sund.ku.dk; Tel.: +45-3532-7879. Received: 4 July 2013; in revised form: 2 August 2013 / Accepted: 6 August 2013 / Published: 14 August 2013 Abstract: In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency. Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus. Bacterial toxins such as staphylococcal enterotoxins (SE) have long been suspected to be involved in the pathogenesis in CTCL. Here, we review links between bacterial infections and CTCL with focus on earlier studies addressing a direct role of SE on malignant T cells and recent data indicating novel indirect mechanisms involving SE- and cytokine-driven cross-talk between malignant- and non-malignant T cells. 1403 Toxins 2013, 5 Toxins 2013, 5 progressive disease die more frequently from infection rather than from the CTCL per se [21,23]. These important findings prompt the question whether the high incidence of infections in CTCL patients is a mere consequence of a compromised skin barrier, a suppressed immune system, or a combination of both. 2. High Prevalence of Infections High incidence of infections is a common clinical experience in CTCL [20–22]. Axelrod et al. examined and quantified different types of infection in 356 CTCL patients [21]. Among the 478 documented infections, 396 were of bacterial origin with the remaining identified as viral, fungal or parasitic. Their study documented that skin was by far the most prevalent site of infection and that risk of infection was intimately associated with the disease stage. Thus, these findings supported the clinical experience that major morbidity and mortality stems from infections and also that patients with 1404 Toxins 2013, 5 In CTCL, the early infiltrating CD4 T cells display a TH1 phenotype and in concert, these immune cells are seemingly capable of controlling CTCL cell expansion via cytokines and cytotoxicity [25–28]. Accordingly, it has been shown that the presence of cytotoxic CD8 T cells within the CTCL lesions is a positive prognostic factor, and several case reports have evidenced that use of the immunosuppressant cyclosporine in treatment of CTCL accelerates disease progression and large cell transformation [10,29,30]. During the disease progression, the concentration of TH1 cytokines decreases in contrast to an increased production of TH2 cytokines and angiogenetic and lymphangiogenetic factors such as VEGF-A and VEGF-C [10,31–35]. This increasing bias towards a TH2 immune response obstructs an effective cellular immune response and can be framed within the immunoediting hypothesis as a process in which the malignancy transitions from an equilibrium phase to a tumor escape phase. Indeed, as the disease progresses, the malignant T cells display an increased expression of B lymphoid tyrosine kinase (BLK), and cyclooxygenase 2 (COX-2) as well as activation of signal transducers and activators of transcription-3 (STAT3) and STAT5 [36–39], which in turn drive expression of TH2 cytokines, oncomiRs (miR-155), and the suppressor of cytokine signaling 3 (SOCS3) [37,40]. The enhanced expression of SOCS3 has been shown to protect malignant T cells from growth-inhibition by pro-inflammatory cytokines such as interferon-alpha (IFNα) [41]. Because IFNα is used for treatment of CTCL, the development of IFNα resistance comprises a pressing clinical problem [41]. Furthermore, direct diversion of anti-tumor immune response been attributed to the malignant T cells in CTCL. Several studies have demonstrated forkhead box P3 (FOXP3) expression in malignant T cells in a subset of patients [42–45] and upregulation of cytotoxic T-lymphocyte antigen 4 (CTLA-4) in a stage-dependent manner [46]. Likewise the interaction of programmed death protein 1 (PD-1) and its ligand, PD-L1 has been associated with immune evasion in CTCL [47,48] as these cell surface molecules are involved in the induction and maintenance of peripheral T cell tolerance. The increased expression of PD-L1 on neoplastic T cells has been hypothesized to involve the aberrant and constitutive activation of the janus associated kinase 3 (JAK3)-dependent STAT3 cell signaling pathway which is also allegedly a key player in sustaining tissue inflammation while antagonizing tumor immunity [49]. Furthermore, the constitutively active STAT3 induces the secretion of the two potent immunosuppressants; IL-10 and transforming growth factor-beta (TGFβ) [44,45,50]. 3. Immunopathogenesis CTCL progression is typically associated with immune suppression. The malignant cells normally exhibit a mature memory CD4 T cell phenotype and express a range of skin-homing receptors in the initial disease stages, which contribute to the characteristic epidermotropism of malignant T cells [6,10]. The immunopathogenesis in CTCL is characterized by a gradual shift of cytokine profile in lesional tissue. Early lesions contain a large proportion of non-malignant cells, which primarily consist of dendritic cells, macrophages and tumor-infiltrating cytotoxic CD8 and CD4 T cells [6,10,24]. CD4 T cells may display several different phenotypes depending on their specific activation as illustrated in Figure 1. While the CD4 T cell helper type 1 (TH1) is crucial in promoting an effective cellular immune response and as such beneficial in an anti-tumor response, the TH2 phenotype is on the contrary promoting a humoral immune response. The more recently recognized TH17 cell is believed to be important in certain microbial infection while the T regulatory phenotype is paramount in establishing and sustaining peripheral tolerance. Figure 1. Schematic illustration of the antigen presenting cells (APC) antigen presentation and cytokine release together with the subsequent induction of different lymphocyte helper subsets. (1) The APC delivers three signals required for successful lymphocyte activation; antigen presentation, co-stimulation and cytokine release with cytokines being the major determinant of lymphocyte subset induction; (2) Additionally dendritic cells DC are able to induce a regulatory phenotype either by the absence of co-stimulation (immature DC’s lack CD80/86) or by activation of lymphocytes in a regulatory cytokine environment (tolerogenic DC’s). 1405 Toxins 2013, 5 Collectively, the expression and secretion of the above mentioned molecules supports the model originally proposed by Berger and colleagues who suggest that CTCL T cells maintain dendritic cell immaturity by the release of regulatory cytokines. Further, according to their hypothesis, this results in polarization of the DC’s towards a tolerogenic phenotype, rather than an activating phenotype. In turn, this subtype of DC should promote malignant T cell proliferation and the acquisition of immunosuppressive charactheristics [50,51]. Finally, the immunodeficiency in late stage CTCL could also caused by a gradual displacement of non-malignant T cells by the expanding malignant T cell clones; in other words, that the malignant T cells eventually outcompete and substitute the non-malignant T cell population, which results in a state reminiscent of advanced AIDS with a lack of functional CD4 T helper cells and severe immunosuppression [1,2,10,52]. Figure 2 shows an illustration of our current view of the dynamic immunological changes during disease progression. Thus, the interplay between malignant T cells, dendritic cells and infiltrating Toxins 2013, 5 malignant T cells with capacities resembling either regulatory T cells or different T helper subsets. Two members of the IL-17 family of cytokines: IL-17A and IL-17F, have recently been implicated in CTCL pathogenesis [54–56]. Expression of IL-17A and IL-17F is increased in skin lesions and comparable to the expression levels in lesional psoriatic skin [55]. Noteworthy, heterogeneity in IL-17A and IL-17F expression existed among CTCL patients with some patients having normal or near normal expression whereas others had highly increased levels of IL-17 cytokines. Importantly, an elevated expression of IL-17F correlated with progressive disease [55]. Given the observations of increased IL-17 expression in CTCL patients with bacterial infections [54], we propose a link between bacterial infection, expression of IL-17F and the disease progression. However, it remains to be determined whether IL-17F and other IL-17 family cytokines are fostering disease progression via induction of angiogenesis or other as yet unidentified mechanisms or if an increased expression of these cytokines is a sign of a “frustrated” immune response unable to combat the bacterial infection. Toxins 2013, 5 1406 Toxins 2013, 5 and/or skin-resident, non-malignant T cells change dramatically as the disease progress from an indolent condition to an aggressive cancer. In early stages and non-progressive disease, dendritic cells produce interferon-alpha (IFNa), non-malignant T cells produce TH1 cytokines (such as IL-12 and IFNg), and CD8 cytotoxic T cells produce granzymes and mediate direct killing of malignant T cells. These events generate a hostile environment inhibiting malignant proliferation—yet without eradicating the malignant T cell clone—i.e., the tumor lesion is kept in a “state of equilibrium” without expansion and spreading (Figure 2(1)). As malignant T cells change and begin expressing immune modulatory molecules and cytokines (which inhibit the immune control), a “tumor immunological privilege” is established. This “immune privilege” shelter malignant T cells from inhibitory signals allowing for malignant proliferation and induction of immunosuppression and eventually, immunodeficiency (Figure 2(2)). Figure 2. Schematic illustration of the transition from a state of tumor equilibrium to a state of tumor immune privilege. The tumor equilibrium state (1) is characterized by T cell- and cytokine-mediated control of tumor progression. Conversely, the state of tumor immune privilege (2) is predominated by regulatory signals and cytokines allowing for immune evasion and tumor progression and metastasis. (Yellow: DC; blue: nonmalignant T cell; red: malignant T cell). Malignant T cells in CTCL display a considerable degree of phenotypic heterogeneity, which ongst other things, is believed to impact disease aggressiveness and response to treatment [6,24,53]. ent studies indicate that this heterogeneity is highly dependent upon crosstalk between malignant ells and the tumor environment, in that malignant T cells have been shown to secrete a wide array cytokines, which collectively may activate keratinocytes and surrounding stromal cells and thus ain tissue inflammation. In return, the activated microenvironment impregnates the malleable Malignant T cells in CTCL display a considerable degree of phenotypic heterogeneity, which amongst other things, is believed to impact disease aggressiveness and response to treatment [6,24,53]. Recent studies indicate that this heterogeneity is highly dependent upon crosstalk between malignant T cells and the tumor environment, in that malignant T cells have been shown to secrete a wide array of cytokines, which collectively may activate keratinocytes and surrounding stromal cells and thus sustain tissue inflammation. In return, the activated microenvironment impregnates the malleable 1407 Toxins 2013, 5 Toxins 2013, 5 1408 chronic expansion of Chlamydia-specific T cells and the combination of SAF and chronic T cell activation was hypothesized to lead steadily to the development of CTCL [76]. Borrelia burgdorferi has also been implicated and in 2006 Bonin and colleagues [77] reported on a minor association between Borrelia burgdorferi and CTCL in a population endemic for Borrelia infection. Later they suggested that Borrelia in conjunction with HTLV-1 (or other infectious agents) can provide a persistent antigenic stimulation, which contributes to the transformation and expansion of T lymphocytes [20]. However, subsequent studies failed to detect a significant presence of Chlamydia pneumonia and Borrelia burgdorferi in CTCL skin specimens [78,79]. Sporadic case reports [80–82] describe other infectious agents of various types but generally they may reflect the findings by Axelrod and colleagues, of a very high degree of diversity in infectious agents present in CTCL patients [21]. As mentioned above, a multitude of pathogens have been isolated from CTCL patients and recurrent infections comprise a large clinical challenge in the care of CTCL patients. The pathogens most frequently associated with CTCL are listed in Tables 1 and 2. Table 1. Prevalence of the most frequent bacterial and viral pathogens associated with cutaneous T-cell lymphoma (CTCL) disease. Patient cohort included 356 CTCL patients. Modified from Axelrod et al. 1992 [21]. Bacteria Number of infections Frequency Staphylococcus aureus 117 33%–38% * Enterobacteriaceae 38 10.7% Beta-hemolytic streptococci 35 9.8% Pseudomonas aeruginosa 12 3.4% Viruses Herpes zoster 34 9.6% Herpes simplex 30 8.4% * A general study by Axelrod et al. (1992) [21] reports that 33% of infections in CTCL are Staphylococcus aureus. Jackow et al. (1997) [83] detects Staphylococcus aureus in 38% of examined CTCL patients. Table 2. Complications associated with infections in CTCL. Table 2. Complications associated with infections in CTCL. Table 2. Complications associated with infections in CTCL. Co-morbidity from infections Bacterial infections bacteremia, pneumonia, intra-abdominal infections Viral infections ulcerative skin lesions, dissemination (Kaposi varicelliform eruption) 4. Infectious Etiology It has previously been hypothesized that infectious agents (such as a retrovirus) were responsible for the outgrowth of neoplastic T cells and as such are a primary etiological factor in CTCL. MacKie originally launched this hypothesis in 1981 by proposing that CTCL arises from an initial viral infection of epidermal antigen presenting cells [57]. Mackie was inspired by observations of distinctive aggregates of epidermal dendritic cells and T cells in MF patients called Pautrier’s abscesses and also from reports of retrovirus-like particles observed in malignant CTCL T cell cultures [58]. Furthermore, viral antigens have the potential to induce loss of T cell receptor (TCR) diversity, which is a characteristic feature of CTCL [59,60]. This may occur when cross-reactivity exists between viral and auto antigens. According to the hypothesis, autoantigen can sustain proliferation and activation of an autoreactive T cell population following virus eradication thereby resulting in a narrowing of the TCR repertoire [61]. The idea of an infectious etiologic agent gained momentum by the earlier discovery of HTLV-1 and its association with adult T cell lymphoma (ATL) [62]. The distinction between the two diseases was not initially recognized due to the clinical, pathological, and histological similarities—although ATL was later established as an unique HTLV-1 induced entity [63]. However, the analogy between CTCL and ATL and the—at the time newly discovered—T-lymphotropic retrovirus, HIV-1 seemed conspicuous [64] and motivated researchers to search for a retroviral culprit in CTCL. Concordantly, retroviral activity and HTLV-like particles in peripheral blood mononuclear cells derived from CTCL patients [65], and successful polymerase chain reaction (PCR) amplification of HTLV-1 sequences was also reported in CTCL skin biopsy specimens [66]. However, controversies arose and later, well-performed, controlled studies failed to associate HTLV-1 with CTCL and the hypothesis of HTLV-1 as the etiological factor in CTCL was put to rest [67–69]. Other viruses such as Epstein-Barr, herpes virus 6-8, and cytomegalovirus were later suspected to be involved in CTCL but so far, the associations have been relatively weak and not (yet) convincingly reproduced [70–74]. In addition, bacterial agents have been assigned a direct role in the etiology of CTCL. One candidate was Chlamydia pneumonia, which was suggested to foster CTCL through the secretion of a Sézary T cell activating factor (SAF) [75]. Chronic infection with Chlamydia was believed to facilitate Toxins 2013, 5 malignant CTCL cells responded to SE in a TCR variable β chain (TCRVβ) restricted manner suggesting a possible involvement in the disease [87]. Later, Duvic and colleagues [83] examined 42 CTCL patients with advanced disease (SS or advanced MF with erythrodermia) for bacterial colonization in skin and blood and found that 76% of the patients harbored staphylococci, of which 50% were SE-producing strains of S. aureus. Moreover, all patients with toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus infections had an expansion of TSST-1 specific T cells expressing the appropriate Vβ2 T cell receptors [83]. This observation suggests that superantigens such as SE may be involved in CTCL pathogenesis, as these toxins can facilitate the observed Vβ-restricted T cell expansion [88]. It was hypothesized that SE provide a persistent antigen stimulus for T lymphocytes driving malignant T cell expansion. This notion has been propelled by multiple reports of skewed or diminished T cell receptor repertoire in CTCL patients as discussed below. However, as these studies examined only patients with advanced disease, they actually do not provide evidence for a key role of SE in the etiology and early stage of CTCL. 5. Staphylococcus Staphylococcus aureus is a major source of morbidity in CTCL causing chronic or recurrent skin infections and life-threatening systemic infections such as sepsis, pneumonia and intra-abdominal infections [21–23,84]. S. aureus is renowned for its ability to produce staphylococcal enterotoxins (SE) (also known as superantigens) [85,86]. Superantigens are characterized by their ability to activate large fractions of T lymphocytes by crosslinking MHC class 2 molecules and T-cell receptors (independently of antigen specificity of the TCR and the antigen-peptide-binding groove in the MHC) thereby circumventing normal antigen processing and presentation. In 1992, Tokura et al. showed that 1409 6. TCRVβ Restriction By spectratyping the variable regions of the TCR’s β-chain Yawalkar et al. [60] demonstrated that half of all early-stage patients and all late-stage patients exhibited a highly diminished complexity of the TCR repertoire compared to the diverse repertoire displayed by normal peripheral T cells [60]. The shrunken TCR repertoire could not reflect a simple monoclonal expansion, as multiple Vβ-families were overrepresented while others were underrepresented or completely absent. In short, Vβ-family distribution failed to follow a normal Gaussian distribution pattern. This skewing of the TCR repertoire was hypothesized to be the result of superantigens such as SE. Superantigens may skew the TCR Vβ repertoire by two mechanisms: (1) One involves the previously mentioned direct mechanism by polyclonal activation and proliferation of Vβ-specific T cells following TCR ligation [83,85,86]. Such Vβ-specific expansion by superantigens was suggested by Linneman [89], based on an early-stage CTCL patient, who displayed a dominant Vβ5 T cell population in the skin biopsies. The idea is that superantigen responsive malignant T cells receive activation-signals and thus obtain a growth advantage allowing them to out-compete non-transformed cells. (2) The other mechanism involves polyclonal expansion followed by activation-induced cell death of superantigen reactive T cells, which results in a relative expansion of the remaining Vβ-families and thus induces a reciprocal superantigen Vβ-signature. This mechanism has been demonstrated by McCormack and colleagues in a series of mouse studies [90] and subsequently expanded to humans by Vonderheid and colleagues [91] in a cohort of 49 CTCL patients in which a majority of whom exhibited increased Vβ5 usage relative to other Vβ families, usually predominant in normal CD4 T cells. By investigating the TCRα and TCRβ gene rearrangement in 29 CTCL patients Van der Fits [92] concluded that the absence of an unambiguous similarity in the complementarity-determining regions argues against the notion of a single ordinary antigen delivering persistent and pathogenic antigen stimulation in CTCL. However, the skewed Vβ and Jβ gene usage suggested the possibility that also here superantigens may be responsible for the restricted TCR repertoire. It remains to be definitely demonstrated by which mechanism superantigens induce polyclonal T cell proliferation in CTCL. 1410 Toxins 2013, 5 However, since Fas receptor expression has been shown to be effectively down-regulated [25,93,94] and anti-apoptotic pathways such as B-cell lymphoma 2 (Bcl-2) and programmed cell death protein 10 (PDCD10) are enhanced in the CTCL clones [95,96], it is tempting to speculate that malignant T cells can evade Fas induced apoptosis after superantigen activation whereas non-malignant T cells expressing the corresponding Vβ TCR families are deleted. Removal by apoptosis of TH1 T cell subsets producing interferon and other inflammatory cytokines, which keep malignant T cells in check, might indirectly promote expansion of malignant T cells. Although several studies provide circumstantial evidence of superantigen-induced Vβ TCR-associated oligo/poly-clonality in CTCL patients, other groups fail to see “Vβ-signatures” indicative of superantigen involvement. In contrast, these studies observe a monoclonal expansion of malignant T cells [97–100]. This discrepancy might, amongst other factors, depend in part on the disease-stage of the examined patients, as studies tend to show increasing monoclonality of T cell populations with progression [1,2,4,60]. Indeed, it may also simply rely on the inherent heterogeneity of CTCL; i.e., the disease may in some patients manifest itself as an oligoclonal or skewed polyclonal expansion of T cells while in others it develops as a monoclonal entity. Collectively the above mentioned observations fail to clarify whether infections and infectious superantigens such as SE, function as a primary causative factor—or if they are a secondary event resulting from a weakened immune system and compromised skin barriers. Therefore, further studies are required in order to ascertain whether or not superantigens directly facilitate early expansion of pre-malignant T cells in CTCL, and it is justified to conclude that definitive evidence for an etiological role of superantigens in CTCL is currently still lacking. Toxins 2013, 5 activation of ZAP70/p72syk, PLCg1, and expression of the IL-2RA subunit [108–113]. Combined, these findings suggest a novel mechanism of tumor growth promotion by SE involving an indirect stimulation of malignant cell proliferation involving LFA-3/CD2-mediated cell-cell contact and soluble growth factors such as IL-2 and other as yet unidentified factors provided by the toxins-activated non-malignant T cells [104]. Figure 3. Schematic illustration of SE-mediated cross-talk between malignant and non-malignant T cells. Malignant T cells often display deficient expression and function of the TCR/CD3 complex and may not respond directly to bacterial superantigens such as staphylococcal enterotoxins (SE). Instead, malignant T cells often express MHC class II molecules, which are high-affinity receptors for SE (1). Non-malignant T cells with the appropriate Vb TCR respond to SE presented by malignant T cells (2, 3) or by antigen presenting cells (APC) (not shown). SE-mediated cross-talk between malignant and non-malignant T cells triggers cell-to-cell contact and production of growth factors, which in turn promote proliferation of malignant T cells (3) [104]. If this mechanism is operating in patients, it indicates that bacterial infections, and especially infections with superantigen-producing bacteria, indirectly activate malignant T cells through help from non-malignant T cells. The activation is not restricted by the Vβ-family on the malignant T cells, but only by their expression of MHC class II molecules or MHC class II expression by other surrounding cells types and their presentation of superantigens to Vβ-specific non-malignant T cells. The ability of non-malignant T cells to inadvertently promote tumor expansion is not unique to CTCL and has previously been reported in other malignancies. Specifically, in a squamous cell carcinoma model, deletion of non-malignant CD4 T cells decreased neoplastic cell progression and tumor incidence [114] underscoring the intimate relationship between inflammation and cancer. In CTCL, the indirect mechanism by which bacterial superantigens activate otherwise non-specific or TCR-deficient malignant T cells predicts that bacterial infections promote expansion of malignant T cells in an inflammatory setting with non-malignant T cells. In contrast, this model does not imply (but does not exclude) that bacterial superantigens have an etiological role in CTCL but does suggest a critical role If this mechanism is operating in patients, it indicates that bacterial infections, and especially infections with superantigen-producing bacteria, indirectly activate malignant T cells through help from non-malignant T cells. 7. Indirect Mechanism of Action The mechanism of oligoclonal expansion of premalignant T cells, should it occur by superantigen stimulation, is confounded by the fact that several studies of T cells from CTCL patients (including the early patch-plaque stage) show that malignant T cells often display deficient function and/or deficient expression of CD3 TCR complex [101–103]. Recently, our group has proposed a novel role of bacterial toxins in disease progression [104]. As illustrated in Figure 2, we observed that whereas malignant T cells did not respond directly to bacterial superantigens, they proliferated vigorously in response to SE in co-cultures with non-malignant T cells indicating an indirect mechanism for growth promotion by SE [104]. Noteworthy, malignant T cells often express major histocompatibility complex (MHC) class II molecules [104], which are high-affinity ligands for bacterial toxins such as SE [105]. Thus, even with defective TCR/CD3 complex, malignant T cells are able to bind SE (Figure 3(2)) and stimulate non-malignant T cells to produce growth factors such as interleukin-2 (IL-2), which in turn promotes growth of the malignant T cells (Figure 3(3)). In addition, toxin-induced cell-to-cell contact between malignant and non-malignant T cells also triggers growth-promoting signals via lymphocyte function associated antigen 3 (LFA-3)/CD2- dependent mechanism (Figure 3(3)) [104]. Importantly, both growth factor and cell-to-cell contact dependent growth stimulation of malignant T cells require MHC class II ligation by the bacterial toxin and expression of a functional TCR/CD3 complex by the non-malignant T cells [104]. In this regard, it is worth mentioning that MHC class II ligation by SE enhances cell-to-cell adhesion [106,107] and IL-2-induced T cell proliferation through an increased 1411 Toxins 2013, 5 Toxins 2013, 5 1412 of superantigens in the progression of CTCL. In keeping, it substantiates the general assumption that in progressive disease, malignant T cells in conjunction with the tumor environment are driven towards promoting a diverted inflammatory response, which boosts malignant T cell growth, exacerbates disease and likely increases susceptibility to additional infection. 9. Conclusions In conclusion, bacterial infections are a major clinical problem in CTCL and an important driver of morbidity and mortality in this disease. Despite much effort, definitive evidence supporting a direct etiological role of bacterial toxins is still lacking but other evidence suggests that toxins may also promote malignant T-cell expansion through a mechanism involving cross-talk between the malignant and non-malignant T cells. Given the proposed model for toxins as drivers of disease progression and the promising clinical data showing a beneficial effect of antibiotics on both morbidity and disease progression, we propose that an aggressive strategy for anti-bacterial therapy should be considered in all patients with clinically relevant and verified infections with S. aureus. 8. Clinical Improvement after Antibiotic Treatment Because many patients are suffering from recurrent bacterial infections, clinicians are often reluctant to undertake an aggressive treatment of skin infections with antibiotics due to the risk of increasing resistance to antibiotics. However, important clinical findings lend support to the hypothesis, that bacterial infections complicate and promote disease progression in CTCL. In several small series and case studies, elimination of S. aureus infection with antibiotics was associated with a rapid clinical improvement: in some patients treatment resulted in complete clinical response with no residual skin involvement by CTCL [83,115,116]. In an early study by Tokura and colleagues, clinical improvement in skin disease was observed after treating two CTCL patients with antibacterial agents [116]. In addition, Duvic and co-workers reported that in patients infected with SE-producing S. aureus, treatment with antibiotics resulted in clear clinical improvement [83]. Another study [115] reported on a high degree of S. aureus colonization in CTCL patients with an increased incidence in advanced erythrodermic SS patients when compared to non-erythrodermic MF patients. Eradication of S. aureus from the nostrils with oral and topical antibiotics was achieved in 85% of cases and similar treatment of skin lesions was effective in 91%. Consequently, after 4–8 weeks significant clinical improvement was seen in a majority of the treated patients [115]. Collectively, these observations speak in favor of an aggressive antibiotic treatment of bacterial infections in CTCL patients. Moreover, they are in support of the mechanism proposed above that bacterial toxins promote CTCL disease progression in CTCL. Toxins 2013, 5 The activation is not restricted by the Vβ-family on the malignant T cells, but only by their expression of MHC class II molecules or MHC class II expression by other surrounding cells types and their presentation of superantigens to Vβ-specific non-malignant T cells. The ability of non-malignant T cells to inadvertently promote tumor expansion is not unique to CTCL and has previously been reported in other malignancies. 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Murinometric measurements and retroperitoneal adipose tissue in young rats exposed to the high-fat diet: Is there correlation?
Brazilian Journal of Biology
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Abstract Aim: This study aimed to verify the correlation between murine measurements and retroperitoneal adipose tissue in rats exposed to the high-fat diet. Material and methods: Wistar male adult rats, descendants of mothers who consumed a high-fat diet during pregnancy and lactation and fed the same diet after weaning were used. At 60 days of life, body weight, longitudinal axis and waist circumference (WC) were measured. The Body Mass Index (BMI) and the Lee Index were calculated for a posterior analysis of the correlation with the amount of retroperitoneal adipose tissue dissected on the same day. For analysis of the data, the Pearson correlation test was used, considering statistical significance for p <0.05. Results: Body weight had a weak correlation (r= 0.31; p= 0.38) with retroperitoneal adipose tissue. While the longitudinal correlated moderately and negative (r= -0.40; p= 0.25). Abdominal circumference (r= 0.62; p= 0.05), body mass index (r= 0.61; p= 0.03) and Lee (r= 0.69; p= 0.03) correlated moderately and positively with adipose tissue. Conclusion: Among the measured murine measurements, weight and longitudinal axis were not good indicators to represent accumulation of retroperitoneal adipose tissue in rats. However, Lee’s index seems to be the best murine marker to diagnose the accumulation of retroperitoneal fat. BMI, CA and Lee index were murine parameters with higher correlation. Keywords: high-fat diet, obesity, rats, murinometric index, retroperitoneal adipose tissue. Brazilian Journal of Biology https://doi.org/10.1590/1519-6984.221405 Original Article https://doi.org/10.1590/1519-6984.221405 Original Article https://doi.org/10.1590/1519-6984.221405 A. P. A Macêdoa* , G. S. Cordeirob , L. S. Santosb , D. A. E. Santob , G. S. Perezb , R. D. Coutoa,c , M. E. P. C. Machadob,d , J. M. Barreto Medeirosb,d  A. P. A Macêdoa* , G. S. Cordeirob , L. S. Santosb , D. A. E. Santob , G. S. Perezb , R. D. Coutoa,c , M. E. P. C. Machadob,d , J. M. Barreto Medeirosb,d  bPrograma de Pós-Graduação em Alimento, Nutrição e Saúde, Escola de Nutrição, Universidade Federal da Bahia, Avenida Araújo Pinho, 32, Canela, Código Postal 40110-150, Salvador, BA, Brasil. cDepartamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal da Bahia, Rua Barão de Jeremoabo, s/n, Ondina, Código Postal 41170-290, Salvador, BA, Brasil. *e-mail: paulamacedo.nut@gmail.com Brazilian Journal of Biology ISSN 1519-6984 (Print) ISSN 1678-4375 (Online) Brazilian Journal of Biology ISSN 1519-6984 (Print) ISSN 1678-4375 (Online) 2. Material and Methods For analysis of the correlation of the murinometric indicators with retroperitoneal adipose tissue, we used the Pearson correlation test, considering the following classification: weak (0 <r <0.4); moderate (0.4 ≤ r < 0.7) and strong (r≥ 0.7). Correlations with statistical significance were considered those with a moderate or strong correlation degree and p≤0.05 (Siqueira and Tibúrcio, 2011). This study was approved by the Ethics Committee on Animal Experimentation, Faculty of Veterinary Medicine and Animal Science, UFBA, according to protocol n° 59/2017. Young rats (Rattus norvegicus), of the Albinus variety, of the Wistar strain at the Laboratory of Experimental Nutrition of the Federal University of Bahia (UFBA), were carried out, in which all animals were kept under the same conditions, with a temperature of 23 ± 2 °C and light / dark cycle of 12 hours. 2.2. High-fat diet This diet consisting of a mixture of commercial ration (Nuvilab® CR1) with roasted peanuts, milk chocolate, and Maria type biscuit, in a ratio of 3: 2: 2: 1. These ingredients were milled, mixed and heated in an oven at 60 °C. Subsequently, they were offered in the form of pellets, containing the percentage of lipids of 23% (Oliveira et al., 2011). It is well established in the literature that the ingestion of hypercaloric and high-fat diets are directly related to the development of obesity in rodents (Souza et al., 2018; Lima et al., 2018). Thus, in recent years, health professionals and researchers in the field have developed experimental studies to understand this clinical condition (Mittwede et al., 2016). 1. Introduction used between 90 and 100 days, with a minimum body weight of 220 g and a maximum of 280 g.i Obesity is considered a public health problem due to the high prevalence and its comorbidities (Banik and Rahman, 2018; Luz et al., 2017). The accumulation of fat in the abdominal region has been reported as a determining factor for the development of chronic non-transmissible diseases (Almeida et al., 2016). The onset of gestation was confirmed by the vaginal smear test (Perez et al., 2015). On the first day of gestation, it was offered to the fat high diet (n = 4). The females maintained the consumption of the high-fat diet until the end of the lactation (21st day of life of the descendants). After weaning, which occurred on the 22nd day of life of the offspring, the high-fat diet was offered to the offspring (n = 10) until the sixtieth day of life of the animal. The increasing number of obese individuals is associated with excessive consumption of high-calorie and high-fat diets, especially saturated fats (Nasreddine et al., 2018). Animal scientific research has sought to reproduce the human dietary habit using high-fat diets, inducing the accumulation of body fat, to similarly reflect the metabolic alterations found in human obesity (West and York, 1998; Casper et al., 2008). 3. Results The descriptive analysis of the murine parameters and the amount of retroperitoneal adipose tissue of the high-fat offspring on the sixtieth day of life is shown in Table 1. The body weight presented a weak correlation (r = 0.31, p=0.38), while the longitudinal correlated moderately 2.3. Data collect On the sixtieth day of life of the offspring, measurements were measured as: weight, longitudinal axis, abdominal circumference and weight of retroperitoneal adipose tissue. And then calculated the BMI, the Lee Index. In experimental studies with rodents exposed to the high-fat diet the murinometric parameters such as body weight, longitudinal axis, and circumference of the abdomen (CA) have been used extensively; and Lee and body mass indexes (BMI) to identify adiposity and diagnose obesity (Licholai et al., 2018; Mcnay and Speakman, 2013). However, it is known that the measurement of body fat is the most sensitive indicator to diagnose obesity (Hariri and Thibault, 2010). The animals’ body weight was recorded using a digital electronic scale -Mart, model S-4000, with a capacity of 4 kg and a sensitivity of 0.001g. Body length was assessed by measuring the longitudinal axis of the naso-anal. With the aid of a pen, the points between the muzzle and the base of the tail were marked, and the distance between them was evaluated by a digital caliper of 0.01 (BTS digital caliper) accuracy. The abdominal circumference was determined by the circumference of the midpoint between the anterior and posterior legs, a method used by Pini et al. (2016). Despite its accuracy, analyzing adipose tissue is a difficult and costly method when compared to basic murine measurements, which makes it usual to measure these measurements in most of the work in rats. Thus, it is relevant to evaluate whether there is a correlation between the murine metric indicators and the amount of retroperitoneal adipose tissue. Therefore, the purpose of this study is to verify the correlation between murine measurements and retroperitoneal adipose tissue in high‑fat diet exposed rats. BMI was determined by body weight (g) / length2 (cm) (Novelli et al., 2007) and for the Lee Index, proposed by Lee in 1928, the formula consists of dividing the cubic root weight in grams by the naso-anal length in centimeters (Bernardis and Patterson, 1968). For the collection of retroperitoneal adipose tissue, the adipose tissue was dissected, sterilized in saline and heavy. Resumo Objetivo: Este estudo teve como objetivo verificar a correlação entre medidas murinométricas e tecido adiposo retroperitoneal em ratos expostos à dieta hiperlipídica. Material e métodos: Foram utilizados ratos Wistar machos adultos, descendentes de mães que consumiram dieta hiperlipídica durante a gestação e lactação e alimentados com a mesma dieta após o desmame. Aos 60 dias de vida, foram medidos o peso corporal, o eixo longitudinal e a circunferência da cintura (CC). O Índice de Massa Corporal (IMC) e o Índice de Lee foram calculados para posterior análise da correlação com a quantidade de tecido adiposo retroperitoneal dissecado no mesmo dia. Para análise dos dados, utilizou‑se o teste de correlação de Pearson, considerando significância estatística para p <0.05. Resultados: O peso corporal apresentou uma correlação fraca (r= 0,31; p= 0,38) com o tecido adiposo retroperitoneal. Enquanto o longitudinal correlacionou moderadamente e negativo (r= -0,40; p= 0,25). A circunferência abdominal (r = 0,62; p = 0,05), índice de massa corporal (r= 0,61; p= 0,03) e Lee (r= 0,69; p= 0,03) correlacionaram-se moderada e positivamente com o tecido adiposo. Conclusão: Entre as medidas murinométricas, o peso e o eixo longitudinal não foram bons indicadores para representar o acúmulo de tecido adiposo retroperitoneal em ratos. No entanto, o índice de Lee parece ser o melhor indicador murinométrico para diagnosticar o acúmulo de gordura retroperitoneal. O IMC, índice de Lee e CA foram parâmetros murinométricos com maior correlação. lavras-chave: dieta hiperlipídica, obesidade, ratos, índice murinométrico, tecido adiposo retroperitoneal. Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 246 Murinometric measurements and retroperitoneal adipose tissue Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 4. Discussion The studies that investigated, the longitudinal axis, another murinometric parameter, are few. It is mainly measured to perform the calculation of BMI and the Lee index (Sospedra et al., 2015). The results of this analysis showed a moderate and negative correlation with adipose tissue, without statistical significance. It may be that the longitudinal growth of the animal has contributed to a greater distribution of body fat (Nascimento et al., 2008). In the present study, the high-fat diet-induced accumulation of retroperitoneal adipose tissue in rats and the correlation with the commonly used murine measurements was used to diagnose obesity. Among the measured murine measurements, BMI, CA and Lee index were murine parameters with a higher correlation. However, weight and longitudinal axis were not good indicators to represent the accumulation of retroperitoneal adipose tissue in rats. The consumption of foods high in fat and high energy density has increased worldwide (Kratz et al., 2013; Santos et al., 2019). This excessive intake causes an increase in body adiposity and has a strong association with chronic non-transmissible diseases (Kratz et al., 2013). In the present study, a moderate correlation was found between the abdominal circumference and adipose tissue, but no statistical significance. suggesting that this is a good parameter to evaluate the accumulation of retroperitoneal fat. Simillary, Angéloco et al. (2012) identified a moderate and positive correlation for abdominal circumference with carcass fat in rodents submitted to a high-fat or high- sucrose diet. Differently, Gerbaix et al. (2010) considered abdominal circumference as a useful tool after having correlated strongly and significantly with the amount of adipose tissue evaluated by radiographic dual-energy absorptiometry (DEXA) and by dissection of the perirenal and peri-epididymal tissues. According to the results found in this study, the weak correlation of body weight with retroperitoneal adipose tissue, without statistical significance. May be related to the fact that weight does not evaluate body composition (Gerbaix et al., 2010). Considering that body weight is determined by bone, muscle and adipose tissues, it may be that changes in the amount of bone and muscle tissues have influenced the results found. Corroborating with the findings in this study, Lac et al. (2008), rodents fed a high-fat diet in adulthood showed lower levels of bone minerals, bone mineral density, and skeletal area. Similarly, Pomar et al. 2.1. Experimental design The animals were from primiparous females fed with high-fat during pregnancy and lactation. Wistar rats were 247 Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 Macêdo, A.P.A. et al. Table 1. Murine measurements and amount of retroperitoneal adipose tissue in young rats submitted to the high-fat diet. N Mean Standard deviation Weight (g) 10 199.52 12.54 Longitudinal axis (cm) 10 20.34 0.57 Abdominal Circumference (cm) 10 14.81 0.59 Body Mass Index (g.cm-2) 10 0.48 0.03 Lee Index ( g.cm-1) 10 0.29 0.01 Retroperitoneal adipose tissue (g) 10 4.50 0.95 Table 2. Correlation coefficient between the murinometric indicators and the amount of retroperitoneal adipose tissue in young rats submitted to the high-fat diet. r p-value Weight (g) 0.31 0.38 Longitudinal axis (cm) -0.40 0.25 Abdominal Circumference (cm) 0.62 0.05 Body Mass Index (g.cm-2) 0.61 0.06 Lee Index ( g.cm-1) 0.69* 0.03 * Statistically significant correlation, p <0.05. and negative (r = -0.40, p=0.25) with the amount of retroperitoneal adipose tissue. Abdominal circumference (r = 0.62, p=0.05), body mass index (r = 0.61, p=0.03) and Lee (r = 0.69, p=0.03) correlated moderately and positively with adipose tissue. Only Lee’s index showed a statistically significant correlation (Table 2). and negative (r = -0.40, p=0.25) with the amount of retroperitoneal adipose tissue. Abdominal circumference (r = 0.62, p=0.05), body mass index (r = 0.61, p=0.03) and Lee (r = 0.69, p=0.03) correlated moderately and positively with adipose tissue. Only Lee’s index showed a statistically significant correlation (Table 2). Obesity causes chronic inflammation that can affect the integrity and function of tissues. In muscle tissue, there is a greater deposition of adipose tissue. This promotes specific changes in the metabolism of muscle proteins, impairing protein synthesis in the muscle (Tardif et al., 2014; Collins et al., 2016). This mechanism justifies the possible loss of lean mass in the animals studied, and, consequently, the loss of weight. Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 Murinometric measurements and retroperitoneal adipose tissue Current Obesity Reports, vol. 7, no. 4, pp. 1-7. http://dx.doi. org/10.1007/s13679-018-0323-x. PMid:30349968. Current Obesity Reports, vol. 7, no. 4, pp. 1-7. http://dx.doi. org/10.1007/s13679-018-0323-x. PMid:30349968. a strong and positive correlation. However, in the work of Novelli et al. (2007), the result of the classification obtained in his study was a very strong correlation. This difference in the intensity of classification can be attributed to the fact that the researchers used a different classification for the correlation values and because they evaluated the carcass fat. And BMI accurately comprises calculating body mass, not retroperitoneal mass. BERNARDIS, L. L. and PATTERSON B. D. 1968, Correlation between ‘Lee index’ and carcass fat content in weanling and adult female rats with hypothalamic lesions. Journal of Endocrinology, vol. 40, no. 4, pp. 527-528. https://doi.org/10.1677/joe.0.0400527 CASPER, R.C., SULLIVAN, E.L. and TECOTT, L., 2008. Relevance of animal models to human eating disorders and obesity. Psychopharmacology, vol. 199, no. 3, pp. 313-329. http://dx.doi. org/10.1007/s00213-008-1102-2. PMid:18317734. Contrary to body mass index, the Lee index was developed specifically for rats to evaluate obesity in these animals (Bernardis and Patterson, 1968). As the Lee index is a specific indicator for rats, this specificity may have resulted in a moderate correlation with statistically significant found with the amount of adipose tissue. Angelóco et al. (2012) showed a positive and strong correlation between the Lee index and carcass fat, suggesting that the Lee index is a good indicator to evaluate body fatness. The difference in the intensity of the classification, as well as the BMI, is because Angelóco et al. (2012) analyzed the total fat of the carcass of the animal. COLLINS, K.H., HART, D.A., REIMER, R.A., SEERATTAN, R.A., WATERS‐BANKER, C., SIBOLE, S.C. and HERZOG, W., 2016. High‐fat high‐sucrose diet leads to dynamic structural and inflammatory alterations in the rat vastus lateralis muscle. Journal of Orthopaedic Research, vol. 34, no. 12, pp. 2069-2078. http:// dx.doi.org/10.1002/jor.23230. PMid:26990324. DUTRA, M.T., MARTINS, K.G., VIEIRA, D.D.R., OLIVEIRA, A.S. and MOTA, M.R., 2018. Association between adiposity indices and blood pressure is stronger in sarcopenic obese women. Current Hypertension Reviews, vol. 14, no. 1. http://dx.doi.org/1 0.2174/1573402114666181031145341. PMid:30381084. DUTRA, M.T., MARTINS, K.G., VIEIRA, D.D.R., OLIVEIRA, A.S. and MOTA, M.R., 2018. Association between adiposity indices and blood pressure is stronger in sarcopenic obese women. Current Hypertension Reviews, vol. 14, no. 1. http://dx.doi.org/1 0.2174/1573402114666181031145341. PMid:30381084. GERBAIX, M., METZ, L., RINGOT, E. and COURTEIX, D., 2010. Murinometric measurements and retroperitoneal adipose tissue Visceral fat mass determination in rodent: validation of dual-energy X-ray absorptiometry and anthropometric techniques in fat and lean rats. Lipids in Health and Disease, vol. 9, no. 1, pp. 140. http://dx.doi.org/10.1186/1476-511X-9-140. PMid:21143884. 5. Conclusion The high-fat diet was able to promote the accumulation of retroperitoneal fat in animals exposed to the diet until adult life. Considering the results of the present study, it can be concluded that the weight and the longitudinal axis did not present a good correlation with retroperitoneal adipose tissue in young rats, but the BMI, CA and the Lee index were murine parameters with higher correlation. Lee’s index seems to be the best murine marker to diagnose retroperitoneal fat accumulation. Therefore, scientific research using rodents to simulate human obesity should investigate the murine measurements more carefully and associate these with other indicators to ensure the reliability of the results reported. HARIRI, N. and THIBAULT, L., 2010. High-fat diet-induced obesity in animal models. Nutrition Research Reviews, vol. 23, no. 2, pp. 270-299. http://dx.doi.org/10.1017/S0954422410000168. PMid:20977819. KRATZ, M., BAARS, T. and GUYENET, S., 2013. The relationship between high-fat dairy consumption and obesity, cardiovascular, and metabolic disease. European Journal of Nutrition, vol. 52, no. 1, pp. 1-24. http://dx.doi.org/10.1007/s00394-012-0418-1. PMid:22810464. LAC, G., CAVALIE, H., EBAL, E. and MICHAUX, O., 2008. Effects of a high fat diet on bone of growing rats. Correlations between visceral fat, adiponectin and bone mass density. Lipids in Health and Disease, vol. 7, no. 1, pp. 16. http://dx.doi. org/10.1186/1476-511X-7-16. PMid:18442361. 4. Discussion (2017), the offspring of rats fed a diet with high lipid content during lactation had lower body weight and lower lean mass, but a greater amount of adipose tissue. Regarding BMI, a moderate and positive correlation was evidenced, with no statistical significance. It is important to consider, during the BMI analysis, that this indicator was formulated to assess the nutritional status of humans (Dutra et al. 2018). With this in mind, great care must be taken in adapting this parameter to animals. In agreement with the results of this work, Angéloco et al. (2012) observed Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 248 Murinometric measurements and retroperitoneal adipose tissue Acknowledgements To the Foundation for Research Support of the State of Bahia (FAPESB), the Coordination of Improvement of Higher Education Personnel (CAPES) and the National Research Council (CNPq) for financial support. LICHOLAI, J.A., NGUYEN, K.P., FOBBS, W.C., SCHUSTER, C.J., ALI, M.A. and KRAVITZ, A.V., 2018. Why do mice overeat high‐fat diets? how high‐fat diet alters the regulation of daily caloric intake in mice. Obesity, vol. 26, no. 6, pp. 1026-1033. http://dx.doi.org/10.1002/oby.22195. PMid:29707908. References LIMA, M.S., PEREZ, G.S., MORAIS, G.L., SANTOS, L.S., CORDEIRO, G.S., COUTO, R.D., DEIRÓ, T.C.B.J., LEANDRO, C.G. and BARRETO-MEDEIROS, J.M., 2018. Effects of maternal high fat intake during pregnancy and lactation on total cholesterol and adipose tissue in neonatal rats. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 78, no. 4, pp. 615-618. http://dx.doi.org/10.1590/1519-6984.166788. PMid:29319751. ALMEIDA, J.B., KIAN, K.O., LIMA, R.C. and SOUZA, M.C.C., 2016. Total and abdominal adiposity and hypertension in indigenous women in Midwest Brazil. PLoS One, vol. 11, no. 6, pp. 1-20. http://dx.doi.org/10.1371/journal.pone.0155528. PMid:27294870. ANGÉLOCO, L.R.N., DEMINICE, R., LEME, I.A., LATARO, R.C. and JORDÃO, A.A., 2012. Bioelectrical impedance analysis and anthropometry for the determination of body composition in rats: effects of high-fat and high-sucrose diets. Revista de Nutrição, vol. 25, no. 3, pp. 331-339. http://dx.doi.org/10.1590/ S1415-52732012000300003. LUZ, F. Q, SAINSBURY, A., MANNAN H., TOUYZ, S., MITCHISON,D. and HAY, P., 2017. Prevalence of obesity and comorbid eating disorder behaviors in South Australia from 1995 to 2015. International Journal of Obesity, vol. 41, pp. 1148-1153. http://dx.doi.org/10.1038/ijo.2017.79. PMid:28337025. MCNAY, D.E.G. and SPEAKMAN, J.R., 2013. High fat diet causes rebound weight gain. Molecular Metabolism, vol. 2, no. BANIK, S. and RAHMAN, M., 2018. Prevalence of overweight and obesity in Bangladesh: A systematic review of the literature. Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 249 Macêdo, A.P.A. et al. 2, pp. 103-108. http://dx.doi.org/10.1016/j.molmet.2012.10.003. PMid:24199147. 2, pp. 103-108. http://dx.doi.org/10.1016/j.molmet.2012.10.003. PMid:24199147. POMAR, C.A., VAN NES, R., SÁNCHEZ, J., PICÓ, C., KEIJER, J. and PALOU, A, 2017. Maternal consumption of a cafeteria diet during lactation in rats leads the offspring to a thin-outsidefat-inside phenotype. International Journal of Obesity, vol. 41, no. 8, pp. 1279-2017. http://dx.doi.org/10.1038/ijo.2017.42. PMid:28190874. MITTWEDE, P.N., CLEMMER, J.S., BERGIN, P.F. and XIANG, L., 2016. Obesity and critical illness: insights from animal models. Shock, vol. 45, no. 4, pp. 349-358. https://dx.doi.org/10.1097%2 FSHK.0000000000000512 SANTOS, L.S., CORDEIRO, G.S., PEREZ, G.S., SANTO, D.A.E., MACÊDO, A.P.A., LIMA, M.S., CARNEIRO, I.B.C., MACHADO, M.E.P.C., DEIRÓ, T.C.B.J. and BARRETO-MEDEIROS, J.M., 2019. Influence of mother nutrition during pregnancy and/or lactation on offspring food preference in experimental models. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 79, no. 2, pp. 220-232. http://dx.doi.org/10.1590/1519- 6984.179134. PMid:30156603. NASCIMENTO, A.F., SUGIZAKI, M.M., LEOPOLDO, A.S., LIMA-LEOPOLDO, A.P., NOGUEIRA, C.R., NOVELLI, E.L., PADOVANI, C.R. and CICOGNA, A.C., 2008. Misclassification probability as obese or lean in hypercaloric and normocaloric diet. Biological Research, vol. 41, no. 3, pp. 253-259. http:// dx.doi.org/10.4067/S0716-97602008000300002. PMid:19399338. References NASREDDINE, L.M., KASSIS, A.N., AYOUB, J.J., NAJA, F.A. and HWALLA, N.C., 2018. Nutritional status and dietary intakes of children amid the nutrition transition: the case of the Eastern Mediterranean Region. Nutrition Research, vol. 57, pp. 12-27. http://dx.doi.org/10.1016/j.nutres.2018.04.016. SIQUEIRA, A.L. and TIBÚRCIO, J.D. 2011, Estatística na área da saúde: conceitos, metodologia, aplicações e prática computacional. Belo Horizonte, MG: Coopmed. SOSPEDRA, I., MORAL, R., ESCRICH, R., SOLANAS, M., VELA, E. and ESCRICH, E., 2015. Effect of high fat diets on body mass, Oleylethanolamide plasma levels and oxytocin expression in growing rats. Journal of Food Science, vol. 80, no. 6, pp. H1425-H1431. http://dx.doi.org/10.1111/1750-3841.12890. PMid:25976631. NOVELLI, E.L.B., DINIZ, S., GALHARDI, C.M., EBAID, G.M.X., RODRIGUES, H.G., MANI, F., FERNANDES, A.A., CICOGNA, A.C. and NOVELLI FILHO, J.L., 2007. Anthropometrical parameters and markers of obesity in rats. Laboratory Animals, vol. 41, no. 1, pp. 111-119. http://dx.doi. org/10.1258/002367707779399518. PMid:17234057. SOUZA, R.M., SOUZA, L., MACHADO, A.E., DE BEM ALVES, A.C., RODRIGUES, F.S., AGUIAR JUNIOR, A.S., SANTOS, A.R.S., DE BEM, A.F. and MOREIRA, E.L.G., 2018. Behavioural, metabolic and neurochemical effects of environmental enrichment in high-fat cholesterol-enriched diet-fed mice. Behavioural Brain Research, vol. 359, pp. 648-656. http://dx.doi.org/10.1016/j. bbr.2018.09.022. PMid:30287273. OLIVEIRA, T.W.S., LEANDRO, C.G., JESUS DEIRÓ, T.C.B., PEREZ, G.S., SILVA, D.F., DRUZIAN, J.I., COUTO, R.D. and BARRETO-MEDEIROS, J.M., 2011. A perinatal palatable high- fat diet increases food intake and promotes hypercholesterolemia in adult rats. Lipids, vol. 46, no. 11, pp. 1071-1074. http://dx.doi. org/10.1007/s11745-011-3604-7. PMid:21847693. TARDIF, N., SALLES, J., GUILLET, C., TORDJMAN, J., REGGIO, S., LANDRIER, J.F., GIRAUDET, C., PATRAC, V., BERTRAND-MICHEL, J., MIGNE, C., COLLIN, M.L., CHARDIGNY, J.M., BOIRIE, Y. and WALRAND, S., 2014. Muscle ectopic fat deposition contributes to anabolic resistance in obese sarcopenic old rats through eIF2α activation. Aging Cell, vol. 13, no. 6, pp. 1001-1011. http://dx.doi.org/10.1111/ acel.12263. PMid:25139155. PÉREZ, G.S., SANTOS, L.S., CORDEIRO, G.S., PARAGUASSÚ, G.M., ATHANAZIO, D.A., COUTO, R.D., DEIRÓ, T.C.B.J., CASTRO, R.M. and BARRETO-MEDEIROS, J.M., 2015. Maternal and postweaning exposure to a high fat diet promotes visceral obesity and hepatic steatosis in adult rats. Nutrición Hospitalaria, vol. 32, no. 4, pp. 1653-1658. http://dx.doi. org/10.3305/nh.2015.32.4.9607. PMid:26545531. PINI, R.T.B., VALES, L.D.M.F., COSTA, T.M.B.C. and ALMEIDA, S.S., 2016. Effects of cafeteria diet and high fat diet intake on anxiety, learning and memory in adult male rats. Nutritional Neuroscience, vol. 20, no. 7, pp. 396-408. http://dx.doi.org/10.1 080/1028415X.2016.1149294. PMid:28277186. WEST, D. B. and YORK, B., 1998. Dietary fat, genetic predisposition, and obesity: Lessons from animal models. Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 References The American journal of clinical nutrition, vol. 67, no. 3, pp. 505S-512S. https://doi. org/10.1093/ajcn/67.3.505S 250 Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250
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http://jurnal.fk.unand.ac.id/index.php/jka/article/download/859/723
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Injeksi Kortikosteroid Intratimpani Sebagai Salvage Therapy pada Pasien Tuli Mendadak
Jurnal kesehatan Andalas/Jurnal Kesehatan Andalas
2,018
cc-by-sa
4,403
http://jurnal.fk.unand.ac.id Laporan Kasus Injeksi Kortikosteroid Intratimpani Sebagai Salvage Therapy pada Pasien Tuli Mendadak Ricko Mariza Putra1, Jacky Munilson2, Yan Edward2, Nirza Warto2, Rossy Rosalinda2 Abstrak Pendahuluan : Tuli mendadak merupakan salah satu kegawatdaruratan di bagian telinga hidung tenggorok bedah kepala dan leher (THT-KL). Tuli mendadak atau sudden sensorineural hearing loss (SSNHL) adalah tuli sensorineural lebih dari 30 dB pada 3 frekuensi berturut turut secara mendadak dalam waktu 3 hari. Etiologi tuli mendadak hingga saat ini belum diketahui dengan pasti, namun penyebab tersering tuli mendadak, yaitu idiopatik (71%). Penatalaksanaan tuli mendadak meliputi terapi konservatif, salah satunya dengan pemberian kortikosteroid secara sistemik dan lokal. Pemberian lokal dapat dilakukan dengan cara injeksi langsung intratimpani. Terapi kortikosteroid secara lokal dapat diberikan sebagai terapi primer, terapi adjuvan (kombinasi) dan salvage therapy. Laporan kasus : seorang pasien perempuan berusia 36 tahun dengan diagnosis tuli mendadak pada telinga kanan yang dilakukan salvage therapy dengan penyuntikan deksametason intratimpani sebanyak empat kali secara selang 3 hari setelah gagal dengan terapi sistemik. Kesimpulan : Injeksi kortikosteroid intratimpani digunakan sebagai salvage therapy dapat menjadi pilihan untuk pasien yang gagal diterapi dengan kortikosteroid sistemik. Kata kunci: tuli mendadak; salvage therapy; deksametason; intratimpani Abstract Introduction: Sudden deafness is one of the emergency in otorhinolaryngology head and neck surgery (ORLHNS). Sudden deafness or sudden sensorineural hearing loss (SSNHL) is sensorineural deafness of more than 30 dB at 3 frequencies within 3 days. The etiology of sudden deafness is recently remain unknown, but the most common cause of deafness is idiopathic (71%). Management of sudden deafness includes conservative therapy, one of which is corticosteroids systemically and locally. Local delivery is done by intratympanic injection. Local corticosteroid therapy can be administered as primary therapy, adjuvant therapy (combination) or salvage therapy. Case report : a female patient aged 36 years old with diagnosis of sudden deafness at right ear which is performed salvage therapy with dexamethasone intratympanic injection four times with interval of 3 days after systemic therapy was failed. Conclusion : corticosteroid intratympanic injection as salvage therapy may be an option for patients who failed therapy with systemic corticosteroids. Keywords: sudden deafness, salvage therapy, dexamethasone, intratympanic Affiliasi penulis : 1Bagian THT-KL Fakultas Kedokteran Universitas Andalas, 2Bagian THT-KL Fakultas Kedokteran Universitas Andalas Korespondensi : Ricko Mariza Putra, dr.rickomp@yahoo.co.id Telp: 08111891391 PENDAHULUAN Tuli mendadak merupakan salah satu kegawatdaruratan yang memerlukan penanganan segera, efektif dan tepat. Walaupun pada beberapa kepustakaan menyatakan bahwa tuli mendadak dapat mengalami pemulihan secara spontan, sekitar 32-70 %.1 Etiologi tuli mendadak hingga saat ini belum diketahui dengan pasti, namun terdapat beberapa kemungkinan penyebab tuli mendadak, yaitu idiopatik (71%), penyakit infeksi (12,8%), penyakit telinga (4,7%) seperti penyakit Meniere, otosklerosis, penyakit autoimun telinga bagian dalam, operasi telinga atau dasar otak, trauma (4,2%), vaskular dan hematologik (2,8%), neoplasma (2,3%), serta penyebab lainnya (2,2%).2,3 Derajat ketulian dapat bervariasi mulai dari ringan, sedang, sedang berat, berat dan sangat berat. Telinga yang terkena biasanya unilateral, hanya 1-5% kasus bilateral.4 Penatalaksanaan tuli mendadak meliputi terapi konservatif dengan beberapa modalitas. Penanganan harus dilakukan sedini mungkin karena penanganan yang terlambat akan menyebabkan tuli yang permanen. Pemberian kortikostreroid dapat dilakukan secara sistemik dan lokal. Kortikosteroid lokal dapat diberikan sebagai terapi primer, adjuvan terapi dan salvage therapy.5,6 Anatomi vaskularisasi telinga dalam Vaskularisasi telinga dalam berasal dari a. labirintin yang merupakan cabang dari a. cerebelaris anteroinferior. Arteri ini masuk ke meatus akustikus internus dan bercabang menjadi a. vestibularis anterior dan a. koklearis communis yang kemudian akan bercabang menjadi a. kohlearis dan a. vestibulokohlearis. Arteri vestibularis anterior memperdarahi n. vestibularis, urtikulus dan sebagian duktus semisirkularis. Sedangkan a. vestibulokoklearis sampai di mediolus daerah putaran basal koklea bercabang menjadi cabang terminal vestibularis dan cabang kohlear.7 Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 96 http://jurnal.fk.unand.ac.id Cabang vestibular memperdarahi sakulus, sebagian besar kanalis semisirkularis dan ujung basal kohlea. Cabang kohlear memperdarahi ganglion spiralis, lamina spiralis ossea, limbus dan ligamen spiralis. a. koklearis berjalan mengitari n. akustikus di kanalis akustikus internus dan di dalam kohlea mengitari modiolus7 (Gambar 1). Gambar 1. Vaskularisasi telinga dalam Definisi dan Epidemiologi Tuli mendadak atau sudden deafness atau sudden sensorineural hearing loss (SSNHL) adalah hilangnya pendengaran sensorineural yang lebih dari 30 dB pada 3 frekuensi berturut turut secara mendadak dalam waktu 3 hari, sering unilateral dan idiopatik. Insiden kasus tuli mendadak berkisar 5-20 orang tiap 100.000 orang pertahun.1,4 Penderita tuli mendadak mencapai 1% dari seluruh penderita ketulian di seluruh dunia, dengan 15.000 kasus baru setiap tahunnya.8 Distribusi kejadian antara laki-laki dan perempuan hampir sama, dengan puncak usia 40-54 tahun.4 Sedangkan menurut Fedriani puncak usia 50-60 tahun.8 Telinga yang terkena sebagian besar satu telinga (unilateral), dan hanya 1-5 % kasus yang terjadi pada kedua telinga (bilateral).4 Etiopatogenesis Penyebab tuli mendadak hingga saat ini belum diketahui dengan pasti, namun banyak teori yang mengemukakan faktor resiko penyebab terjadinya tuli mendadak. Sekitar 10 – 15 % penyebab tuli mendadak yang dapat teridentifikasi.4,8 Empat teori utama yang dapat menjelaskan terjadinya tuli mendadak diantaranya infeksi virus, gangguan pembuluh darah, ruptur membran intra koklea dan penyakit autoimun telinga dalam.4 Diagnosis Diagnosis ditegakkan berdasarkan anamnesis, pemeriksaan fisik, audiometri dan pemeriksaan penunjang lainnya. Anamnesis didapatkan hingga 80% datang dengan keluhan telinga penuh. Tinitus terjadi pada sekitar 80% pasien dan vertigo 30%. Pemeriksaan fisik umum, seperti tekanan darah, nadi, nafas, suhu serta pemeriksaan lokalis THT-KL. Pasien dengan tuli sensorineural hampir selalu memiliki pemeriksaan otoskopi yang normal.9,10 Pemeriksan pendengaran dengan penala didapatkan Rinne positif, Weber lateralisasi ke telinga yang sehat, Schwabach memendek. Pada audiometri nada murni didapatkan tuli sensorineural ringan hingga sangat berat.9-11 Tuli mendadak juga dapat dievaluasi dengan speech reception threshold, speech discrimination score, refleks stapedius, timpanogram dan tone decay.11 Pemeriksaan penunjang lainnya dapat juga diperiksa seperti pada (Tabel 1) Tabel 1. Pemeriksaan laboratorium dan pencitraan4 Test Tujuan Darah lengkap Polisitemia, leukemia, dengan trombositosis differensial Tingkat Skrining penyakit auto immun sedimentasi, LED atau inflamasi, diikuti dengan test ANA atau 68 kD Ab Tes koagulasi Koagulopati (jika ada indikasi, atau riwayat) Tes fungsi tiroid Hypothyroidism (jika ada indikasi, atau riwayat) FTA-Abs atau Tes antibodi, Treponema MHA-TP pallidum, kongenital atau didapat MRI Lesi Retrocochlear, multiple sclerosis, perdarahan intra koklea Tomografi kelainan kapsul otic pada pasien komputer pediatrik 68 KD Ab, 68 kiloDalton antibodi; ANA. antibodi antinuclear; FTA-Abs,Fluorescent treponemal anibodyabsoption test ; MHA-TP. microhemagglutinationTreponema pallidum Penatalaksanaan Pengobatan tuli mendadak idealnya didasarkan pada penyebabnya tetapi umumnya bersifat idiopatik, sehingga pengobatan dilakukan secara empiris. Terapi tuli mendadak umumnya berupa steroid sistemik, vasodilator (histamin, papaverin, verapamil, carbogen), hemodilusi (dextran, pentoxifylline, manitol, dan heparin), atau antiviral (acyclovir, valacyclovir). Terapi lainnya dapat berupa oksigen hiperbarik 100% 2 hingga 2,5 ATA selama 90 menit setiap hari selama 10-20 kali terapi. Selain itu, pasien dianjurkan tirah baring selama 2 minggu, diet rendah garam dan rendah kolesterol, penggunaan neurotonik, vitamin C, vitamin E, dan preparat herbal (gingko biloba).8,9 Pemberian kortikosteroid pada kasus tuli mendadak bervariasi dari segi jenis, dosis maupun teknik pemberian (Tabel 2) : a. Terapi kortikosteroid sistemik Kortikosteroid sistemik yang paling sering digunakan antara lain prednison, metilprednisolon dan deksametason. Prednison diberikan dengan dosis 1 mg/kg berat badan/hari atau maksimal 60 mg/hari tapering off selama 12-15 hari. Metilprednisolon diberikan dengan dosis 48 mg tapering off. Deksametason diberikan dengan dosis 10 mg/hari tapering off. Efek samping yang ditimbulkan penggunaan kortikosteroid antara lain insomnia, dizziness, gastritis, kenaikan berat badan, berkeringat, Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 97 http://jurnal.fk.unand.ac.id perubahan mood, hiperglikemia dan fotosensitif. Efek samping yang cukup berat, tetapi jarang ditemukan yakni pankreatitis, perdarahan, hipertensi, katarak, miopati, infeksi oportunistik, osteoporosis, dan osteonekrosis.2 Pasien dengan kondisi medis sistemik, seperti insulin-dependent diabetes mellitus (IDDM), diabetes tidak terkontrol, hipertensi labil, tuberkulosis, dan ulkus peptikum tidak diajurkan terapi kortikosteroid sistemik.2 b. Terapi kortikosteroid intratimpani Injeksi kortikosteroid intratimpani pertama kali dilaporkan penggunaannya oleh Itoh (1991) untuk pengobatan penyakit telinga bagian dalam pada pasien dengan Penyakit Meniere. Pada kasus tuli mendadak terapi kortikosteroid intratimpani pertama kali dilaporkan oleh Silverstein (1996). Meskipun efektifitasnya belum terbukti secara definitif, terapi kortikosteroid intratimpani untuk tuli sensorineural menjadi lebih banyak digunakan.5,6 Protokol pengobatan tuli mendadak dengan kortikosteroid intratimpani terdiri dari:5,6,8,9 • Terapi Primer; terapi intratimpani sebagai pengobatan pertama untuk tuli mendadak, tanpa kortikosteroid sistemik • Terapi Adjuvan; terapi diberikan bersamaan dengan sistemik kortikosteroid • Salvage therapy: terapi dimulai setelah terapi kortikosteroid sistemik gagal. Pemberian kortikosteroid intratimpani sebagai terapi primer biasanya pada kelompok pasien yang tidak bisa mentolerir efek samping sistemik dosis tinggi dari terapi kortikosteroid sistemik, seperti pasien diabetes atau tekanan darah tinggi yang sulit untuk dikontrol. Sebuah studi mengenai terapi kombinasi kortikosteroid sistemik dosis tinggi dan kortikosteroid intratimpani menunjukkan hasil perbaikan fungsi pendengaran secara signifikan. Namun, studi lainnya tidak menghasilkan perbedaan pemulihan pendengaran antara terapi kombinasi kortikosteroid oral dan intratimpani dengan terapi kortikosteroid oral saja.12 Kebanyakan penggunaan kortikosteroid intratimpani dilakukan setelah pengobatan tuli mendadak menggunakan kortikosteroid sistemik gagal. Jumlah yang disuntikkan antara 0,3 – 0,5 ml, sesuai dengan perkiraan volume ruang telinga tengah. Normalnya pada dewasa volume telinga tengah 0,6 1,5 ml. Periode waktu dan jumlah penyuntikan juga berbeda-beda yang bervariasi mulai dari 3 hari sampai 7 hari selama 3 sampai 4 kali pemberian (Tabel 2).8,9 Metoda yang dilakukan juga bervariasi : penyuntikan melalui membran timpani langsung dengan jarum suntik (Gambar 2a), melalui miringotomi dengan tabung (grommet) (Gambar 2b), menggunakan pipa (wick) yang diletakkan pada miringotomi (Gambar 2c), atau melalui pompa yang ditanamkan untuk mengantarkan steroid secara konstan (Gambar 2d).13 Tabel 2. Pedoman umum terapi kortikosteroid pda tuli mendadak Oral Intratimpani Kortikosteroid Kortikosteroid Waktu Segera, ≤ 14 Segera, pengobatan hari pertama. Salvage (rescue) Keberhasilan setelah pengobatan dilaporkan sistemik gagal hingga 6 minggu onset tuli mendadak Dosis Prednison Dexamethasone 1 mg/kg/hr 24 mg/ml atau 16 (maks 60 mg/hr) mg/ml (ditambah), atau atau 10 mg/ml Methylprednisol (stok) one 48 mg/hr Methylprednisolone atau 40 mg/ml atau 30 Deksametason mg/ml 10 mg/hr Durasi/ Dosis penuh Injeksi 0,4-0,8 ml frekuensi selama 7-14 ke telinga tengah hari, dilanjutkan setiap 3-7 hari dengan untuk 3-4 sesi tappering Teknik Jangan Diberikan obat bius membagi dosis topikal, lalu penyuntikkan ke kuadran posterior inferior dengan jarum suntik untuk mengisi ruang telinga tengah. Kepala posisi otologic (telinga yang sakit berada diatas) selama 1530 menit Pemantauan Audiogram : Audiogram : Setelah selesai Sebelum setiap pengobatan dan suntikan, setelah pada delayed selesai pengobatan interval dan pada delayed interval Periksa membran timpani (TM) untuk memastikan penyembuhan setelah selesai pengobatan dan pada delayed interval Modifikasi Efek samping Dapat dipasang seperti insomnia tabung bertekanan Memantau jika direncanakan hiperglikemia, beberapa kali dan hipertensi suntikan, tetapi pada pasien meningkatkan risiko yang rentan perforasi membran timpani Dapat juga dipertimbangkan menambahkan jalur transportasi jendela bundar Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 98 http://jurnal.fk.unand.ac.id Gambar 2. Metoda penyuntikan (a) membran timpani langsung dengan jarum suntik (b) melalui tabung (grommet), (c) menggunakan pipa (wick), (d) melalui pompa yang ditanamkan. Prognosis Prognosis dan keberhasilan penatalaksanaan tuli mendadak dipengaruhi oleh beberapa faktor diantaranya usia, onset kehilangan pendengaran, derajat ketulian, jenis audiogram pertama ada tidaknya vertigo, ada tidaknya tinnitus, respon 2 minggu pengobatan pertama, dan adanya faktor predisposisi.2,4,11 Derajat ketulian menurut ISO diklasifikasikan sebagai ringan (>25-40), sedang (>40-55), sedang berat (>55-70), berat (>70-90), sangat berat (>90), sesuai rata-rata nada murni pada frekuensi 500Hz, 1000Hz, 2000Hz, 4000Hz.10 Semakin berat derajat ketulian maka prognosis menjadi lebih rendah.14 Tipe kehilangan pendengaran diklasifikasikan menjadi 4 kelompok yaitu ascending type (frekuensi 250-500Hz), descending type (frekuensi 4000-8000), flat type (<20dB perbedaan antara yg terbaik dan terburuk), total atau sub total (>85dB).14 Ascending type memiliki prognosis yang lebih baik bila dibandingkan dengan descending, flat serta total atau sub total. Sedangkan menurut Atay pembagian tipe kehilangan pendengaran terdiri dari upward-sloping type (gangguan lebih berat pada frekuensi rendah), downward-sloping type (gangguanlebih berat pada frekuensi tinggi), flat type (tidak lebih dari 10dB dari ambang dengar) dan profound type (lebih dari 70dB pada semua frekuensi).1 Awal mulai pengobatan oleh Kasaplogu dkk dibagi menjadi 2 kelompok yaitu early (5 hari pertama), late (hari ke 5-15).14 Pengobatan dimulai kurang dari lima hari akan memberikan hasil yang lebih baik. Vertigo dapat digunakan sebagai indikator tingkat keparahan lesi dan berhubungan dengan prognosis pengobatan yang rendah. Menurut Kasaplogu menyimpulkan hasil penelitian Danino bahwa adanya gejala tinitus mempengaruhi reversibilitas hingga 80%. Usia lanjut, hipertensi, diabetes dan hiperlipidemia berkaitan dengan disfungsi mikrovaskuler di koklea yang akan memperburuk prognosis kesembuhan.1,11,14 Namun pasien tuli mendadak yang tidak diobati dapat juga mengalami pemulihan spontan. Menurut Jones seperti yang dikutip oleh Conlin menyatakan tuli mendadak dapat mengalami pemulihan spontan sekitar 32-70%.15 Menurut Matox seperti yang dikutip oleh Kasapoglu menyatakan sekitar 65% kasus tuli mendadak terjadi pemulihan spontan, sedangkan Battaglia menyatakan sekitar 32-64% tuli mendadak dapat mengalami pemulihan spontan.14 Evaluasi fungsi pendengaran dilakukan setiap minggu selama satu bulan. Kriteria perbaikan pendengaran menurut Kallinen: Sangat baik, apabila perbaikan lebih dari 30 dB pada 5 frekuensi. Sembuh, apabila perbaikan ambang pendengaran kurang dari 30 dB pada frekuensi 250 Hz, 500 Hz, 1000Hz, 2000 Hz dan di bawah 25 dB pada frekuensi 4000 Hz. Baik, apabila rerata perbaikan 10-30 dB pada 5 frekuensi. Tidak ada perbaikan, apabila terdapat perbaikan kurang dari 10 dB pada 5 frekuensi.11 Evaluasi menurut Ho seperti yang dikutip oleh Filipo menyatakan pembagian evaluasi menjadi 4 kelompok yaitu pulih total dengan ambang dengar kurang dari 25 dB, pulih bermakna dengan peningkatan ambang dengar lebih dari 30 dB, pulih minimal dengan peningkatan ambang dengar sekitar 10-30 dB, dan tidak terjadi pemulihan dengan peningkatan ambang dengar kurang dari 10 dB.16 Sedangkan Siegel seperti yang dikutip oleh Ferri dkk membagi kriteria perbaikan pendengaran setelah 3 bulan terapi menjadi : sembuh sempurna bila perbaikan lebih dari 30 dB dan pendengaran lebih baik dari 25 dB, penyembuhan parsial bila perbaikan lebih dari 15 dB dan pendengaran antara 25–45 dB, penyembuhan ringan (slight) bila perbaikan lebih dari 15 dB tetapi pendengaran lebih buruk dari 45 dB, tidak ada perbaikan bila perbaikan kurang dari 15 dB dan pendengaran lebih buruk dari 75 dB.17 LAPORAN KASUS Seorang pasien perempuan usia 36 tahun datang ke IGD RSUP dr. M. Djamil Padang dengan keluhan telinga kanan tiba-tiba tidak dapat mendengar sejak 2 hari yang lalu. Keluhan ini dirasakan saat pasien bangun tidur. Telinga kanan dirasakan berdenging kadang bergemuruh sejak kejadian dan terus menerus. Pasien sudah berobat ke Spesialis THT-KL di klinik swasta di kota Padang dan telah dilakukan pemeriksaan audiometri serta disarankan untuk dirawat. Riwayat kedua telinga berair sebelumnya tidak ada, nyeri pada kedua telinga tidak ada, riwayat trauma tidak ada, riwayat terpapar bising tidak ada. Riwayat demam, batuk dan pilek tidak ada. Pusing berputar tidak ada. Riwayat sakit kepala hebat tidak ada. Riwayat diabetes dan tekanan darah tinggi tidak ada. Riwayat stroke dan penyakit jantung tidak ada. Pasien bekerja sebagai staf di kantor notaris. Satu minggu terakhir pasien bekerja lembur. Pemeriksaan status generalis didapatkan keadaan umum sedang, kesadaran komposmentis kooperatif, frekuensi nafas 18 kali permenit, frekuensi nadi 80 kali permenit, tekanan darah 120/80 mmHg Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 99 http://jurnal.fk.unand.ac.id dan suhu 36,9° C, Berat badan 65 Kg. Pemeriksaan telinga kanan dan kiri didapatkan liang telinga lapang, membran timpani utuh, refleks cahaya positif. Tes penala didapatkan Rinne positif pada telinga kanan dan kiri, Weber lateralisasi ke kiri, Schwabach telinga kanan memendek, telinga kiri sama dengan pemeriksa. Kesan tuli sensorineural pada telinga kanan. Pada pemeriksaan hidung dan tenggorok dalam batas normal. Tes keseimbangan sederhana dapat dilakukan. Pemeriksaan audiometri nada murni didapatkan kesan telinga kanan tuli sensorineural derajat sangat berat dengan ambang dengar 103,75 dB dan telinga kiri normal dengan ambang dengar 11,25 dB (Gambar 3). spesialis penyakit dalam untuk hiperkoagulasi dan dari bagian penyakit dalam pasien diterapi dengan heparin 2 x 5000 sc. Hari ke-4 rawatan, pasien masih mengeluhkan kurang pendengaran, telinga berdenging belum berkurang, demam tidak ada, pusing berputar tidak ada, mual muntah tidak ada, nyeri ulu hati tidak ada. Tanda vital dalam batas normal. Tes penala 512 Hz didapatkan Rinne positif pada telinga kanan dan telinga kiri, Weber lateralisasi ke kiri, Schwabach telinga kanan memendek dan kiri normal. Kesan tuli sensorineural pada telinga kanan. Terapi diteruskan. Hari ke-10 rawatan, pasien masih mengeluhkan kurangnya pendengaran, telinga berdenging berkurang, pusing tidak ada, demam tidak ada, mual muntah tidak ada, nyeri ulu hati tidak ada, merah pada wajah tidak ada. Tanda vital dalam batas normal. Tes penala didapatkan Rinne positif pada telinga kanan dan telinga kiri, Weber lateralisasi ke kiri, Schwabach telinga kanan memendek dan kiri normal. Kesan tuli sensorineural pada telinga kanan. Pemeriksaan audiometri nada murni didapatkan telinga kanan tuli sensorineural derajat sangat berat dengan ambang dengar 97,5 dB (gambar 4). Gambar 3. Audiogram saat pasien masuk, Tuli sensorineural derajat sangat berat dengan ambang dengar 103,75 dB pada telinga kanan Hasil pemeriksaan laboratorium darah lengkap didapatkan Hb 12,6 gr/dl, leukosit 9100/mm3, trombosit 279.000/mm3, hematokrit 39%, PT 8,9’’, APTT 31,2’’, INR 0,8, gula darah sewaktu 136 mg/dl, kolesterol total 185 mg/dl, HDL 55 mg/dl, LDL 120 mg/dl, trigliserida 52 mg/dl, ureum 24 mg/dl, kreatinin 0,6 mg/dl, natrium 139 mmol/dl, kalium 3,6 mmol/dl, klorida 105 mmol/dl, SGOT 11 u/L, SGPT 10 u/L. Kesan : PT memendek. Pasien didiagnosis dengan tuli mendadak telinga kanan dengan hiperkoagulasi. Pasien dirawat dan diberi terapi sesuai protap tuli mendadak di bagian THT-KL FK Unand RSUP. Dr. M. Djamil Padang yaitu oksigen intranasal 4 liter permenit selama 15 menit tiap 6 jam, metilprednison tablet 16mg peroral tappering off dengan dosis hari IIII dosis 1 x 4 tablet, hari IV-VI dosis 1 x 3 tablet, hari VII-IX dosis 1 x 2 tablet, hari X-XII dosis 1 x 1 tablet. Drip pentoksifilin 300 mg dalam RL 500 cc selama 8 jam, injeksi mekobalamin 3 x 500 mcg IV, injeksi vitamin C 3 x 100 mg IV, gingko biloba 2 x 80 mg peroral, lanzoprazol 1 x 1 tablet peroral. Pasien dianjurkan istirahat total. Pasien dikonsulkan ke dokter Gambar 4. Audiogram hari ke-10 rawatan, Tuli sensorineural derajat sangat berat dengan ambang dengar 97,5 dB pada telinga kanan Hasil audiometri mengambarkan adanya perbaikan sebesar 6,25 dB. Hasil laboratorium ulang PT 9,9’, APTT 29,3’, INR 0,93, kesan normal. Pasien minta rawat jalan karena alasan keluarga. Terapi metilprednison tapering off diteruskan, mekobalamin 3 x 500 mg peroral, pentoksifilin 3 x 400 mg peroral, neurobion 1 x 1 tablet peroral, vitamin C 3 X 100 mg peroral, gingko biloba 2 x 80mg peroral. Terapi heparin dihentikan. Pasien disarankan tetap istirahat total dirumah dan dianjurkan kontrol 5 hari lagi. Pengobatan hari ke-15, pasien kontrol ke poli THT-KL. Pasien masih merasakan kurangnya Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 100 http://jurnal.fk.unand.ac.id pendengaran pada telinga kanan, kemudian dianjurkan untuk menjalani terapi penyuntikan deksametason intratimpani sebanyak 4 kali 0,3 ml (1,5 mg) selang 3 hari. Prosedur penyuntikan sebagai berikut: Pasien tidur telentang dengan kepala diputar sekitar 30o menjauhi telinga yang sakit, telinga dibersihkan, lalu dilakukan aseptik / antiseptik menggunakan betadin, kemudian dilakukan anestesi lokal menggunakan xylocain spray yang disemprotkan ke kapas gulung lalu dimasukkan ke liang telinga sampai ke membran timpani, dibiarkan selama 10 menit lalu kapas diangkat. 0,3 ml deksametason 5 mg/ml disuntikkan ke kavum timpani melalui kuadran posterorinferior membran timpani menggunakan spuit 1 ml dengan jarum no. 23G yang ujungnya dibengkokkan, dengan panduan mikroskop. Sebelumnya larutan deksametason dipanaskan sesuai suhu tubuh (37oC). Setelah penyuntikan, posisi pasien dipertahankan selama 1 jam. Prosedur penyuntikan selesai, evaluasi respon pasien. Pasien kontrol ulang setelah 1 minggu tindakan dengan membawa hasil audiometri, yaitu tuli sensorineural derajat berat dengan ambang dengar 77,5 dB (Gambar 5). Pasien merasakan pendengaran lebih baik dibandingkan sebelum diterapi, telinga berdengung berkurang, pusing berputar tidak ada, nyeri pada telinga tidak ada, keluar cairan dari telinga tidak ada. Tes penala didapatkan Rinne telinga kanan dan kiri positif, Weber lateralisasi ke kiri, Schwabach telinga kanan memendek dan kiri sama dengan pemeriksa. Pasien disarankan kontrol 1 bulan lagi untuk evaluasi perbaikan pendengaran, tetapi pasien tidak kontrol dikarenakan masalah pekerjaan. Evaluasi keluhan pasien melalui telepon mengatakan pendengaran sudah lebih baik dari kontrol terakhir. Gambar 5. Audiogram 1 minggu setelah tindakan, tuli sensorineural derajat berat dengan ambang dengar 77,5 dB pada telinga kanan PEMBAHASAN Telah dilaporkan satu kasus pasien perempuan usia 36 tahun dengan diagnosis tuli mendadak telinga kanan. Tuli mendadak dapat terjadi pada semua usia dengan usia rata-rata 50-60 tahun. Insiden pada laki-laki sama dengan perempuan. Pada kasus ini pasien mendapat terapi kortikosteroid oral selama dua minggu kemudian dilanjutkan dengan terapi penyuntikan deksametason intratimpani sebanyak empat kali secara selang 3 hari, dikarenakan tidak terdapat perbaikan atau gagal terapi. Sebelum diterapi ambang dengar 103,75 dB setelah terapi kortikonsteroid oral menjadi 97,5 dB. Pasien ini menjalani salvage therapy, terapi penyuntikan deksametason intratimpani sebanyak empat kali, (selang 3 hari) dengan dosis 0,3 ml deksametason 5 mg/ml. Hal ini sesuai dengan yang dilakukan Wang pada penelitian di China. Besar dosis yang digunakan tergantung dari volume telinga tengah pasien sebesar 0,66 ml. Penelitian Zhou juga melakukan penyuntikan intratimpani sebanyak 4 kali secara selang hari tetapi dengan metilprednisolon. Efek samping penyuntikan yang dirasakan pada pasien adalah nyeri setelah penyuntikan, pasien mendapat terapi asam mefenamat 500 mg bila nyeri.18,19 Penelitian Lim menyimpulkan bahwa antara terapi oral kortikosteroid, injeksi intra timpani dan terapi kombinasi memberikan hasil yang relatif sama, sehingga dapat digunakan sebagai terapi pertama dalam pengobatan tuli mendadak.20 Penelitian Lee menyimpulkan bahwa terapi kombinasi antara injeksi intra timpani dengan kortikosteroid siistemik lebih baik dari pada kortikosteroid sistemik saja.21 Sedangkan menurut Dispenza dkk menyarankan salvage therapy menggunakan injeksi deksametason intratimpani pada pasien yang mengalami kegagalan terapi pertama kortikosteroid sistemik.22 Pada pasien ini terdapat perbaikan pendengaran dibandingkan awal terapi kortikosteroid 4 minggu setelah tindakan sebesar 26,25 dB. Pada awal terapi 103,75 dB, 2 minggu setelah oral kortikosteroid tindakan 97,5 dB dan 2 minggu setelah tindakan terdapat perbaikan pendengaran menjadi 77,5 dB.. Menurut kriteria Siegel pasien ini termasuk perbaikan ringan.17 Menurut kriteria Kallinen : Baik, apabila rerata perbaikan 10-30 dB pada 5 frekuensi. Pada pasien ini masing masing frekuensi terdapat perbaikan 40 dB (250Hz), 35 dB (500Hz), 35 dB (1000Hz), 20 dB (2000Hz), dan 25 dB (4000Hz).11 Sedangkan menurut Ho seperti yang dikutip oleh Filipo; pulih minimal dengan peningkatan ambang dengar sekitar 10-30 dB.16 Pada pasien ini perbaikan ambang dengar 26,25 dB. Prognosis tergantung pada usia, onset kehilangan pendengaran, ada tidaknya vertigo, jenis audiogram pertama, respon 2 minggu pengobatan pertama, derajat ketulian, adanya tinnitus dan faktor predisposisi. Pasien ini berusia 36 tahun, dengan onset kehilangan pendengaran 2 hari, Tidak ada Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 101 http://jurnal.fk.unand.ac.id vertigo, tetapi terdapat tinitus. Derajat ketulian pasien ini menurut ISO adalah tuli sangat berat (>90dB), yaitu 103,75dB, dengan tipe pendengaran menurut Atay adalah profound (>70dB pada masing-masing frekuensi).1 Mulai pengobatan pasien ini termasuk early treatment dikarenakan kurang dari 5 hari dari onset pertama. Setelah terapi kortikosteroid sistemik dari ambang dengar 103,75 dB menjadi 97,5 dB, dengan makna tidak ada perbaikan dikarenakan peningkatan 6 dB. Setelah dilanjutkan dengan penyuntikan deksametason intra-timpani ambang dengar menjadi 77,5 dB, terdapat peningkatan 20 dB. Menurut Haynes keberhasilan injeksi intratimpani pada salvage therapy apabila terdapat peningkatan minimal 20 dB.6 Pada pasien ini sesudah terapi terdapat peningkatan 20dB, dapat dikatakan bahwa terapi pada pasien ini berhasil. 8. 9. 10. 11. SIMPULAN Pemberian kortikosteroid intratimpani sebagai salvage therapy bermanfaat sebagai penatalaksanaan pada pasien tuli mendadak yang setelah pengobatan oral kortikosteroid gagal. Salvage therapy dikatakan berhasil apabila terdapat peningkatan minimal 20 dB setelah terapi 12. 13. DAFTAR PUSTAKA 1. 2. 3. 4. 5. 6. 7. Atay G, Kayahan B, Cınar BC, Sarac S, Sennaroglu L. Prognostic Factors in Sudden Sensorineural Hearing Loss. Balkan Med J. 2016; 33:87-93. Novita S, Yuwono N. Diagnosis dan Tata Laksana Tuli Mendadak. Cermin Dunia Kedokteran. 2013; 40(11):820-6. Chau JK, Lin JRJ, Atashband S, Irvine RA, Westerberg BD. Systematic review of the evidence for the etiology of adult sudden sensorineural hearing loss. The Laryngoscope. 2010; 120. Oliver ER, Hashisalri GT. Sudden Sensory Hearing Loss. In: Johnson JT, Rosen CA, editors. Bailey's Head & Neck Surgery Otolaryngology. Otology. 2. 5 ed. Philadelphia: Lippincott Williams & WJ.lkins; 2014. p.2589-96. Raymundo IT, Filho JB, Maia A, Pinheiro TG, Oliveira CA. Intratympanic Methylprednisolone as Rescue Therapy in Sudden Sensorineural Hearing Loss. Braz J Otorhinolaryngol. 2010; 76(4):499509. Haynes DS, O’Malley M, Cohen S, Watford K, Labadie RF. Intratympanic Dexamethasone for Sudden Sensorineural Hearing Loss After Failure of Systemic Therapy. The Laryngoscope. 2006; 117:1-15. Weber PC, Khariwala S. Anatomy and Physiology of Hearing. In: Johnson JT, Rosen CA, editors. Bailey's Head and Neck Surgery Otolaryngology. Otology 2. 5 ed. Philadelphia: Lippincott Williams & WJ.lKins; 2014. p.2253-73. 14. 15. 16. 17. 18. 19. Fedriani J. Steroid Intratimpani untuk Penanganan Tuli Mendadak. Cermin Dunia Kedokteran. 2015; 42(2):149-51. Stachler RJ, Chandrasekhar SS, Archer SM, Rosenfeld RM, Schwartz SR, Barrs DM, et al. Clinical Practice Guideline: Sudden Hearing Loss. Otolaryngology–Head and Neck Surgery. 2012; 146(1S):1-35. Soetirto I, Hendarmin H, Bashiruddin J. Gangguan Pendengaran (Tuli). 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Early transtympanic steroid injection in patients with ‘poor prognosis’ idiopathic sensorineural sudden hearing loss. ORL. 2011; 73:31-7. Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 102 http://jurnal.fk.unand.ac.id 20. Lim HJ, Kim YT, Choi SJ, Lee JB, Park HY, Park K, et al. Efficacy of 3 Different Steroid Treatments for Sudden Sensorineural Hearing Loss: A Prospective, Randomized Trial. Otolaryngology– Head and Neck Surgery. 2012; 148(1):121-7. 21. Lee JB, Choi SJ. Potential Benefits of Combination Therapy as Primary Treatment for Sudden Sensorineural Hearing Loss. Otolaryngology–Head and Neck Surgery. 2016; 154(2):328-34. 22. Dispenza F, Stefano AD, Costantino C, Marchese D, Riggio F. Sudden Sensorineural Hearing Loss: Results of intratympanic steroids as salvage treatment. Am J Otolaryngol Head Neck Med Surg. 2013; 34:296-300. Jurnal Kesehatan Andalas. 2018; 7(Supplement 3) 103
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Absence of vaccinia virus detection in a remote region of the Northern Amazon forests, 2005-2015
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To cite this version: Galileu Barbosa Costa, Anne Lavergne, Edith Darcissac, Vincent Lacoste, Betania Paiva Drumond, et al.. Absence of vaccinia virus detection in a remote region of the Northern Amazon forests, 2005-2015. Archives of Virology, 2017, 162 (8), pp.2369 - 2373. ￿10.1007/s00705-017-3353-x￿. ￿pasteur-01585618￿ Galileu Barbosa Costa, Anne Lavergne, Edith Darcissac, Vincent Lacoste, Betania Paiva Drumond, Jônatas S. Abrahão, Erna Geessien Kroon, Benoît de Thoisy, Giliane de Souza Trindade Distributed under a Creative Commons Attribution 4.0 International License HAL Id: pasteur-01585618 https://riip.hal.science/pasteur-01585618v1 Submitted on 18 Sep 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Title: Absence of Vaccinia virus detection in a remote region from Northern Amazon forests 1 2005-2015 2 Galileu Barbosa Costa1†, Anne Lavergne2, Edith Darcissac2, Vincent Lacoste2, Betânia Paiva 3 Drumond1, Jônatas Santos Abrahão1, Erna Geessien Kroon1, Benoit de Thoisy2, Giliane de Souza 4 Trindade1† 5 6 1 – Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas 7 Universidade Federal de Minas Gerais. Av Antônio Carlos, nº 6627, Pampulha, Belo Horizonte 8 Minas Gerais, Brazil. CEP: 31270-901. 9 2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP 10 6010, 97306 Cayenne, French Guiana. 11 †Corresponding authors. E-mail address: galileuk1@gmail.com; gitrindade@yahoo.com.br 12 giliane@icb.ufmg.br; Tel: +553134092747; Fax: +553134436482. Full mailing address: Avenida 13 Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901 14 15 16 Abstract 17 Vaccinia virus (VACV) circulates in Brazil and other South America countries, being responsible 18 for a zoonotic disease that usually affects dairy cattle and humans, causing economic losses and 19 impacting animal and human health. Furthermore, VACV was already detected in wild areas from 20 Brazilian Amazon. To better understand the natural history of VACV, we investigated it 21 circulation in wildlife from French Guiana, a remote region in Northern Amazon forests. ELISA 22 and plaque reduction neutralization test were performed to detect anti Orthopoxvirus antibodies 23 1 – Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, 7 Universidade Federal de Minas Gerais. Av Antônio Carlos, nº 6627, Pampulha, Belo Horizonte, 8 Minas Gerais, Brazil. CEP: 31270-901. 9 2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP 10 6010, 97306 Cayenne, French Guiana. 11 2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP 10 6010, 97306 Cayenne, French Guiana. HAL Id: pasteur-01585618 https://riip.hal.science/pasteur-01585618v1 Submitted on 18 Sep 2017 11 2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP 10 6010, 97306 Cayenne, French Guiana. 11 †Corresponding authors. E-mail address: galileuk1@gmail.com; gitrindade@yahoo.com.br; 12 giliane@icb.ufmg.br; Tel: +553134092747; Fax: +553134436482. Full mailing address: Avenida 13 Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901 14 †Corresponding authors. E-mail address: galileuk1@gmail.com; gitrindade@yahoo.com.br; 12 giliane@icb.ufmg.br; Tel: +553134092747; Fax: +553134436482. Full mailing address: Avenida 13 Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901 14 Vaccinia virus (VACV) circulates in Brazil and other South America countries, being responsible 18 for a zoonotic disease that usually affects dairy cattle and humans, causing economic losses and 19 impacting animal and human health. Furthermore, VACV was already detected in wild areas from 20 Brazilian Amazon. To better understand the natural history of VACV, we investigated its 21 circulation in wildlife from French Guiana, a remote region in Northern Amazon forests. ELISA 22 and plaque reduction neutralization test were performed to detect anti-Orthopoxvirus antibodies. 23 Real-time and standard PCR targeting C11R, A56R and A26L were applied to detect VACV DNA 24 in serum, saliva and tissue samples. VACV was not evidenced in any samples tested. These 25 2 findings provide additional information on the VACV epidemiological puzzle. The virus could 26 nevertheless be circulating at low levels that were not detected in areas with no human or cattle 27 presence. 28 29 30 Vaccinia virus (VACV) is the prototype of the Orthopoxvirus (OPV) genus, Poxviridae 31 family, and was used as a live vaccine around the world to eradicate smallpox. VACV circulates in 32 zoonotic cycles involving bovine herd and dairy workers in rural areas from Brazil, causing a 33 disease named Bovine Vaccinia (BV) which accounts for several economical losses and increasing 34 the public health concerns [1]. 35 In the last seventeen years, VACV spread throughout all Brazilian territory [1], though its 36 occurrence varies among different regions. Furthermore, VACV has been detected in countries 37 bordering Brazil, such as Argentina [2], Uruguay [3], and Colombia [4]. Although since 1999, 38 several BV outbreaks affecting mainly dairy cattle and humans have been reported [1], natural 39 VACV circulation had already been described in a rodent of Oryzomys genus captured in the 60´s in 40 the Amazon region [5]. HAL Id: pasteur-01585618 https://riip.hal.science/pasteur-01585618v1 Submitted on 18 Sep 2017 41 During these last years in which reports of BV outbreaks have been observed, VACV 42 circulation has been described in the Amazon region, in mammal species such as bovines, primates, 43 coatis, and also humans [6–12]. Nevertheless, there is little information on a possible VACV 44 sylvatic cycle and on its natural reservoirs since the origin of VACV is still unknown and its natural 45 reservoir is still unidentified [13–16]. Given that VACV has a broad host range and that wild 46 animals might be implicated in its transmission chain, we decided to search for VACV circulation 47 in wildlife animals (rodents, marsupials, and bats) captured in pristine forest areas with the absence 48 or limited anthropogenic impacts of Northern Amazon forests. 49 3 We analyzed a total of 2,365 wild mammals from French Guiana, captured between 2005 50 and 2015 in tropical upland rain forest, Amazon region [17, 18]. Captured sites were located in 51 remote forests, slightly disturbed forests (facing selective logging, hunting pressure, and low 52 fragmentation level), secondary forests and edge between forest and open habitats (Figure 1). Wild 53 mammals included rodents (n = 420), marsupials (n = 606) and Chiroptera (n = 1,339) (Table 1). 54 All samples underwent molecular testing for VACV. To detect VACV DNA we used real-time PCR 55 targeting the C11R and A56R genes, and semi-nested PCR targeting C11R and A26L genes [19]. A 56 molecular approach was performed in serum and tissue samples from rodents and marsupials, and 57 in DNA extracted from saliva and blood clots of bats. The positive controls used on molecular assays 58 were DNA extracted from serum and liver of experimental infected Balb/c mice with Vaccinia virus Western 59 Reserve (VACV-WR) strain and Brazilian Vaccinia (Br-VACV) group 1 and 2 viruses [20]. For those for 60 which we were not able to have an experimental infected animal (marsupials and bats), we provided 61 an experimentally contamination of serum and liver samples as in the protocol previously described 62 by Dutra and co-authors [21]. Briefly, it was added different concentrations of VACV-WR and Br- 63 VACV strains (with a range from 104 pfu/µL to 1 pfu/µL, respectively). The experimentally 64 contaminated samples were tested by PCR, compared to the Balb/c controls, and then used as 65 positive controls for marsupials and bats. HAL Id: pasteur-01585618 https://riip.hal.science/pasteur-01585618v1 Submitted on 18 Sep 2017 66 50 In addition, serum samples from the rodents and marsupials were screened for anti-OPV IgG 67 by ELISA and neutralizing antibodies by ≥50% plaque reduction neutralization test (PRNT50) [19]. 68 Sera were grouped into pools of 2–5 serum samples from animals belonging to the same species 69 that were from the same collection area. No serum samples from bats were available for serological 70 tests. For serological assays, the positive controls were also those provided by experimental infected 71 mouse with VACV-WR and Br-VACV strains [20]. For the ELISA assay particularly, we used 72 purified recombinant A/G protein peroxidase conjugated as secondary antibody. We run rodent and 73 4 marsupial samples together in the same plate (for ELISA and PRNT), comparing the results with 74 known positive and negative rodents’ serum samples. 75 Most of the rodents captured belong to the species Proechimys guyannensis (46,4%), 76 followed by Proechimys cuvieri (11,2%) and Zygodontomys brevicauda (9,5%). The main 77 marsupials studied were Didelphis marsupialis (34,1%) and Marmosa murina (26,2%). For bat 78 species, most of them were Desmodus rotundus (32,3%), Carollia perspicillata (17,2%) and 79 Pteronotus spp. (14,0%). All animals tested by both serological and molecular techniques were 80 negative for VACV. 81 In this study, we assessed numerous forest mammal species and found no serological or 82 molecular evidence for VACV circulation among them. Increasing efforts have been done regarding 83 VACV ecology in South America, and some studies have highlighted the importance of wild 84 animals and peridomestic rodents as VACV carriers [7, 22, 23, Miranda JB, personal 85 communication/in peer review]. Our negative results contrast with the above-mentioned ones and 86 some hypotheses can be considered in explaining the lack of evidence for VACV circulation in wild 87 animals in French Guiana during the 2005-2015 period. One hypothesis is that wildlife was 88 investigated in a highly preserved Northern Amazon region, where natural geographic barriers of 89 unfavorable forest habitats exist, preventing VACV dissemination from Brazil to French Guiana. 90 This could also be emphasized by the “dilution effect” hypothesis, taking into account that in a 91 highly preserved environment, the maintenance of a highly diverse mammal community may 92 prevent infection of putative more sensitive species. HAL Id: pasteur-01585618 https://riip.hal.science/pasteur-01585618v1 Submitted on 18 Sep 2017 A great diversity of mammal species was 93 studied, but with only a small sampling of species from which VACV has been detected in Brazil, 94 such as Mus musculus [20], Oryzomys sp [5], and Caluromys philander [Miranda JB, personal 95 communication/in peer review]. Furthermore, the species Didelphis albiventris from which VACV 96 was recently detected in São Paulo State, Southeast region of Brazil [24], is not present in French 97 5 Guiana. Nevertheless, two closely related species Didelphis imperfecta (n = 2) and D. marsupialis 98 (n = 207) were tested. 99 Guiana. Nevertheless, two closely related species Didelphis imperfecta (n = 2) and D. marsupialis 98 (n = 207) were tested. 99 Another explanation for the absence of VACV detection in this study could be the extremely 100 low number of bovines in French Guiana. One hypothesis for the broad circulation of VACV in 101 Brazil is that bovines are acting as VACV amplifiers, excreting high loads of viable particles, being 102 a source of infection for themselves and other animals [25]. VACV can be detected in animals 103 without clinical disease since it has been detected in blood samples and feces in animals without 104 lesions but presenting neutralizing antibodies [25]. Due to the potential role of cattle as a viral 105 amplifier, the presence of bovines could facilitate VACV persistence and dissemination into the 106 environment. 107 Another explanation for the absence of VACV detection in this study could be the extremely 100 low number of bovines in French Guiana. One hypothesis for the broad circulation of VACV in 101 Brazil is that bovines are acting as VACV amplifiers, excreting high loads of viable particles, being 102 a source of infection for themselves and other animals [25]. VACV can be detected in animals 103 without clinical disease since it has been detected in blood samples and feces in animals without 104 lesions but presenting neutralizing antibodies [25]. Due to the potential role of cattle as a viral 105 amplifier, the presence of bovines could facilitate VACV persistence and dissemination into the 106 environment. 107 VACV has recently been identified at the borders of Brazil, in countries such as Argentina 108 [2], Uruguay [3], and Colombia [4]. HAL Id: pasteur-01585618 https://riip.hal.science/pasteur-01585618v1 Submitted on 18 Sep 2017 Even though the circumstances of VACV detection in South 109 American countries are still uncertain, the cattle trade and translocation has to be considered, as 110 highlighted in Amazon region (Mato Grosso and Rondônia states) [9]. Furthermore, some authors 111 have hypothesized that the seroprevalence of OPV-antibodies in human populations from the 112 Amazon basin (Acre state) could be linked to agriculture and livestock practices, reinforcing the 113 role of cattle on VACV maintenance [8]. 114 VACV has recently been identified at the borders of Brazil, in countries such as Argentina 108 [2], Uruguay [3], and Colombia [4]. Even though the circumstances of VACV detection in South 109 American countries are still uncertain, the cattle trade and translocation has to be considered, as 110 highlighted in Amazon region (Mato Grosso and Rondônia states) [9]. Furthermore, some authors 111 have hypothesized that the seroprevalence of OPV-antibodies in human populations from the 112 Amazon basin (Acre state) could be linked to agriculture and livestock practices, reinforcing the 113 role of cattle on VACV maintenance [8]. 114 Compared to other South American countries where VACV has been detected, a different 115 situation is observed in French Guiana where a lower deforestation rate is observed and where only 116 a few cattle or horses are present [26] (http://www.globalforestwatch.org/) (Figure 1). Nevertheless, 117 our findings do not definitively establish that VACV is not circulating, and also do not exclude the 118 possibility that small mammals can act as its reservoir in nature. However, data presented here 119 strongly suggest the absence of circulation of VACV, or at low levels that were not detected, in 120 remote Amazonian areas with no human impact over a long period of time. The monitoring and 121 6 surveillance of VACV circulation is important to ensure early warning of zoonotic outbreaks for 122 vulnerable human populations. 123 124 125 Legend for figure: 126 An overview of French Guiana with the location of the studied regions, where were implemented 127 several trapping sites. The background of the map highlights the human footprint index expected to 128 summarize the anthropic threats on biodiversity. These threats (accessibility, land use, human 129 density) are summed and allowed producing an index with lower pressures strenght in light and 130 higher pressures darker. For each studied region, the dominant habitat is given. HAL Id: pasteur-01585618 https://riip.hal.science/pasteur-01585618v1 Submitted on 18 Sep 2017 131 132 Acknowledgements 133 We thank colleagues from the Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la 134 Guyane for their excellent technical support. We also thank Pró-Reitoria de Pesquisa from 135 Universidade Federal de Minas Gerais (PRPq/UFMG), CAPES, FAPEMIG and CNPq. JS Abrahão, 136 EG Kroon, and GS Trindade are researchers from CNPq. 137 138 Funding 139 This study was funded by two European projects, CAROLIA and RESERVOIRS, through ERDF 140 funds and a European Commission "REGPOT-CT-2011-285837-STRonGer" grant within the FP7. 141 This work also benefited from an "Investissements d’Avenir' grant managed by Agence Nationale 142 de la Recherche, France (CEBA, ref. ANR-10-LABX-25-01). 143 144 Conflict of interest 145 Authors declare no conflict of interest. 146 surveillance of VACV circulation is important to ensure early warning of zoonotic outbreaks for 122 vulnerable human populations. 123 Acknowledgements 133 Acknowledgements 133 We thank colleagues from the Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la 134 Guyane for their excellent technical support. We also thank Pró-Reitoria de Pesquisa from 135 Universidade Federal de Minas Gerais (PRPq/UFMG), CAPES, FAPEMIG and CNPq. JS Abrahão, 136 EG Kroon, and GS Trindade are researchers from CNPq. 137 138 Funding 139 This study was funded by two European projects, CAROLIA and RESERVOIRS, through ERDF 140 funds and a European Commission "REGPOT-CT-2011-285837-STRonGer" grant within the FP7. 141 This work also benefited from an "Investissements d’Avenir' grant managed by Agence Nationale 142 de la Recherche, France (CEBA, ref. ANR-10-LABX-25-01). 143 144 Conflict of interest 145 Authors declare no conflict of interest. 146 We thank colleagues from the Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la 134 Guyane for their excellent technical support. We also thank Pró-Reitoria de Pesquisa from 135 Universidade Federal de Minas Gerais (PRPq/UFMG), CAPES, FAPEMIG and CNPq. JS Abrahão, 136 EG Kroon, and GS Trindade are researchers from CNPq. 137 7 147 147 Ethical approval 148 Captures of rodents, bats and marsupials do not require ethic committee in French Guiana. 149 150 References 151 1. Kroon EG, Mota BE, Abrahão JS, da Fonseca FG, de Souza Trindade G (2011) Zoonotic 152 Brazilian Vaccinia virus: from field to therapy. Antiviral Res 92:150-63. 153 154 2. Franco-Luiz AP, Fagundes-Pereira A, Costa GB, Alves PA, Oliveira DB, Bonjardim CA, 155 Ferreira PC, Trindade GS, Panei CJ, Galosi CM, Abrahão JS, Kroon EG (2014) Spread of vaccinia 156 virus to cattle herds, Argentina, 2011. Emerg Infect Dis 20:1576-8. 157 158 3. Franco-Luiz AP, Oliveira DB, Pereira AF, Gasparini MC, Bonjardim CA, Ferreira PC, Trindade 159 GS, Puentes R, Furtado A, Abrahão JS, Kroon EG, (2016a) Detection of Vaccinia virus in dairy 160 cattle serum samples from 2009, Uruguay. Emerg Infect Dis 22:2174-2177. 161 162 4. 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Acknowledgements 133 Trindade GS, Emerson GL, Sammons S, Frace M, Govil D, Fernandes Mota BE, Abrahão JS, 214 de Assis FL, Olsen-Rasmussen M, Goldsmith CS, Li Y, Carroll D, Guimarães da Fonseca F, Kroon 215 E, Damon IK (2016) Serro 2 Virus Highlights the Fundamental Genomic and Biological Features of 216 a Natural Vaccinia Virus Infecting Humans. Viruses 8:328. 217 208 15. Medaglia ML, Moussatché N, Nitsche A, Dabrowski PW, Li Y, Damon IK, Lucas CG, Arruda 209 LB, Damaso CR (2015) Genomic Analysis, Phenotype, and Virulence of the Historical Brazilian 210 Smallpox Vaccine Strain IOC: Implications for the Origins and Evolutionary Relationships of 211 Vaccinia Virus. J Virol 89:11909-25. 212 213 16. Trindade GS, Emerson GL, Sammons S, Frace M, Govil D, Fernandes Mota BE, Abrahão JS, 214 de Assis FL, Olsen-Rasmussen M, Goldsmith CS, Li Y, Carroll D, Guimarães da Fonseca F, Kroon 215 E, Damon IK (2016) Serro 2 Virus Highlights the Fundamental Genomic and Biological Features of 216 a Natural Vaccinia Virus Infecting Humans. Viruses 8:328. 217 10 218 17. de Thoisy B, Bourhy H, Delaval M, Pontier D, Dacheux L, Darcissac E, Donato D, Guidez A, 219 Larrous F, Lavenir R, Salmier A, Lacoste V, Lavergne A, (2016) Bioecological Drivers of Rabies 220 Virus Circulation in a Neotropical Bat Community. PLoS Negl Trop Dis 10:e0004378. 221 222 18. de Thoisy B, Lacoste V, Germain A, Muñoz-Jordán J, Colón C, Mauffrey JF, Delaval M, 223 Catzeflis F, Kazanji M, Matheus S, Dussart P, Morvan J, Setién AA, Deparis X, Lavergne A, 224 (2009) Dengue infection in neotropical forest mammals. Vector Borne Zoonotic Dis 9:157-70. 225 226 19. Geessien Kroon E, Santos Abrahão J, de Souza Trindade G, Pereira Oliveira G, Moreira Franco 227 Luiz AP, Barbosa Costa G, Teixeira Lima M, Silva Calixto R, de Oliveira DB, Drumond BP (2016) 228 Natural Vaccinia virus infection: diagnosis, isolation, and characterization. Curr Protoc Microbiol 229 42:14A.5.1-14A.5.43. 230 231 20. Oliveira G, Assis F, Almeida G, Albarnaz J, Lima M, Andrade AC, Calixto R, Oliveira C, 232 Diomedes Neto J, Trindade G, Ferreira PC, Erna G, Abrahão J (2015) From lesions to viral clones: 233 biological and molecular diversity amongst autochthonous Brazilian vaccinia virus. Viruses 7:1218- 234 37. 235 236 21. Dutra LA, de Freitas Almeida GM, Oliveira GP, Abrahão JS, Kroon EG, Trindade GS (2017) 237 Molecular evidence of Orthopoxvirus DNA in capybara (Hydrochoerus hydrochaeris) stool 238 samples. 22. Abrahão JS, Guedes MI, Trindade GS, Fonseca FG, Campos RK, Mota BF, Lobato ZI, Silva- 241 Fernandes AT, Rodrigues GO, Lima LS, Ferreira PC, Bonjardim CA, Kroon EG (2009) One more 242 26. Hansen MC, Potapov PV, Moore R, Hancher M, Turubanova SA, Tyukavina A, Thau D, 259 Stehman SV, Goetz SJ, Loveland TR, Kommareddy A, Egorov A, Chini L, Justice CO, Townshend 260 JR (2013) High-resolution global maps of 21st-century forest cover change. Science 342:850-3. 261 Acknowledgements 133 Arch Virol 162:439-48. 239 240 22. Abrahão JS, Guedes MI, Trindade GS, Fonseca FG, Campos RK, Mota BF, Lobato ZI, Silva- 241 Fernandes AT Rodrigues GO Lima LS Ferreira PC Bonjardim CA Kroon EG (2009) One more 242 218 11 piece in the VACV ecological puzzle: could peridomestic rodents be the link between wildlife and 243 bovine vaccinia outbreaks in Brazil? PLoS One 4:e7428. 244 245 23. Peres MG, Bacchiega TS, Appolinário CM, Vicente AF, Allendorf SD, Antunes JM, Moreira 246 SA, Legatti E, Fonseca CR, Pituco EM, Okuda LH, Pantoja JC, Ferreira F, Megid J (2013) 247 Serological study of vaccinia virus reservoirs in areas with and without official reports of outbreaks 248 in cattle and humans in São Paulo, Brazil. Arch Virol 158:2433-41. 249 250 24. Peres MG, Barros CB, Appolinário CM, Antunes JM, Mioni MS, Bacchiega TS, Allendorf SD, 251 Vincent AF, Fonseca CR, Megid J (2016) Dogs and Opossums Positive for Vaccinia virus during 252 Outbreak Affecting Cattle and Humans, São Paulo State, Brazil. Emerg Infect Dis 22:271-3. 253 254 25. Guedes MI, Rehfeld IS, de Oliveira TM, Assis FL, Matos AC, Abrahão JS, Kroon EG, Lobato 255 ZI, (2013) Detection of Vaccinia virus in Blood and Faeces of Experimentally Infected Cows. 256 Transbound Emerg Dis 60:552-5. 257 258 26. Hansen MC, Potapov PV, Moore R, Hancher M, Turubanova SA, Tyukavina A, Thau D, 259 Stehman SV, Goetz SJ, Loveland TR, Kommareddy A, Egorov A, Chini L, Justice CO, Townshend 260 JR (2013) High-resolution global maps of 21st-century forest cover change. Science 342:850-3. 261 piece in the VACV ecological puzzle: could peridomestic rodents be the link between wildlife and 243 bovine vaccinia outbreaks in Brazil? PLoS One 4:e7428. 244
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NMR Profiling of Reaction and Transport in Thin Layers: A Review
Polymers
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Document license: CC BY Document license: CC BY DOI: 10.3390/polym14040798 Document status and date: Published: 18/02/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There c important differences between the submitted version and the official published version of record. Peop interested in the research are advised to contact the author for the final version of the publication, or vi DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication DOI: 10.3390/polym14040798 Document status and date: Published: 18/02/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. p • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. 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Oct. 2024 NMR Profiling of Reaction and Transport in Thin Layers Citation for published version (APA): Nicasy, R., Huinink, H., Erich, B., & Olaf, A. (2022). NMR Profiling of Reaction and Transport in Thin Layers: A Review. Polymers, 14(4), Article 798. https://doi.org/10.3390/polym14040798 Citation for published version (APA): Nicasy, R., Huinink, H., Erich, B., & Olaf, A. (2022). NMR Profiling of Reaction and Transport in Thin Layers: A Review. Polymers, 14(4), Article 798. https://doi.org/10.3390/polym14040798 polymers polymers polymers   Citation: Nicasy, R.; Huinink, H.; Erich, B.; Olaf, A. NMR Profiling of Reaction and Transport in Thin Layers: A Review. Polymers 2022, 14, 798. https://doi.org/10.3390/ polym14040798 Article Ruben Nicasy 1 , Henk Huinink 1,* , Bart Erich 1,2 and Adan Olaf 1,2 Ruben Nicasy 1 , Henk Huinink 1,* , Bart Erich 1,2 and Adan Olaf 1,2 1 Applied Physics Department, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands; r.j.k.nicasy@tue.nl (R.N.); s.j.f.erich@tue.nl (B.E.); o.c.g.adan@tue.nl (A.O.) 2 Organization of Applied Scientific Research, TNO The Netherlands, P.O. Box 49, 2600 AA Delft, The Netherlands 2 Organization of Applied Scientific Research, TNO The Netherlands, P.O. Box 49, 2600 AA D lft Th N th l d Abstract: Reaction and transport processes in thin layers of between 10 and 1000 µm are important factors in determining their performance, stability and degradation. In this review, we discuss the potential of high-gradient Nuclear Magnetic Resonance (NMR) as a tool to study both reactions and transport in these layers spatially and temporally resolved. As the NMR resolution depends on gradient strength, the high spatial resolution required in submillimeter layers can only be achieved with specially designed high-gradient setups. Three different high-gradient setups exist: STRAFI (STRay FIeld), GARField (Gradient-At-Right-angles-to-Field) and MOUSE (MObile Universal Surface Explorer). The aim of this review is to provide a detailed overview of the three techniques and their ability to visualize reactions and transport processes using physical observable properties such as hydrogen density, diffusion, T1- and T2-relaxation. Finally, different examples from literature will be presented to illustrate the wide variety of applications that can be studied and the corresponding value of the techniques. Keywords: high-resolution nuclear magnetic resonance (NMR); thin layer; transport; reaction; GARField; STRAFI; MOUSE   Citation: Nicasy, R.; Huinink, H.; Erich, B.; Olaf, A. NMR Profiling of Reaction and Transport in Thin Layers: A Review. Polymers 2022, 14, 798. https://doi.org/10.3390/ polym14040798 Academic Editor: Andrea Mele Received: 1 November 2021 Accepted: 14 February 2022 Published: 18 February 2022   Citation: Nicasy, R.; Huinink, H.; Erich, B.; Olaf, A. NMR Profiling of Reaction and Transport in Thin Layers: A Review. Polymers 2022, 14, 798. https://doi.org/10.3390/ polym14040798 Academic Editor: Andrea Mele Received: 1 November 2021 Accepted: 14 February 2022 Published: 18 February 2022 www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 1. Introduction Thin layers (10–1000 µm) are very important in a broad range of applications [1] that can be found in building materials [2], energy storage [3,4], photovoltaic devices [5,6], art [7,8], flexible electronics [9], optics [10] and most importantly in coatings [11–13]. The reason to incorporate thin layers can be manifold, for example, down-scaling [14], intro- ducing novel technologies [15], increasing performance and stability. Another important feature when using thin polymer films is the ability to incorporate a huge variety of func- tional molecules which allow electrical [16–18], magnetic [19] or even color-like [20] like features in the thin layer. Performance and stability of these thin layers will be determined by their final structure and underlying physical processes. In literature, the study of these processes is mostly categorized in different groups belonging to polymer or non-polymer thin layers. Here, we also differentiate between polymer and non-polymer films. Within the polymer films, we identify two main groups of processes, firstly, the ones during film formation and, secondly, processes that occur when the film is formed. The process of film formation is a multistage process which in literature can be defined in different ways. In this review, we categorize two types of film formation, both starting with the evaporation of the solvent followed by either curing or coalescence. After the film has formed, other processes such as transport of liquid and chemical reaction take place. In non-polymer films, such as cements or printing paper, liquid uptake and deformation are the most important processes. A more fundamental understanding of these processes will lead to more cost-effective, efficient and stable applications. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/polymers Polymers 2022, 14, 798. https://doi.org/10.3390/polym14040798 Polymers 2022, 14, 798 2 of 32 Film formation [21,22] is crucial in determining the final structure of the film and its stability. If film formation happens incorrectly, the layer can suffer from non-uniformities, skin formation [23,24], precipitation [25], chemical differences and other mechanical mal- functions [26] that all reduce performance. In most cases, applying thin layers starts with a liquid solution that covers the surface, containing both the film material (polymer, latex) and a solvent [21]. 1. Introduction In the mid-1900s, volatile organic components were mostly used as solvent. However, since 1950, concerns about their toxicity [27], flammability and envi- ronmental effects [28,29] have led to a change towards using water as solvent [30]. When the layer is applied, film formation happens in a two-stage process where first the solvent has to evaporate, allowing close contact between the polymers, followed by a type of mechanism to overcome their mutual repulsion and form a rigid layer. The rate of solvent evaporation is critical for film formation and depends on multiple factors [31,32]. At first, the rate of evaporation will depend on vapor pressure, temperature, surface area and air flow. In a second stage, the release of solvent becomes limited by transport through the thin layer [31,33]. If the solvent evaporates too quickly, the polymer will not have time to adhere to the surface or form a continuous film. In contrast, a slow evaporation rate will induce overwetting in the subsurface. When enough solvent has evaporated, a continuous layer is formed by curing (cross-linking) or coalescence in the case of latex [34,35]. Both cross-links and coalescence will be responsible for the strength and characteristics of the initial thin layers. A wide variety of studies focus on cross-linking [36–38], and coalescence [22] to gain a better understanding and improve the final structure. g g p Processes after film formation such as liquid transport [39,40] and reactions within these films are responsible for performance, degradation and instabilities. A wide variety of degradation processes can be identified, including biodegradation [41], photodegradation by light [42] and chemical degradation. A more fundamental understanding of the transport of liquids and chemical reactions within the thin layer will contribute to better performance and stability of the thin layer. Studies on the penetration and diffusion of liquids and the corresponding effect on the film morphology are the focus of a large group of studies and will be discussed in this review [43]. Other types of non-polymer thin layers can be found in the printing industry [44–46] (printing paper with a thickness of 100 µm). In these printing papers, the penetration of ink is of great importance as it influences the final print quality [47,48]. The wide variety of coatings [49], hydrophobicity [50] and basis weight found in these papers will all influence the printing process and the resulting print quality. 1. Introduction Another type of thin layer can be found in the human skin [51,52] where layers such as the stratum corneum and viable epidermis, both around 50 µm thick, can be found. In these skin layers, processes such as the penetration of skin care products [53] or oils take place. As a final example, in cement pastes [54,55] used in building applications, strength is based upon the internal morphology and transport of ions. gy Experimental techniques able to study film formation, reaction and transport processes are necessary to gain the insights allowing improvement of performance and stability. Since many processes in thin layers such as diffusion, curing and chemical reactions will happen with a so-called front, gaining spatial and time-dependent information is a crucial requirement. Conventional techniques are not able to gain both spatial and time-resolved information coherently. Techniques such as weight measurements [56], microscopy or ASA (Automatic Scanning Absorptometer) measurements [57] can only measure global properties such as mass or liquid uptake in the case of ASA, but will not gain spatial information. High-spatial-resolution MRI has proven to be a versatile tool for studying these processes. Nuclear Magnetic Resonance (NMR) is a well-established technique that started in 1946 where for the first time NMR was used for the detection of magnetic properties of atoms by Bloch [58,59] and Purcell [60]. The method was later extended to two different areas, namely, nuclear Magnetic Resonance Imaging (MRI) and NMR spectroscopy, which led to Nobel Prizes in Medicine [61] (Paul C. Lauterbur and Sir Peter Polymers 2022, 14, 798 3 of 32 3 of 32 Mansfield) and Chemistry [62] (Richard R. Ernst and Kurt Wuthrich). In this review, the focus lies on the first one (MRI). MRI has made its way into material research, as well as in polymer research [22,63–66]. NMR can be used to measure density profiles, atoms’ mobility, and probe their environment which enables to study reactions and transport processes in thin layers. Reactions in thin films, such as cross-linking [67–69], glass transitions, curing and many more chemical reactions, will all influence the mobility of the atoms, which is visible in the NMR signal. Additionally, transport processes can be studied by measuring time-dependent density profiles. We will discuss different studies to demonstrate how chemical and physical information of thin polymer films between 10 and 1000 µm can be extracted using high- spatial-resolution NMR setups. 1. Introduction Characterizing polymers with NMR has been the focus of many studies. Most research is conducted with NMR spectroscopy which is already well known and described in many books [70–73]. Here, the focus lies upon the different high-resolution NMR setups that are able to extract information with high spatial resolution. Three different high-gradient NMR setups exist with resolution in the micrometer range that are able to extract physical and chemical information about thin films: STRAFI (STRay FIeld), GARField (Gradient-At- Right-angles to Field) and MOUSE (MObile Universal Surface Explorer). This paper will begin by explaining the basic theory behind NMR and how hydrogen atoms can be used to extract spacial information. p In Section 3, the GARField [74], STRAFI [75] and MOUSE [76] setup will be discussed, where an overview will be given about their similarities and differences. Lastly, in Section 4, different thin layer studies will be discussed in order to provide a better understanding of the methods’ possibilities. 2.1. NMR Spatial Encoding The frequency #»f [MHz] depends on the magnetic field and is given by #»f = #»ω 2π = γ 2π # » B0 (4) (4) where #»ω [rad s−1] is the Larmor frequency.# » where #»ω [rad s−1] is the Larmor frequency.# » #» By applying a second magnetic field » B1 perpendicular to the main magnetic field ( »B0), the hydrogen atoms will precess along the new established magnetic field which allows manipulation of the hydrogen atoms. The excitation is best when the radio-frequency (RF) pulse exactly coincides with the Larmor frequency #»ω from Equation (4). In most cases, this manipulation is used to bring the magnetic moments of the hydrogen atoms in the xy-plane. In an NMR measurement, the total magnetisation # » M along this xy-plane can be measured, which is a sum of all the magnetic moments, # » M = ∑ i #» µi (5) (5) Measuring this signal is mostly conducted in the form of spin echoes, firstly introduced by Hahn [80,81] in 1950. After a combination of pulses that will be discusses in Section 2.3, the signal is recorded at some echo time te[s]. Figure 1. Left: Larmor frequency, middle: conventional NMR setup (Helmholtz coils) with condition |B|/G ≫1 where the gradient field −→ G and applied magnetic field −→ B0 are shown and approximated by straight lines because the radii of curvature in this case are negligible. Depicted in red is the measurement area used in NMR experiments. Right: The same Helmholtz coils as the middle, but with |B|/G ≪1. In this particular case, the radius of curvature cannot be neglected which can be seen in the field lines from −→ B0. −→ G is not drawn because it lies in correspondence with the middle figure, not in one direction, which would make the graph too complicated. Figure 1. Left: Larmor frequency, middle: conventional NMR setup (Helmholtz coils) with condition |B|/G ≫1 where the gradient field −→ G and applied magnetic field −→ B0 are shown and approximated by straight lines because the radii of curvature in this case are negligible. Depicted in red is the measurement area used in NMR experiments. Right: The same Helmholtz coils as the middle, but with |B|/G ≪1. In this particular case, the radius of curvature cannot be neglected which can be seen in the field lines from −→ B0. 2.1. NMR Spatial Encoding The existence of a nuclear spin was demonstrated by Bloch and Purcell in 1946 when they measured for the first time nuclear magnetic resonance (NMR) [58,77]. They received the Nobel Prize in Physics for this work in 1952. The basics of NMR are often explained using a classical vector model [65]. In this classical model, the nuclei can be described by a small magnetic dipole with magnetic moment #»µ [Am2], also known as “spin”. The nuclear spin #»µ is the consequence of a moving charge within the atoms nucleus. A relation exists between the angular momentum #»b and the magnetic moment of the nucleus #»µ, #»µ = γ#»b (1) (1) where the proportionality constant γ [rad s−1 T−1] is the gyromagnetic ratio. The most studied nucleus (also abundant in polymers) is hydrogen (1H) where γ/2π = 42.58 MHz/T. where the proportionality constant γ [rad s−1 T−1] is the gyromagnetic ratio. The most studied nucleus (also abundant in polymers) is hydrogen (1H) where γ/2π = 42.58 MHz/T. We want to emphasize that although hydrogen is the most abundant and well known atom, there also exist studies on other elements such as fluorine [78] (40.08 MHz/T) or carbon-13 (10.71 MHz/T) [79]. # » We want to emphasize that although hydrogen is the most abundant and well known atom, there also exist studies on other elements such as fluorine [78] (40.08 MHz/T) or carbon-13 (10.71 MHz/T) [79]. » ( / ) [ ] When these nuclear spins enter an external applied magnetic field # » B0 [T], they will experience a torque #»τ [Nm] related to the magnetic field by #»τ = #»µ × # » B0 (2) (2) As the torque equals the time derivative of the angular momentum #»b , Equations (1) and (2) can be combined to: d#» As the torque equals the time derivative of the angular momentum #»b , Equations (1) and (2) can be combined to: d#»µ dt = γ#»µ × # » B0 (3) (3) Since all the nuclei will experience this same time dependence in their magnetic moment #»µ, they will start to align and precess around the applied magnetic field # » B0, which by normal convention is pointing in the z-direction. This precession is called Larmor Polymers 2022, 14, 798 4 of 32 precession. A schematic picture of this precession is shown in Figure 1 left. 2.2. NMR Relaxation In the previous section, the formula for the signal intensity (Equation (7)) introduced two characteristic time scales that described signal loss after excitation. This signal loss is known as relaxation and will be described in this section. There are two typical char- acteristic relaxation processes called T1- or spin–lattice relaxation and T2- or spin–spin relaxation. In the first one, energy between the hydrogen spins and the surroundings is exchanged. This energy exchange will relax the spins back to their original z-direction. How fast Mz is restored is characterized by this T1-time. The energy transfer will be most effective when surrounding atoms vibrate at the Larmor frequency. The second relaxation process will account for losses in the transversal component of the magnetization vector (Mxy). The rate at which this relaxation occurs is defined by a T∗ 2 -relaxation time that is strongly correlated to the molecular motion and the local environment. For example, dipolar interactions between neighboring atoms induces a small difference in magnetic field [82], which results in dephasing and a faster signal decay. Both relaxation times will be influenced by different local factors that enable to extract physical information about their surroundings and their mobility. To understand how these times can be used to gain physical information, a deeper look at both relaxation times is provided. p The total T∗ 2 - and T1-relaxation time can be written as the contribution of different factors as [83] p The total T∗ 2 - and T1-relaxation time can be written as the contribution of different 1 T∗ 2 = 1 T2 + 1 T2i = 1 T2dip + 1 T2pores + 1 T2di f + 1 T2i (8) 1 T1 = 1 T1dip + 1 T1pores (9) (8) (9) where T∗ 2 and T1 are the relaxation times found in Equation (7). Here, T∗ 2 is a combination of both T2 and T2i, where the second one is attributed to inhomogeneities in the magnetic field introduced by the setup [84]. When using Hahn [80,81], CPMG [85,86] or other pulse sequences (Section 2.3), one can compensate for field inhomogeneities and the T2i contribution can be neglected. Therefore, T∗ 2 can be replaced by T2. 2 The first relaxation mechanism comes from neighboring dipoles (T1dip, T2dip). Dipoles will introduce small changes in the local magnetic field that lead to a faster relaxation. 2.1. NMR Spatial Encoding −→ G is not drawn because it lies in correspondence with the middle figure, not in one direction, which would make the graph too complicated. One of the advantages of NMR is the ability to achieve spatial resolution along the y-direction, perpendicular to the thin layer. Encoding spatial information can be achieved by applying a magnetic field gradient #»G[T/m] ≡(∂Bz/∂x, ∂Bz/∂y, ∂Bz/∂z) perpendicular to the # » B0 field, see Figure 1 middle. Depending on the strength of the gradient (G) and magnetic field (|B0|), there exist two different scenarios, |B|/G ≫1 and |B|/G ≪1. The rest of this section will cover the special case where |B|/G ≫1, found in conven- tional NMR setups. In this particular case, the curvature of the magnetic field lines is so small that the following assumptions can be made: the magnetic field is constant, pointing in the z-direction, and the gradient along the y-direction can be assumed to be constant and equal to ∂Bz/∂y (Figure 1 middle). Both assumptions will simplify the explanation. How- ever, for most high-gradient NMR setups used for thin film profiling where |B|/G ≪1 [74], these assumptions are not valid anymore. This will be discussed in Section 2.4. In the case where |B|/G ≫1, the gradient will cause a change in magnetization along the y-direction, indicated by the black arrows in Figure 1 middle, which leads to a change in Larmor frequency, (6) ω(y) = 2π f (y) = γ(B0 + Gy · y) (6) ω(y) = 2π f (y) = γ(B0 + Gy · y) Polymers 2022, 14, 798 5 of 32 with Gy—the gradient of the z-component of the magnetic field along the y-direction (∂Bz/∂y ≈∂B0/∂y) and f—the frequency amplitude. The total measured NMR signal is a combination of all different nuclei that resonate with different Larmor frequencies. When applying a Fourier transform of the recorded echo, the separate contributions of all different Larmor frequencies can be extracted and linked to a specific y-position through Equation (6), providing the spatial density distribution ρ(y). After excitation, the total signal intensity S(y, te) at position y will decay over a time due to environmental effects and is given by S(y, te) ∝ρ(y) 1 −exp −tr T1 ! exp  −te T∗ 2 ! (7) (7) where T1 and T∗ 2 are characteristic timescales that define the signal decay. 2.1. NMR Spatial Encoding More information about these times and the underlying processes can be found in the next section. 2.2. NMR Relaxation In most cases, this relaxation mechanism can be linked to the molecular mobility. This molecular mobility is quantified by a motional correlation time (τc) which is defined as the average time necessary for a molecule to rotate one radian. The influence of dipole–dipole interaction was described by Bloembergen, Purcell and Pound (BPP) in 1948 [87], where Polymers 2022, 14, 798 6 of 32 they established relationships between T1dip and T2dip and the motional correlation time (τc), Figure 2 left: 2 1 T1dip = 3 10 γ4¯h2 r6 [ τc 1 + ω2 0τ2c + τc 1 + 4ω2 0τ2c ] (10) 1 T2dip = 3 20 γ4¯h2 r6 [3τc + 5τc 1 + ω2 0τ2c + 2τc 1 + 4ω2 0τ2c ] (11) (10) 1 T2dip = 3 20 γ4¯h2 r6 [3τc + 5τc 1 + ω2 0τ2c + 2τc 1 + 4ω2 0τ2c ] (11) (11) These relaxation times can be used to discriminate between different polymer motional regimes inside a thin film, providing information about different structures, mobility, chemical reactions and mechanical responses. In a rigid environment, such as solids or polymers below their glass transition temperature, hydrogen atoms will experience the same deviations in magnetic field for a longer time, leading to a larger phase difference and faster decay. On the contrary, when molecules are mobile, for example in liquid water, the differences in magnetization will be averaged out over time leading to a longer relaxation time. This was used to observe for example different polymer substances [66], crosslinked and non-crosslinked materials and skin layers within thin polymer films [88,89], see also Section 4. This could ultimately be used to observe any difference between materials where the molecular mobility of the studied atoms is different. The second term appearing in both relaxations is the relaxation caused by a porous matrix (T1pores, T2pores) which was described in detail by Brownstein and Tarr [90]. When atoms diffuse through a porous matrix they will encounter the pore surface, where re- laxation due to dipoles or other effects occurs. How effectively this surface relaxation occurs depends on the particular medium and is quantified by the surface relaxivity σ [m/s] [91–95]. Depending on the diffusion constant (D [m2/s]), pore radius (r[m]) and surface relaxation, different regimes can be excluded [96]. 2.2. NMR Relaxation There is a fast diffusion regime with σr/D ≪1, where the surface relaxation will become dominant, and a slow diffusion regime, with σr/D ≫1 where the surface relaxation can be neglected. Here, the factor r/D is related to the number of encounters with the surface. Therefore, the surface relaxation will become important when the surface relaxivity is high or many encounters are present. In the fast diffusion regime, Brownstein and Tarr found that 1/T1pores and 1/T2pores can be approximated by 1 T2pores = σ2 S V (12) 1 T1pores = σ1 S V (13) (12) (13) where the factor S/V is the pore surface-to-volume ratio. These relaxation times can be used to gain useful information about the porous matrix [83,91–94,97] and possible changes over time when subjected to different environmental factors. A third parameter only found in T2-relaxation comes from diffusion of molecules. When atoms diffuse, they will encounter a different field strength due to the setups gradient. This leads to dephasing and an accelerated decay of Mxy. This diffusion-induced decay will also influence the resulting T2-time. This effect will become larger with increased gradient strength, diffusion constant (D[m/s2]) and diffusion time (te), which is described by the following formula [98,99], 1 T2di f = αγ2G2t2 e D (14) (14) Probing relaxation times can also be useful for studying different pools of atoms at the same time, a trick commonly used in thin film studies [100–104]. When the relaxation times of different hydrogen groups are far enough apart, their relative contributions to the signal can be separated. Figure 2 right demonstrates this principle by showing a decay of a latex film [105]. The decay clearly shows a two-component exponential decay. From the BPP-theory we know that the latex can be attributed to short relaxation time while the long T2-relaxation time can be linked to free water. 7 of 32 Polymers 2022, 14, 798 Figure 2. Left: Dependence of T1 and T2 upon τc (Reproduced from Chem. Soc. Rev., 2014,43, 1627–1659 with permission from the Royal Society of Chemistry.) [106]. Right: Signal decay of a latex film measured with a GARField NMR. Both soft and hard-type latex are visible and show a multi-exponential decay where the short and long T2– component are marked. The short relaxation time is attributed to hydrogen atoms within the polymer, while the long relaxation time comes from free water. 2.2. NMR Relaxation (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder, Paul Venema and Olaf Adan, Water mobility during drying of hard and soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from Elsevier [107]). Figure 2. Left: Dependence of T1 and T2 upon τc (Reproduced from Chem. Soc. Rev., 2014,43, Figure 2. Left: Dependence of T1 and T2 upon τc (Reproduced from Chem. Soc. Rev., 2014,43, 1627–1659 with permission from the Royal Society of Chemistry.) [106]. Right: Signal decay of a latex film measured with a GARField NMR. Both soft and hard-type latex are visible and show a multi-exponential decay where the short and long T2– component are marked. The short relaxation time is attributed to hydrogen atoms within the polymer, while the long relaxation time comes from free water. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder, Paul Venema and Olaf Adan, Water mobility during drying of hard and soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from Elsevier [107]). By exploiting this method, the relative contribution and relaxation time of different pools of hydrogen atoms can be followed over time using the same experiment. This is by far the most powerful tool in studying thin films over time [108]. Using this technique, M.R.Halse [109] showed that they could follow three groups of hydrogen atoms in a decane/rubber system at the same time: dry rubber (T2 = 0.7 ms), swollen (T2 = 1.8 ms) rubber and solvent (T2 = 200 ms). 2.3. Pulse Sequences In the beginning, the magnetization vector is in equilibrium, pointing in the same direction as the main magnetic field #»B0. Bringing the magnetisation out of equilibrium and creating an NMR signal is achieved by using a radio frequency (RF) wave. This RF irradiation will have a magnetic component (#»B1) along the xy-plane exerting a torque on the magnetization (Equation (2)). Applying this RF-field for a certain amount of time is called an “RF-pulse”. The angle by which the magnetization will rotate is called the “flip angle” (α[◦]) and depends on both the pulse time (tp[s]) and the magnitude of the RF-field (B1[T]) given by (15) α = γB1tp (15) Applying different RF-pulses is referred to as a pulse sequence and can be used to measure all the above mentioned parameters such as signal intensity, relaxation times and diffusion. As the focus of this review lies on the techniques, only the main sequences and some basics will be discussed. More details and explanations on the different types of pulse sequences can be found in more advanced studies [43,88,110] and reviews [83,111]. q Measurements of the signal intensity are mostly performed using Hahn spin echoes [80,81] with the following pulse sequences [90◦-τ-180◦-τ-echo] [111]. At t = 0, a 90◦pulse rotates the spins into the xy-plane. After this pulse, the transverse magneti- zation starts to dephase due to the environment, field inhomogeneity and the gradient according to Equation (7). At t = te/2, a second 180◦is given that refocuses the spins. At t = te, the spins are refocused and a so-called spin echo is created from which the transverse magnetization can be measured. g To measure the T2-relaxation, the Carr–Purcell–Meiboom–Gilll sequence (CPMG) [85,86] is performed. The sequence start exactly the same as the Hahn spin echo, followed by a train of 180◦pulses [90◦-τ-(180◦-τ-echo-τ)n]. Every 180◦pulse refocuses the spins, resulting Polymers 2022, 14, 798 8 of 32 in a series of spin echoes. The echo intensity drops due to T2-relaxation. The signal intensity at the nth echo is given by S(nte) = S0exp(−nte/T2) (16) (16) from which the T2-relaxation time can be calculated. In literature, adjustments to this well-known sequence are reported. A specific pulse sequence that should be mentioned here is the Ostroff–Waugh (OW) pulse sequence where the 180◦pulse is exchanged by another 90◦pulse [112]. from which the T1-relaxation time can be calculated. from which the T1-relaxation time can be calculated. from which the T1-relaxation time can be calculated. 2.3. Pulse Sequences In high-gradient measurements, this sequence is chosen above the more conventional CPMG sequence, for reasons that will be explained in Section 2.5. To measure the T1-relaxation time, conventional NMR setups use mainly two se- quences, the saturation recovery and inversion recovery sequence. However, in the high- resolution setups described in this review, the saturation recovery sequence is chosen for reasons that will be explained in Section 2.5. The pulse sequence of the saturation recovery sequence is given by [(α)m-τ2-90◦-τ-180◦-τ-echo] [113]. The measurement starts by saturating the nuclear spins, setting the magnetization in the transverse plane to zero. This is achieved by m pulses with a certain angle α. After complete saturation, Hahn spin echoes will be measured at different time intervals specified by τ2. The measured echo intensity is then given by S(τ2) = S0(1 −exp(−τ2/T1)) (17) (17) m which the T1-relaxation time can be calculated. from which the T1-relaxation time can be calculated. 2.4. High Resolution Spatial Encoding To profile thin films with a high-enough resolution, a high gradient is required, see Equation (6). Three NMR setups are able to generate high-enough gradients to profile films between 10–1000 µm, namely, STRAFI (STRAy FIeld) [114], GARField (Gradient-At- Right-angles-to-Field) [74] and MOUSE (MObile Universal Surface Explorer) [115]. In these high-gradient setups, where |B|/G ≪1, Equation (6) cannot be used anymore because the curvature in the magnetic field lines is significant, see Figure 1 right. g g g g To understand this, we need to consider Maxwell in free space with a static magnetic field and no current, » ∇× #»B = 0. (18) (18) According to this condition, the following relation should hold for the gradient in the y-direction (Gy = ∂Bz/∂y), ∂Bz ∂y = ∂By ∂z (19) (19) Therefore, by introducing a gradient in the y-direction (∂Bz/∂y), there must be a gradient in another direction (∂By/∂z) resulting in a curvature into the mean magnetic field, which is of the order of |B|/G [74]. In conventional NMR setups with a low gradient, this curvature is around 102m and can be neglected as in the previous section. When the gradient becomes much larger, the assumptions made in the previous section are not valid anymore. Consequently, it follows from Equation (6) that #»f (#»r ) = γ 2π (#»B0 + #»G · #»r ) (20) (20) where we use a vector notation to account for the fact that the Larmor frequency is different along the same horizontal plane. As signals are excited and resolved according to their respective Larmor frequency, this inhomogeneous Larmor frequency will lead to some problematic effects within high-resolution setups. The RF-pulses that normally excite a rectangular region will now excite curved slices that depend on the shape of the magnetic field lines. The curvature in STRAFI, for example, is around 0.1 m [74], much lower than in conventional NMR setups (102 m). Figure 3 shows the sensitive region of an original NMR-MOUSE where a clear curvature can be observed [116]. 9 of 32 Polymers 2022, 14, 798 Figure 3. Left: sensitive region of an original NMR-MOUSE. 2.5. Resolution and Field of View In the previous section, we discussed how a gradient is used to encode spatial informa- tion in a measurement. The resulting resolution is mostly determined by the high gradient and its ability to encode and read out spatial information. However, there are some other limiting factors that determine the maximum achievable resolution of the experimental setup. Another important parameter is field of view (FOV) which provides the maximum area that can be measured with the NMR setup. This section will cover some of the most important factors that determine the final resolution and FOV. Both parameters will be discussed for two measurement strategies—a Fourier measurement and a slice selective pulse measurement. In the first one, the spatial information is collected via a Fourier transformation of the NMR signal, mostly found in GARField [105,117] and sometimes in STRAFI [118]. In the slice selective pulse measurement, a slice selective pulse will collect the NMR signal of a thin slice without the need for a Fourier transformation. Here, the profile is built up slice-by-slice which requires a mechanical movement of the sample or magnet, mostly found in STRAFI [119] and MOUSE [120]. In a Fourier transformation measurement, the maximum theoretical resolutions that can be achieved are determined by the lowest frequency difference (∆f [Hz]) that can be differentiated. This difference will depend on the window width or acquisition time (∆ta [s]) of the measurement (∆f ≈1/∆ta) [121]. The dimension that corresponds to this frequency difference is determined by the gradient [121] and is given by ∆z = 1 γGz∆ta (21) (21) Thus, for a gradient of 40 T/m and a window width of 100 µs, the maximum achievable resolution becomes 5.9 µm. It can therefore be seen that both a higher gradient or acquisition time will increase the maximum theoretical resolution. However, ∆ta should always be lower than the T∗ 2 found in Equation (8). When ∆ta > T∗ 2 , the signal is limited by a fast T2-relaxation and not by the acquisition time. In this particular case, ∆ta in Equation (21) should be replaced by T∗ 2 . As the typical ∆ta of high-gradient setups are around 100 µs, much lower then conventional NMR setups, these cases almost never appear. In the slice selective measurements, the resolution is determined by the frequency bandwidth (∆f) of the RF-pulse. The frequency bandwidth from the RF-pulse is inversely proportional to the pulse length (tp). 2.4. High Resolution Spatial Encoding Right: Slice thickness of the signal amplitude for a conventional NMR-MOUSE (Reprinted from Magnetic Resonance Imaging, Volume 23, Issue 2, Bernhard Blümich, Federico Casanova, Juan Perlo, Sophia Anferova, Vladimir Anferov, Kai Kremer, Nicolae Goga, Klaus Kupferschläger, Michael Adams, Advances of unilateral mobile NMR in nondestructive materials testing, pages 197–201, Copyright 2005, with permission from Elsevier. [116]). Figure 3. Left: sensitive region of an original NMR-MOUSE. Right: Slice thickness of the signal Figure 3. Left: sensitive region of an original NMR-MOUSE. Right: Slice thickness of the signal amplitude for a conventional NMR-MOUSE (Reprinted from Magnetic Resonance Imaging, Volume 23, Issue 2, Bernhard Blümich, Federico Casanova, Juan Perlo, Sophia Anferova, Vladimir Anferov, Kai Kremer, Nicolae Goga, Klaus Kupferschläger, Michael Adams, Advances of unilateral mobile NMR in nondestructive materials testing, pages 197–201, Copyright 2005, with permission from Elsevier. [116]). amplitude for a conventional NMR-MOUSE (Reprinted from Magnetic Resonance Imaging, Volume 23, Issue 2, Bernhard Blümich, Federico Casanova, Juan Perlo, Sophia Anferova, Vladimir Anferov, Kai Kremer, Nicolae Goga, Klaus Kupferschläger, Michael Adams, Advances of unilateral mobile NMR in nondestructive materials testing, pages 197–201, Copyright 2005, with permission from Elsevier. [116]). 2.5. Resolution and Field of View Thus, for a 10 µs pulse time, the excited frequency bandwidth (∆f) is around 0.1 MHz which for a gradient of 40 T/m would excite a slice of 58.7 µm = (∆r ≈1/tpγG). p A problem arises in high-gradient fields because of the curvature induced on the magnetic field (see Section 2.1) that limits the homogeneity of |#»B0| and therefore the resolution. In the STRAFI and MOUSE, these inhomogeneities will be the limiting factor for the resolutions. Determining these resolutions can be achieved experimentally by measuring the profile of a thin slice, containing NMR active atoms, and analyzing the Polymers 2022, 14, 798 10 of 32 10 of 32 resulted profile [3]. An example of this limitation is shown in Figure 3, where on the left the sensitive area for a normal NMR-MOUSE is shown. The corresponding depth profile can be viewed on the right. In most cases, the experimental resolution is determined by taking the half-width of this profile which in this case would be around 1 mm and limits the maximum achievable resolution. As will be explained later, GARField introduces specially designed poles in order to make |#»B0| homogeneous [74]. For this reason, the achievable resolution in GARField is not limited by inhomogeneities and is mostly higher than in a STRAFI and MOUSE setup. p The high gradients will also induce a large spread in resonant frequencies and unlike a conventional NMR imaging setup, the gradients cannot be switched off. The spread in resonance frequencies can be of the order of 25 MHz cm−1 [121] which severely limits the single-shot field of view (FOV) that can be achieved with one RF-pulse. In a Fourier measurement, the theoretical FOV is determined by the slice selective pulse (∆r ≈1/tpγG) which can be increased by lowering the pulse time. Now, we can explain why in a high- gradient setup, an OW-sequence and saturation recovery sequence are chosen above the more conventional CPMG sequence and inversion recovery sequence. Since the OW- and saturation recovery sequences use only 90◦pulses, always the same volume will be excited. In a CPMG- and inversion-recovery sequence where 90◦and 180◦pulses are used, this is not the case. Another problem in the case of the inversion recovery arises in the first pulse, used to invert the magnetization from Mz to M−z, which will not be exactly 180◦ throughout the sample which can interfere with the T1-measurement. 3. Methods In this section, the three mentioned high-resolution NMR setups (STRAFI, GARField and MOUSE) will be discussed in more detail. The goal is to provide a general guideline that helps in deciding which setup is most suitable for studying a specific application or material. 2.5. Resolution and Field of View However, in real measurements, limitations arise because the sensitivity drops significantly when moving away from the RF-coil, limiting the FOV for a GARField setup to around 500 µm depending on the signal-to-noise ratio. In a slice selective measurement, the sample can be moved through the sensitive area which in essence could result in a limitless FOV. However, the setup design will mostly be the limiting factor leading to a FOV in the order of a few millimeters. The slight differences between the three setups will lead to slight changes into the achievable resolution and FOV. A more detailed explanation can be found in Section 4 where the setups are discusses separately. However, in most cases, the achievable resolution and FOV can be approximated using the above-mentioned formulas. 3.1. STRAFI (STRAy Field Imaging) Stray field imaging was introduced by A. A. Samoilenko et al. [75] in 1988. The STRAFI technique uses the stray field (fringe field) of a superconducting magnet to produce its high gradient. Figure 4 left is a schematic representation of a STRAFI setup. Indicated with black dotted lines are the field lines from the superconducting magnet indicating the direction of the main magnetic field #»B0. The measurement area lies in the stray field just below the superconducting magnet where a large gradient can be found that lies along the same direction as #»B0. Typical gradients that can be achieved are between 30 and 60 [Tm−1] [114]. Indicated with red lines in Figure 4 are the field lines from the RF-pulse. From here there exist mainly two different ways to acquire a complete profile, leading to two types of STRAFI, conventional STRAFI [122,123] and Fourier transform STRAFI [118]. Polymers 2022, 14, 798 11 of 32 Figure 4. Left: schematic representation of STRAFI. Middle: schematic representation of GARField. Right: schematic representation of MOUSE. In all figures, the measurement area is depicted with an orange rectangle. The magnetic field lines are indicated with black for the B0-field while the ones from the RF-pulse are indicated with red. In all setups, the direction of the gradient (G), main magnetic field (B0) and RF-pulse field (B1) in the measurement area are indicated with black arrows. Figure 4. Left: schematic representation of STRAFI. Middle: schematic representation of GARField. Figure 4. Left: schematic representation of STRAFI. Middle: schematic representation of GARField. Right: schematic representation of MOUSE. In all figures, the measurement area is depicted with an orange rectangle. The magnetic field lines are indicated with black for the B0-field while the ones from the RF-pulse are indicated with red. In all setups, the direction of the gradient (G), main magnetic field (B0) and RF-pulse field (B1) in the measurement area are indicated with black arrows. g p p Right: schematic representation of MOUSE. In all figures, the measurement area is depicted with an orange rectangle. The magnetic field lines are indicated with black for the B0-field while the ones from the RF-pulse are indicated with red. In all setups, the direction of the gradient (G), main magnetic field (B0) and RF-pulse field (B1) in the measurement area are indicated with black arrows. In the conventional STRAFI, the profile is acquired by recording the NMR signal slice-by-slice [122,123]. 3.1. STRAFI (STRAy Field Imaging) One method of imaging slice-by-slice is by moving the sample, which can be seen in Figure 4. As slices are imaged separately, no Fourier transform is required, which leads to a resolution that is limited by the frequency bandwidth of the excitation pulse, see Section 2.5. The fact that the resolution is pulse-time-dependent involves some drawbacks and limitation on the resolutions. As the pulse lengths can never be longer than the T2 of the material, the resolution for polymers with very short T2 times is limited. Since the frequency bandwidth of the pulse is inversely proportional to the pulse time, this method allows to have rather good resolutions at the expense of long measurement times. Another time restraint comes from the mechanical movement required in slice-by-slice measurements that has a huge influence on the measurement time. This becomes problematic when measuring fast dynamic processes. However, there are some tricks to lower the time required to measure a single profile. For example, during the repetition time, one can already start to measure other slices which means that the time is limited by the T1 of the sample [114]. As slices are measured separately and changing, some parameters will always influence measurement time. For example, increasing the resolution or sample thickness will also increase measurement time as more slices need to be measured. Therefore, for slow processes, good resolution can be achieved, while for fast processes, the measurement time is too long. Thus, measuring with STRAFI is easier for slow processes and lower resolutions (above 50 µm). p In the special case, when the frequency band with of the excitation pulse is large enough to capture the complete sample, a Fourier transform STRAFI can be implemented [118]. Here, the echo is collected from the whole sample and the profile is reconstructed by a Fourier transform. Using this method, measurements are only limited by T1 and not because of mechanical movement. This technique was mostly used to image thin film below 400 µm. The theoretical resolutions in these measurements are determined by Equation (21). However, the resolution is mostly limited by the shape of the sensitive area and should be determined experimentally. A major drawback of STRAFI is that the sensitive area where the #»B0-field is most homogeneous does not coincide with the optimum gradient position. 3.2. GARField (Gradient-At-Right-Angles-to-Field) 3.2. GARField (Gradient-At-Right-Angles-to-Field) To solve the high-curvature problem found in STRAFI that limits the maximum resolution, the GARField NMR was introduced in 1999 by P. M. Glover et al. [74]. In this setup, the researchers changed from a gradient along one component (y-direction) to a gradient in the magnetic field amplitude |#»B0|. To achieve this, the setup uses electromagnets with specially designed pole tips. The specific shape was calculated by P.M. Glover et al., using a specific solution of the Laplace equation ∆2φ = 0 where φ is the scalar potential defined by B = grad φ. For a detailed calculation, we refer to the original paper [74]. The specially designed pole tips from the electromagnets are able to generate a horizontal plane where |B0| is constant along the xy-plane and perpendicular to the gradient, Figure 4 middle. When making these pole tips, it was found that the ratio G/|B| is always constant and independent of y and z. This allows to operate with different gradient strengths without losing the in-plane uniformity of the magnetic field, allowing for an easy adaptation of the resolution. Shown in Figure 4, middle, is a schematic representation of the setup where the shape of the magnetic pole tips can be seen. Implementing these magnetic poles was only possible when the orientation of the magnetic field and gradient were slightly different compared to STRAFI. Where in STRAFI the B0 and G are aligned, they are perpendicular in the GARField setup. As the RF-pulse should be perpendicular to the main magnetic field, the RF-coil from the STRAFI and GARField will have different orientations. The highest sensitivity is obtained when the sample is placed at the end of the RF-coil. This is only possible in the GARField setup. In STRAFI, this arrangement is impossible and the sample should be placed apart from the RF-coil, which lowers the sensitivity. p p y Compared to conventional STRAFI and MOUSE, a depth profile is measured in one single measurement without repositioning the sample or magnet. The time required to measure a single profile depends on the different parameters used in the pulse sequence but is typically shorter than in STRAFI. A drawback, however, is the limited FOV. Without the possibility to reposition the sample, the FOV is mostly limited by the reduced sensitivity when moving away from the coil, mostly around 500 µm. 3.2. GARField (Gradient-At-Right-Angles-to-Field) As some applications such as coatings are mostly used on metallic surfaces, a special note should be made. Metallic surfaces interfere with the magnetic signals in the setup, which introduces artifacts in the measurement. Artifacts can be the result of differences in magnetic susceptibility between polymer and metal and from eddy currents generated in the metal. To address this problem, H. Zhu et al. [125] investigated the effect of metal substrates on the NMR signal. The researchers found through simulations and experimental work that magnetic susceptibility can be neglected when measuring with a GARField NMR, but that the eddy currents interfere with the pulse field. Therefore, it was calculated that measuring on metallic surfaces required more pulse power to manage these eddy currents. 3.1. STRAFI (STRAy Field Imaging) Therefore, the actual gradient is mostly lower than the maximum gradient that is reported for the particular electromagnet used in the STRAFI setups. After a trade-off between measurement speed and resolution, the most encountered studies reported resolutions between 50 [124]–450 [122] µm. Polymers 2022, 14, 798 12 of 32 12 of 32 3.4. Guideline Choosing the proper setup depends on multiple parameters, such as the sample material, required resolutions and measurement time. When resolution is most important, GARFIeld is the best option. By solving the curva- ture problems found in STRAFI, GARField will have the best resolution of all setups—the most reported resolutions lie somewhere between 5 and 15 µm (Table 1). In addition to a good resolution, the Fourier measurements used in GARField offers faster measurement speed then slice-selective measurements (Table 1). Profiles are mostly measured at times between 1 and 10 min. It should be mentioned that STRAFI also has a Fourier implementa- tion, but with lower resolutions (most reported resolutions between 24–60 µm) and lower sensitivity coming from the RF-coils orientation. Therefore, for a dynamical process or if a high resolution is required, GARField would be the best option. A drawback of GARField is the rather low FOV (<500 µm) with a sensitivity that lowers when moving away from the RF-coil. When larger samples should be measured, STRAFI would be a better choice. Due to the slice-selective excitation, samples can be moved through the sensitive area, making the FOV, in essence, limitless. While at 400 µm the GARField already loses a lot of sensitivity, the STRAFI does not encounter this signal loss. In these measurements, resolution always comes with a trade-off towards measurement time. Higher resolutions require to measure more slices when keeping the FOV constant. When measurements are required on-site or if a small piece of the sample is not available, MOUSE is the only suitable option. For most applications such as walls, oil/water wells [130–132], PE pipes [116] or paintings, samples small enough for the STRAFI or GARField setup are not available, leaving MOUSE as the only option. An overview of the most frequently encountered parameters is given in Table 1. It should be mentioned that in all cases, trade-offs between different parameters are made. Therefore, the best achievable resolution is almost never achieved. However, the table should give an idea about different ranges that are commonly used for the different setups. Table 1. The table summarizes the most important characteristics of the different setups. p p |# » B0| |#» G[T/m]| Measure Time Resolution Portable Sample Size STRAFI 2.3–7 30–60 >3 min 24–60 µm no limitless GARField 0.7–1.4 17–44 1–10 min 5–15 µm no 50–400 µm MOUSE 0.025–0.7 11.5 60 min 50–1000 µm yes 100–4000 µm 3.3. The MOUSE Different from the STRAFI and GARField, the NMR-MOUSE (MObile Universal Surface Explorer) is a portable device invented to investigate large objects in a nondestruc- tive manner [7,8]. The NMR-MOUSE consists of a compact permanent magnet (red and blue in Figure 4), which generates the B0-field. Inside the magnets, the magnetic field is homogeneous but when moving to the fringe field, the field starts to become more inho- mogeneous, creating a rather high gradient of about 22 T/m depending on the specifics of the magnets [7]. The small size and low weight make it suitable for on-site testing in a non-destructive manner. This makes the NMR-MOUSE suitable to carry out measurements on large surfaces such as walls or paintings without the need for collecting samples and destroying the object. This magnetic field is typically quadratic along the x- and z-direction and with a main gradient along the y-direction. This variation in the y-direction can be used to extract depth profiles of a specific material. The B1-field is obtained from a built-in RF-coil, see Figure 4 right. By calculating the exact magnetic field, is has been found that there is a sensitive region just outside the device [126]. Just like a conventional STRAFI, slice-selective pulses Polymers 2022, 14, 798 13 of 32 13 of 32 are used to extract spatial information, therefore, recording a signal is fully equivalent to STRAFI [114]. However, the sensitive region lies outside the magnets, which limits the final depth that can be measured. Different devices are available to measure different depths ranging from 3 to 25 mm [7]. Selecting the best device depends on the application as more depth coincides with a lower sensitivity. A drawback of the simple magnetic design is the fact that the sensitive region is oddly shaped, varying in thickness, see Figure 3. This results in low sensitivity and huge restrictions on the maximum achievable resolution. Spatial resolutions better than half a millimeter are hard to achieve [127,128]. Attempts have been made in order to reduce the resolution. J. Perlo et al. [129] reported resolutions as low as 2.3 µm by making a new magnetic geometry with four permanent magnets. Measuring with these high resolutions in a slice-selective manner is, however, very time consuming. Another drawback of the design is the limitation of the sensitive volume, which limited the FOV to 50 µm. 4.1. Structure and Structural Evolution Studying thin layers starts by identifying their internal structure and structural evolu- tion. Therefore, the first NMR measurements performed on thin layers aimed to determine the moisture content and different polymer states such as crystallinity or cross-linking. Determining the structure of thin layers can be achieved using the signal intensity, dif- fusivity and T1/T2-relaxation times. All these parameters provide information about the hydrogen content, their environment and micro structures, as described in Section 2.2. In the first part of this section, two studies on cement pastes performed by P. J. McDonald et al. [54,55] are used to explain how the relaxation times can be used to obtain structural information, followed by a summary of some important studies using similar principles. In these studies, GARField was only used to measure in one particular slice. In principle, this information could have been obtained via conventional NMR instruments. Nevertheless, we value this particular study as GARField has the potential to perform a similar study, but with high spatial resolution in depth. Lastly, a study performed by B. Voogt et al. [107] is used to explain how diffusivity can be used to determine structural information using the above mentioned setups. In studies performed by P. J. McDonald et al. [54,55], the researchers characterized hydrated cement paste using different T1- and T2-relaxation studies. T1- and T2-relaxation times for different cement paste were determined using a saturation recovery and OW- sequence with the GARField-setup. Using the GARField-setup, a slice selective measure- ment was performed 10 mm below the surface of the sample with a slice thickness of 0.6 mm. When plotted in a T1-T2 correlation spectrum, the researches could relate the relax- ation times to different groups of hydrogen atoms with varying pore radii (Equations (12) and (13)). Such plots for a white cement sample cured under water are shown in Figure 5. They identified that the cement paste had pores with the following length scales: gel pores (T2 = 400 µs) and multiple capillary pores (T2 > 400 µs) with different pore sizes. Observing the different relaxation times, they found that after 6 days, 2 peaks are visible, both with low T1 and T2 times corresponding to gel pores. At day 7, capillary pores with longer relaxation times also start to become visible (Figure 5). Characterizing bulk properties like this can also be performed using a normal NMR spectrometer [133]. 4. Applications The following sections will provide a deeper look into the most common types of measurement performed by these three NMR setups on thin layers. Every section will start Polymers 2022, 14, 798 14 of 32 14 of 32 by discussing the general concepts using a representative study as an example, followed by a summary of related studies on a wide variety of samples and materials. by discussing the general concepts using a representative study as an example, followed by a summary of related studies on a wide variety of samples and materials. 4.1. Structure and Structural Evolution Red corre- sponds to water in capillary-sized pores, green to gel-sized pores and blue for interlayer pores and black is the total amount of moisture. The authors observed that after 1 hour, the moisture uptake already reaches 90% of its total amount. A striking fact is that they saw for the first time that moisture will start to redistribute between the different pores. In this research, they only looked at a particular part in the layer, however, with GARField, MOUSE or STRAFI, the same information at different positions is available at every position in the layer. Studies on cement like-materials such as pastes and Portland cement have also been performed on STRAFI [134–136] and MOUSE [135,137]. In other studies, this separation based on relaxation times was used to separately study water and polymer contribution in latex films [105,107], human skin [138] and another group of polymers such as Polyurethane coatings [139], plasticizing of nylon-6 [140,141], semi-crystalline polyethylene [142] and photo-polymerization of methacrylate [143]. Additionally, medical applications, such as the binding behavior of collagen-binding liposomes, have been studied [144,145]. Even layers with very low relaxation times, such as ice (T2 = 3.5 µs), have been imaged during melting using a STRAFI setup [122]. g g p In addition to T2- and T1-relaxation, another way to characterize the structure of thin layers is via the diffusion coefficient D of the hydrogen atoms. In materials, water can be present in a wide variety of states with different diffusion coefficients such as free water, water confined in pores or water bound to the polymer matrix. When materials undergo a structural transition, the water diffusivity will be altered, giving insight into the internal structure of the layer. A study performed by B. Voogt et al. [107] demonstrates this principle. The researchers used the proton local mobility and diffusivity to characterize the structure of hard and soft latex during drying. By measuring the T2-relaxation time with different echo times, the diffusion coefficient was determined, see Equation (14). Figure 7 shows the diffusion coefficient measured at different times steps during drying. From t = 30 min onward, the diffusion constant was too low to be determined. As a result of drying, the particles’ mobility will be restricted lowering the auto-diffusion D (Figure 7) and the T2 of the atoms. The T2- of latex remained rather constant around 0.1 ms, indicating that the proton mobility is constant. 4.1. Structure and Structural Evolution In this particular case, the researchers identified the correlation maps at a specific location of 10 mm below the surface. The advantage of the GARField NMR could be to perform the correlation maps at different positions and study the different pore fraction at different positions, however, this was not done in this particular study. Figure 5. T1-T2-correlation maps of white cement with water–cement ratio of 0.4. Figures are taken from the curing process under water after 2 days (left), 6 days (middle) and 7 days (right). A development in pore space can be observed by the separation of the single peak into smaller peaks when approaching 6 and 7 days. (Reprinted from Magnetic Resonance Imaging, Volume 25, Issue 4, Peter J. McDonald, Jonathan Mitchell, Michael Mulheron, Luc Monteilhet, Jean-Pierre Korb, Two-dimensional correlation relaxation studies of cement, pages 470–473, Copyrights 2007, with permission from Elsevier [54]). Figure 5. T1-T2-correlation maps of white cement with water–cement ratio of 0.4. Figures are taken from the curing process under water after 2 days (left), 6 days (middle) and 7 days (right). A development in pore space can be observed by the separation of the single peak into smaller peaks when approaching 6 and 7 days. (Reprinted from Magnetic Resonance Imaging, Volume 25, Issue 4, Peter J. McDonald, Jonathan Mitchell, Michael Mulheron, Luc Monteilhet, Jean-Pierre Korb, Two-dimensional correlation relaxation studies of cement, pages 470–473, Copyrights 2007, with permission from Elsevier [54]). Polymers 2022, 14, 798 15 of 32 15 of 32 In a follow-up study [55], the dynamic porosity in cement paste during water up- take and drying was studied, using T2-relaxation. Three main groups were found that correspond to different pore sizes: hydrates inner layers of 1 nm (T2 = 120 µs), gel pores of 3–5 nm (T2 = 360 µs) and capillary pores larger than 5 nm (T2 = 1080 µs). Figure 6, left, shows typical signal decays where the solid line is the total decay and marked with dashed lines are the contributions of the different pores. For completeness, the authors added a dotted line representing the part coming from the crystalline solid. Using this multi-exponential decay, the researchers could follow the different types of hydrogen atoms during evaporation (Figure 6 middle) and moisture uptake (Figure 6 right). 4.1. Structure and Structural Evolution By analyzing the diffusion constants and relaxation times, they found that water changes from a free state towards pore water and finally water that is physically bound to the polymer matrix. The researchers also observed a difference in packing between the soft and hard type of latices. The relative concentration of the polymer increased in the soft type but remained constant in the hard type. This indicates that in the soft type, the particles come closer together, forming a close packed structure, while for the hard type, this is not possible due to the hardness. These measurements clearly show the restricted movement of the hydrogen atoms. In a similar study performed by V. Baukh et al. [141], multilayered coatings were investigated. The authors found that the diffusion coefficient increased with water content and that binding of water to the polymer was stronger at low water concentrations. In other studies, diffusion coefficients were measured in order to determine the amount of bacteria [123] or structural changes in water-swollen cellophane [146]. As the diffusion coefficient depends on the structure of the material, it can also be used to differentiate layers in heterogeneous structures. P. J. McDonald et al. [52] used this method to determine the diffusion constants and profiles of the human skin in vivo. The Polymers 2022, 14, 798 16 of 32 measurements revealed a clear contrast between the stratum corneum and viable epidermis. This contrast was largely attributed to a difference in diffusion constant between both layers. Comparable studies on human skin that characterized the different skin layers were performed on GARField [51,147] and MOUSE [138,148]. In addition to water and polymers, studies on small penetrants such as toluene and n-hexane in PE pellets have also been performed [149]. measurements revealed a clear contrast between the stratum corneum and viable epidermis. This contrast was largely attributed to a difference in diffusion constant between both layers. Comparable studies on human skin that characterized the different skin layers were performed on GARField [51,147] and MOUSE [138,148]. In addition to water and polymers, studies on small penetrants such as toluene and n-hexane in PE pellets have also been performed [149]. Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the total fit. The dashed lines represent the fractions with different T2 times (120 µs, 360 µs and 1080 µs). 4.1. Structure and Structural Evolution Middle and right: Pore water fractions measured 600 µm below the surface during drying (middle) and wetting (right). Red corresponds to water in capillary pores, green to gel sized pores and blue to interlayer spaces. Black represents the total hydrogen content. (Reprinted from Cement and Concrete Research, Volume 133, Peter J. McDonald, Ors Istok, Magdalena Janota, Agata M. Gajewicz-Jaromin, David A. Faux, Sorption, anomalous water transport and dynamic porosity in cement paste: A spatially localised 1H NMR relaxation study and a proposed mechanism [55]). Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the total fit. The dashed lines represent the fractions with different T2 times (120 µs, 360 µs and 1080 µs). Middle and right: Pore water fractions measured 600 µm below the surface during drying (middle) and wetting (right). Red corresponds to water in capillary pores, green to gel sized pores and blue to interlayer spaces. Black represents the total hydrogen content. (Reprinted from Cement and Concrete Research, Volume 133, Peter J. McDonald, Ors Istok, Magdalena Janota, Agata M. Gajewicz-Jaromin, David A. Faux, Sorption, anomalous water transport and dynamic porosity in cement paste: A spatially localised 1H NMR relaxation study and a proposed mechanism [55]). Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the total fit. The dashed lines represent the fractions with different T2 times (120 µs, 360 µs and 1080 µs). Middle and right: Pore water fractions measured 600 µm below the surface during drying (middle) and wetting (right). Red corresponds to water in capillary pores, green to gel sized pores and blue to interlayer spaces. Black represents the total hydrogen content. (Reprinted from Cement and Concrete Research, Volume 133, Peter J. McDonald, Ors Istok, Magdalena Janota, Agata M. Gajewicz-Jaromin, David A. Faux, Sorption, anomalous water transport and dynamic porosity in cement paste: A spatially localised 1H NMR relaxation study and a proposed mechanism [55]). Figure 7. Diffusion coefficient of free water within the hard- and soft-type latex. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder, Paul Venema and OlafAdan, Water mobility during drying of hard and soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from Elsevier [107]). 4.1. Structure and Structural Evolution Figure 7. Diffusion coefficient of free water within the hard- and soft-type latex. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder, Paul Venema and OlafAdan, Water mobility during drying of hard and soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from Elsevier [107]). 4.2. Film Formation—Drying Film formation is the process in which a polymer emulsion or colloidal dispersion will overcome their stabilizing forces to form a continuous film. These stabilizing forces can be overcome when the continuous liquid phase evaporates, forcing the emulsion droplets or polymer particles together. The evaporation of excess liquid is referred to as the drying phase of the film formation process. When enough liquid has evaporated, the polymers or colloidal particles need to form a continuous film. After solvent evaporation, there are two types of processes: curing and coalescence [150]. In the curing process, a stable film is formed by cross-links between reactive polymers. On the other hand, in coalescence, the polymer particles will first coalescence and deform to form a close packed structure. In Polymers 2022, 14, 798 17 of 32 17 of 32 order to form a homogeneous film, the deformation is followed by entanglement of the polymer chains. Particle boundaries disappear, which will lower the film roughness [22]. All these combination of drying, curing and coalescence make film formation a complicated process. Since most applied films and coatings undergo this film formation, this section is dedicated to studies that are focused this topic. First, drying experiments and then the subsequent processes are discussed. During drying, water or solvent evaporates from the polymer film, mostly reducing the overall thickness. The drying process can be monitored with an NMR signal. A representative example of a study on the drying processes of waterborne colloidal films was performed by J.-P. Gorce et al. [151]. Measurements were performed on a GARField setup using an OW-sequence with a 8.7 µm resolution. Profiles of the NMR signal measured at different times are shown in Figure 8. The width of the signal corresponds to the thickness of the polymer film. First of all, evaporation leads to film shrinkage due to the disappearance of hydrogen atoms. When all water has evaporated, the profile reaches its final thickness, corresponding to 150 µm. This decrease in profile thickness can be used to characterize a drying front and a corresponding drying rate. Secondly, the maximum signal intensity reduces over time from 0.7 to 0.4. To understand the reduced signal, we need to introduce ρ = ρa + ρb in Equation (7), S ∝ρaexp(−te/T2a) + ρbexp(−te/T2b) (22) (22) S ∝ρaexp(−te/T2a) + ρbexp(−te/T2b) where we have left out the T1-term. 4.2. Film Formation—Drying Additionally, the water fractions where determined for every profile, where for the left figure the water fraction were 0.48 (62 min), 0.33 (602 min), 0.23 (821 min), 0.15 (956 min) and 0.09 (1061 mi.), while for the right figure, the water fractions were 0.44 (2 min), 0.25 (7 min), 0.15 (13 min) and 0.10 (31 min). (Reprinted by permission from Springer Nature Customer Service Centre GmbH Springer Nature: The European Physical Journal E—Soft Matter, Vertical water distribution during the drying of polymer films cast from aqueous emulsions, J.-P. Gorce et al., 2014 [151]). Figure 8. NMR profiles measured during film drying of an alkyd emulsion in a closed environmen Figure 8. NMR profiles measured during film drying of an alkyd emulsion in a closed environment with Pe = 0.2 (left) and with Pe = 16 (right). Additionally, the water fractions where determined for every profile, where for the left figure the water fraction were 0.48 (62 min), 0.33 (602 min), 0.23 (821 min), 0.15 (956 min) and 0.09 (1061 mi.), while for the right figure, the water fractions were 0.44 (2 min), 0.25 (7 min), 0.15 (13 min) and 0.10 (31 min). (Reprinted by permission from Springer Nature Customer Service Centre GmbH Springer Nature: The European Physical Journal E—Soft Matter, Vertical water distribution during the drying of polymer films cast from aqueous emulsions, J.-P. Gorce et al., 2014 [151]). g p g y g y with Pe = 0.2 (left) and with Pe = 16 (right). Additionally, the water fractions where determined for every profile, where for the left figure the water fraction were 0.48 (62 min), 0.33 (602 min), 0.23 (821 min), 0.15 (956 min) and 0.09 (1061 mi.), while for the right figure, the water fractions were 0.44 (2 min), 0.25 (7 min), 0.15 (13 min) and 0.10 (31 min). (Reprinted by permission from Springer Nature Customer Service Centre GmbH Springer Nature: The European Physical Journal E—Soft Matter, Vertical water distribution during the drying of polymer films cast from aqueous emulsions, J.-P. Gorce et al., 2014 [151]). In a comparable study performed by P. Ekanayake et al. [152], density profiles of drying colloidal films revealed that the particle concentration gradient inside this dense layer of colloidal particles scales with Pe0.8. Furthermore, in this study, the GARField setup was chosen as it has the best resolution, allowing to see this thin dense top layer. 4.2. Film Formation—Drying Here, the signal is split into a term coming from the solvent (ρa, T2a) and one coming from the polymer or colloidal particles (ρb, T2b). When solvent is released, the total active hydrogen atoms ρ is almost unchanged as hydrogen atoms from the water are replaced by the ones from the polymer. Therefore, the signal decrease cannot be attributed to a loss in active hydrogen atoms. However, the relative contributions do change, where during drying ρa reaches zero, ρb reaches its maximum. As the mobility of the polymer is much lower then that of free water, the T2 of the polymer is lower then that of free water by at most one order of magnitude (see Section 2.2). Faster relaxation leads to a lower signal, where the final signal amplitude is closely related to the mobility of the polymer phase which in this particular case was very low. In the beginning, the signal intensity (0.7) is dominated by the solvent (ρaexp(−te/T2a)); after drying, the signal will be determined by the polymer (ρbexp(−te/T2b)) which due to a faster decay will have a lower signal intensity (0.4). g y In the same study, the influence of the Peclet number Pe = vLe f /D was investigated. v[m/s] is the speed of the receding water front, Le f [m]—the thickness of the layer and D[m2/s]—the diffusion coefficient. By controlling the thickness, the speed of the receding water front and the diffusion coefficient, the Pe number could be adjusted. When Pe > 1, advection dominates, but for Pe < 1, diffusion is more important. Figure 8 left shows profiles for the case were Pe = 0.2, whereas right represents Pe = 16. A difference in profile shape was observed that indicates that diffusion towards the surface can keep up with the evaporation (flat profiles) when Pe = 0.2, but not when Pe = 16, resulting in a concentration gradient of water molecules near the surface. The formation of this dense polymer layer to the surface showed limited diffusion towards the surface, slowing down evaporation. 18 of 32 18 of 32 Polymers 2022, 14, 798 Figure 8. NMR profiles measured during film drying of an alkyd emulsion in a closed environment with Pe = 0.2 (left) and with Pe = 16 (right). 4.2. Film Formation—Drying g p y In some cases, drying can induce the formation of a skin layer that can trap water. These skin layers reduce solvent evaporation and significantly slow down the drying process [23,24]. E. Ciampi et al. [23] investigated skin formation upon drying of poly(vinyl alcohol) (PVOH). Profiles were measured with a GARField NMR for an initial PVOH content of 10 WT% and 25 WT%. The profiles for the 10 WT% dried similarly to the ones measured by [151]. In case of high-polymer-weight fractions (25 WT%), a skin layer developed that trapped water inside the profile, leading to slower evaporation. Similar studies of the drying behavior of different polymers, such as alkyd lay- ers [89,117,153,154], latex layers [107], gelatin layers [155] and even dental resins [119,156–158], have been conducted using the GARField, STRAFI and MOUSE NMR setups. 4.3. Film Formation—Curing In case of reactive compounds, a curing reaction can start when enough water evapo- rates and the polymer content is high enough. Measurements with a GARField on drying alkyd coatings [153] illustrate the capability to measure the drying–curing film formation process. In this study performed be S. J. F. Erich et al., NMR profiles on alkyd coatings were measured every 10 min with an OW-sequence. Measured profiles can be seen in Figure 9. The profiles reveal this two-stage process where the polymer layer shrinks and the intensity drops due to solvent evaporation, as explained in previous section. This process is indicated by the arrows in Figure 9 and it accounts for a rapid loss in signal intensity in the beginning. After drying, a (reaction) curing front moves into the polymer film. The signal loss due to curing of polymers can be attributed to the loss in mobility of the polymer. When polymers are cured, they become more rigid, the T2 drops and signal is lost when the T2 approaches te. 19 of 32 19 of 32 Polymers 2022, 14, 798 Figure 9. Hydrogen profiles of a solvent-borne alkyd sample measured during drying. The top of the coating corresponds to the left side, while the cover glass can be found on the right. The vertical dashed line indicates the top of the film after drying. (Reprinted from S. J. F. Erich, J. Laven, L. Pel, H. P. Huinink, and K. Kopinga, “Dynamics of cross linking fronts in alkyd coatings”, Appl. Phys. Lett. 86, 134105 (2005) https://doi.org/10.1063/1.1886913, with permission of AIP Publising [153]). Figure 9. Hydrogen profiles of a solvent-borne alkyd sample measured during drying. The top of the coating corresponds to the left side, while the cover glass can be found on the right. The vertical dashed line indicates the top of the film after drying. (Reprinted from S. J. F. Erich, J. Laven, L. Pel, H. P. Huinink, and K. Kopinga, “Dynamics of cross linking fronts in alkyd coatings”, Appl. Phys. Lett. 86, 134105 (2005) https://doi.org/10.1063/1.1886913, with permission of AIP Publising [153]). In the study of S. J. F. Erich et al. [153], curing experiments are performed on water- borne and solvent-borne alkyds. Both systems showed the same behavior: a clear evapora- tion and curing stage could be distinguished. 4.3. Film Formation—Curing In a related paper [89], NMR and confocal Raman Microscopy were compared, and the curing could be related to the disappearance of double bonds and the formation of oxidative cross-links. These cross-links lowered the mobility and reduced the NMR signal, giving rise to the observed curing front. Tracking these fronts in different environmental conditions revealed that oxygen supply was the limiting factor in the curing front dynamics. g g y Further, the effect of drying on porous media [117], catalysts [159–162] and pigment volume concentrations [163] was studied. Curing also plays a role in dental resins. By exposing the resin to a light source, it will begin to polymerize and shrink. This polymerization shrinkage (PS) is crucial for the durability of the material, as it will determine stresses and strains in the film and the uptake of fluids and bacteria. A polymer that is widely used as a dental bonding agent is dimethacrylic acid. Curing studies on this polymer have been performed with a STRAFI NMR [119,156–158]. In an experiment performed by T. Nunes et al. [119], glass vials were filled with a liquid resin. The liquid resin was exposed to light. Different groups of hydrogen atoms could be discriminated based on their T2- and T1-relaxation times: mobile molecules (free monomers) with long relaxation times and rigid molecules (cured AB2). It was found that oxygen strongly influenced the kinetic behavior. Using STRAFI, the influence of different cements and curing protocols could be determined and all were shown to have an effect on the particular curing process, and they should be chosen with care in medical applications. Other curing measurements can be found for the curing of wood glue layers [154] and gelatin layers (biopolymer) [155]. 4.4. Film Formation—Coalescence In some applications, film formation involves coalescence (e.g., with latex particles). In case of latex dispersions, film formation occurs due to the interdiffusion of polymer chains rather then curing. Well-known applications are water-based paints [164–167] or pressure-sensitive adhesives [24,168]. The formation of dry, homogeneous films from colloidal dispersions such as latex in water can be described by a three-step process [22]. First, water evaporates and the particles concentrate and overcome their colloidal stability [169]. Next, the particles deform [170], Polymers 2022, 14, 798 20 of 32 20 of 32 trying to fill the void volume in the film. Lastly, interdiffusion of individual polymer chains overcomes the particles’ boundaries, forming a continuous and rigid film [171]. The deformation and interdiffusion of these polymers depens on the mobility of the latex particles, reflected by their glass transition temperature (tg) [107]. The sequence of events can overlap in time. The processes can also influence each other. For instance, the water fraction will influence the mobility of the polymer chains [107,172], leading to a decrease in deformation and interdiffusion upon drying [85]. Studying these processes with NMR can give crucial information, leading to a better understanding of unwanted effects such as film cracking [173], trapped water [24] and irregular particle formation [174]. g pp g p To illustrate the use of high-resolution NMR for latex film formation, we use a study by B. Voogt et al. [107] as an example. In this study, drying of both soft (tg < room tem- perature) and hard (tg > room temperature) latices was studied inside a GARField NMR. Measurements were performed with an OW-sequence with te = 2τ = 40 µs. Figure 10 shows profiles measured during the drying of both soft- (left) and hard-type (right) latices at an RH of 80%. As discussed in Section 4.2, a step-wise process is observed where first evaporative drying takes place. This will induce film shrinkage due to the evaporation of water that can be observed via a receding front. This will lead to higher concentration of latex particles. In both films, thickness (d) and maximum signal intensity (Smax) decreased over time. It was observed that the hard-type latex dried much faster. Hard-type latex thickness decreased until 20 min, while for the soft latex, this only happened at 40 min. 4.4. Film Formation—Coalescence The maximum intensity of the hard-type latex also lowered faster and ended lower at around 0.2, while for the soft latex a signal intensity of 0.6 was observed, both leveling off at 40 min. The difference in leveling off between Smax and d for hard latex suggests a continuation of bulk water evaporation, while the latex particles are fixed in position and are not able to keep concentrating. This is understandable as particles below their tg will form brittle and porous networks [22] and are unable to form a continuous film. Figure 10. NMR signal intensity profiles during drying of soft- (left) and hard-type (right) latex. Indicated by Smax and d are the final maximum signal intensities and thicknesses of the coatings. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder, Paul Venema and OlafAdan, Water mobility during drying of hard and soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from Elsevier [107]). Figure 10. NMR signal intensity profiles during drying of soft- (left) and hard-type (right) latex. Indicated by Smax and d are the final maximum signal intensities and thicknesses of the coatings. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder, Paul Venema and OlafAdan, Water mobility during drying of hard and soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from Elsevier [107]). In this study by B. Voogt et al., the protons in free water and polymer where studied simultaneously, using a multi-exponential decay analysis Section 2.2. A typical OW-decay is shown in Figure 2 right. For both latices, a clear multi-exponential decay was observed where the short relaxation time could be linked to the polymer and the long relaxation time to the free water phase. Both short relaxation times are fixed at 0.1 ms, showing that both latices have proton pools with mobilities that are not affected by drying. These are the protons embedded within the latex. Using this multi-exponential decay, the different concentrations could be followed over time. The researchers found that a loss of free water (ρlong) due to evaporation is Polymers 2022, 14, 798 21 of 32 independent of the lattice type. However, the latex fraction shows an increase for the soft latex type (ρshort), indicating a further increase in concentration. 4.4. Film Formation—Coalescence This effect was not observed in the hard latex type. The increase in proton density is the consequence of coalescence of the particles. When film formation happens above the polymers tg, the polymer mobility is high enough that the polymer chains can interdiffuse and form a polymer film. This indicates that the tg of latex has a huge influence on the film formation processes and the coalescence of the particles. This study illustrates that coalescence of latex particles can be made visual inside the NMR setups. This makes it possible to study the effect of multiple parameters on the coalescence kinetics and improve latex film applications. Other studies have focused on the coalescence of particles at the liquid–air interface, leading to a ”skin-layer” [24,175]. In certain compositions, coalescence appears near the liquid–air interface during the drying stage. This is schematically represented in Figure 11 left. This skin-layer will block the evaporation, leading to trapped water within the film. R. Rodriguez et al. [175] studied the film formation for different compositions: acrylic copolymer (SM0), a hybrid latex containing 25% PDMS (SM25), and a blend of the acrylic (SM0) with 11 wt% PDMS emulsion. Drying measurements on two of these compositions are shown in Figure 11, namely, SM0 (b) and SM25 (c). The SM0 polymer has a much lower mobiltiy than the SM25. Therefore, the signal intensity for SM0 is almost zero after drying, while for the SM25 there is still signal. By setting the echo time to 180 µs, the researchers made sure that almost no signal was attributed to the SM0 polymer. The signal could be attributed to free water. In both drying experiments, the signal intensity increases in the depth of the film. This increase was also observed in other studies [24] and was attributed to a gradient in free water. In the neat acrylic dilution (SM0), a deviation from this linear behavior is observed where a layer with lower signal intensity appears at the top. This indicates a step in the free water concentration and a denser packing of latex particles at the surface (Figure 11a). This layer increased in thickness over time. This drastic step in particle density was not observed in the SM25, see Figure 11c. Here, a more uniform profile is observed that reached its final form at 55 min. Figure 11. 4.4. Film Formation—Coalescence (a): schematic representation of latex packing near the surface accompanied by a represen- tation of the corresponding NMR signal. NMR profiles measured over time for (b) acrylic copolymer latex (SM0) and (c) 25 wt% PDMS on the acrylic monomer(SM25) (Reprinted with permission from Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society [175]). Figure 11. (a): schematic representation of latex packing near the surface accompanied by a represen- tation of the corresponding NMR signal. NMR profiles measured over time for (b) acrylic copolymer latex (SM0) and (c) 25 wt% PDMS on the acrylic monomer(SM25) (Reprinted with permission from Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society [175]). Other studies used similar NMR measurements to study the effect of glass transition temperature [176], surfactant [168], tackifying resins (TR) [24], salts [177], different amounts of carboxylic acid functional groups [105] and the Peclet number [151] on the film formation process. GARField NMR seems to be the best technique for these processes, as it has a high-enough FOV to cope with the latex films and can give the best resolution. However, measurements with a MOUSE were also performed on latex films [120]. This, however, led to lower resolutions (30 µm) and was hampered by long measurement times. 4.5. Diffusion/Penetration Liquid uptake and diffusion in thin layers is important for coatings, the printing industry or sustainability of materials. Water and solvents can weaken interactions between neighboring polymers and ultimately lead to failure of the thin layer by softening or cracking. Both the amount of liquid and the speed of penetration are crucial parameters in Polymers 2022, 14, 798 22 of 32 22 of 32 characterizing the stability of thin layer. In this section, it will be shown how NMR profiling has been used to measure liquid penetration over time. A crucial parameter for solvent penetration is the diffusion coefficient. This parameter is, however, difficult to measure because it depends on morphology such as crystallinity [178] or temperature. p p gy y y p To illustrate the capability of high-resolution NMR depth profiling in studying pen- etration and diffusion experiments, a study by N. J. W. Reuvers et al. [179] is used. The water uptake in thin nylon-6 films was measured with a GARField NMR. p y The water uptake was measured using an OW-sequence. A water uptake experiment in a 200 µm thick nylon-6 is shown in Figure 12 left. When going from right to left, the glass plate, glue layer, nylon and water can be observed which can also be seen in the schematic picture of the setup. While the glass plate cannot be imaged by the NMR, a clear difference between the silicon glue, nylon film and water is observed. The observed differences in signal intensities can be linked to varying T1 and T2-times. Shown with a bold line is the signal intensity measured before the experiment. The researchers distinguished three different processes. (1) A liquid fronts develops, traveling towards the bottom of the layer (t < 6 h), (2) water distributes equally over the film (6 h < t < 10 h) and (3) a slower processes occurs where a small signal increase is observed near the glass–polymer interface. Using these profiles, the researchers determined the actual diffusion coefficient within the layer. To be able to do this, the researchers needed to convert the NMR signal intensity to moisture content (θ). They could link the NMR signal to a moisture profile using gravimeter calibration [179]. The resulting relationship was nonlinear and was attributed to plasticization and a change in relaxation of free water. 4.5. Diffusion/Penetration The NMR signal profiles in Figure 12 can be viewed as a supper position of a liquid front and plasticization front. Using the relation between moisture content and NMR signal, the original NMR profiles (Figure 12 left) are converted to moisture content profiles (Figure 12 right). The small signal increase observed before (3) could be attributed to polymers that become more mobile and contribute to the signal. Figure 12. Left: Liquid profiles measured during the uptake of water into 200 µm thick nylon membranes. Right: same profiles as in the middle graph but after replacing the signal intensity with the moisture content. (Reprinted with permission from Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society. [179]). Figure 12. Left: Liquid profiles measured during the uptake of water into 200 µm thick nylon membranes. Right: same profiles as in the middle graph but after replacing the signal intensity with the moisture content. (Reprinted with permission from Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society. [179]). The moisture profiles were used to extract the diffusion coefficient of water within the nylon film. The calculated diffusion coefficients are shown in Figure 13. The diffusion coefficient increases with increasing moisture content. To study the signal change due to plasticizing, the excess water is replaced by D2O. D2O has the same characteristics as water but will not contribute to the NMR signal. It was observed that half the NMR signal in the profiles was linked to hydrogen atoms of water and half of the signal to mobilized polymer. Additionally, the glass transition temperature and T2-relaxation could be linked with moisture content (Figure 13 right). In a follow-up Polymers 2022, 14, 798 23 of 32 study, it was found that the plasticization lags behind the water migration in these nylon-6 thin films [180]. Figure 13. Left: the diffusion coefficient (D) in function of moisture content. Right: The glass transition temperature (tg) and average relaxation time (T2) in function of moisture content. (Reprinted with permission from Macromolecules 2008, 41, 22, 8537–8546. Copyright 2012 American Chemical Society [175]). Figure 13. Left: the diffusion coefficient (D) in function of moisture content. Right: The glass transition temperature (tg) and average relaxation time (T2) in function of moisture content. (Reprinted with permission from Macromolecules 2008, 41, 22, 8537–8546. Copyright 2012 American Chemical Society [175]). Similar penetration studies have been performed on dental resins. Using STRAFI, G. 4.5. Diffusion/Penetration Hunter et al. [158] studied the uptake of water and water/ethanol mixtures into a commercial dental resin. Measurements with different ethanol concentrations revealed that the diffusion coefficient increased with ethanol content. Additionally, transport in ceramic substrates [181], multilayer coatings [141,182], glassy pellets of the starch polymer amylose [124], cement pastes [136] glue lines [154], vulcanized rubber [183] and nylon- 6 [140,141] have been studied. In addition to the penetration of water, the transport of different ions such as Mn2+ and Cu2+ [184], different salt solutions [185] and the ingress of vapours [186,187] have also been studied with the NMR setups. Finally, the influence of stress [188] on the penetration behavior was studied by V. Baukh et al. 4.6. Thin Films on Site: Cultural Heritage In some applications, measurements of thin layers need to be performed outside the lab because it is impossible to collect small samples or to recreate a similar structure. As the STRAFI and GARField setups are located within the lab and require samples of a specific shape and size, measurements on site are performed by NMR-MOUSE. The portability of the NMR-MOUSE makes it possible to measure samples with infinite lateral dimensions where limits are mostly bound because of time constraints. One big category of such applications studied with the NMR-MOUSE is cultural heritage [189]: for example, in case of paintings on wood. A significant issue with cultural heritage is structural damage during conservation. Using the NMR-MOUSE, the internal structure of paint layers can be studied. These studies can help in characterizing different sources of damage and identifying the best conservation environment. Damage of cultural heritage is mainly caused by moisture uptake which, for example, in wooden painting or walls, can cause degradation over time. Measuring the moisture distribution can provide information on the conditions in which the painting should be stored and can help in preserving the cultural heritage [8]. In this section, a closer look at some analysis methods will be given, demonstrating the use of the NMR-MOUSE on cultural heritage. g B. Blumich et al. [7] demonstrated the use of the NMR-MOUSE for studying the layered structure of paintings. They started with a wood panel covered by a primer and one or multiple layers of paint. They tried to mimic the structure found in old paintings. Figure 14 shows a picture of the structure where two positions are marked where either one (1) or two (2) layers of paint are used. Depicted in Figure 14b are the depth profiles measured by an NMR-MOUSE of the two sides shown in Figure 14a. Both paint layers had good signal and the thickness of the layers is also reflected in the width of the NMR signals. Imaging B. Blumich et al. [7] demonstrated the use of the NMR-MOUSE for studying the layered structure of paintings. They started with a wood panel covered by a primer and one or multiple layers of paint. They tried to mimic the structure found in old paintings. Figure 14 shows a picture of the structure where two positions are marked where either one (1) or two (2) layers of paint are used. 4.6. Thin Films on Site: Cultural Heritage Depicted in Figure 14b are the depth profiles measured by an NMR-MOUSE of the two sides shown in Figure 14a. Both paint layers had good signal and the thickness of the layers is also reflected in the width of the NMR signals. Imaging Polymers 2022, 14, 798 24 of 32 24 of 32 different layers was performed both with total amplitude measurement and characterizing the signal decays (CPMG). In Figure 14e, the total NMR signal is measured along the thickness of the painting revealing a layered structure where paint, primer and wood are observed. Using this method, the thickness of the different layers could be monitored at different points, revealing damage at certain positions. The CPMG could give information about changes within the layers over time. g y In the same paper, the authors also demonstrated the ability to characterize dam- aged old paper structures by measuring the hydrogen atoms in the cellulose fibres, time- dependent water uptake and drying in stone. Even old master violins such as a Stradivari were investigated in this way. The study revealed that the master violins’ wood density increased with age, which determined the quality of the instrument. The possibilities of this measurement technique was also used for the ancient Roman fresco and the bricks in the walls of the cryptoporticus at Colle Oppio in Rome [190], the degradation of historical paper [76] and the conservation treatments on paintings [191–193]. 4.7. Conclusions In GARField, the problem with the inhomogenous field is solved by specially designed poles leading Polymers 2022, 14, 798 25 of 32 to the best space and time resolution found in all the setups. Moreover, Fourier STRAFI exists, which can measure much faster then conventional STRAFI. However, due to the arrangement of the magnetic field, a solenoid-shaped coil is needed that compared to the surface coil in GARField will always have lower sensitivity. A drawback of this special design is that GARField lacks the ability to measure samples larger then 400 µm. To measure larger samples, STRAFI would be more appropriate. Finally, the NMR-MOUSE has proven to be valuable in cultural heritage because it is the only mobile device able to measure depth profile on-site in a non-invasive manner. p p A variety of applications such as drying, film formation, penetration and measure- ments on cultural heritage have been reviewed illustrating the potential of these NMR- profiling tools. By measuring hydrogen profiles over time, the setups were able to follow drying, curing and penetration fronts. Using these profiles, the diffusion coefficient and front kinetics could be determined for a wide variety of parameters. Detailed structural information about thin layers could be gathered by measuring T2, T1 relaxation times and signal intensity. The information could be used to follow film formation, film degradation, penetration and diffusion experiments. p p The current measurement techniques lack the ability to measure below 1 min. Future improvements towards higher time resolutions is, for example, useful in the printing industry where the penetration occurs at timescales of 100 ms. Great potential was shown by R. J. K. Nicasy et al. [194] that demonstrated the use of high-speed NMR to profile liquid uptake in non-transparent porous media with time resolutions of 10 ms. This was only shown for liquid penetration but could be extended towards the characterization of chemical structures. In addition to measuring speed, improvement towards the amount of chemical information obtained with the setup can be achieved by combining the above- mentioned techniques with NMR spectroscopy. Currently, no such studies are available, but they could be very valuable to determine chemical components and reactions in much more detail. Lastly, by combining the NMR techniques with other measurement setups, a better understanding of the information can be achieved. 4.7. Conclusions Three different high-resolution NMR setups able to study thin layers between 10 and 1000 µm were reviewed, namely, STRAFI, GARField and MOUSE. These NMR techniques are all able to give spatial and time-resolved information about thin layers. Figure 14. (a): Easel painting model with a wooden background covered by a primer and one (1) or two (2) paint layers. (b): measured depth profiles for the position marked in Figure 14a. A clear difference in thickness is observed between both layers where one layer is thinner than the two layers. (d) Signal decay measurements at position 2 for different depths. (e) Complete depth profile through the painting indicating the different layers. (Reprinted with permission from Acc. Chem. Res. 2010, 43, 6, 761–770. Copyright 2010 American Chemical Society [7]). Figure 14. (a): Easel painting model with a wooden background covered by a primer and one (1) or two (2) paint layers. (b): measured depth profiles for the position marked in Figure 14a. A clear difference in thickness is observed between both layers where one layer is thinner than the two layers. (d) Signal decay measurements at position 2 for different depths. (e) Complete depth profile through the painting indicating the different layers. (Reprinted with permission from Acc. Chem. Res. 2010, 43, 6, 761–770. Copyright 2010 American Chemical Society [7]). The setups used two different acquisition methods: slice-selective pulse and Fourier- based measurements. In the slice-selective pulse measurements performed by MOUSE and in conventional STRAFI, the signal is recorded in a step-wise manner, whereby the pulse excites the region of interest. In this case, the resolution is determined by the pulse length given by ∆r ≈1/tpγG. High resolution can be achieved at the expense of long measurement times. The advantage of the step-wise acquisition is that sample size is only limited by the aperture used. Unfortunately, the resolution is limited by the large curvatures found in the main magnetic field # » B0 of both STRAFI and MOUSE and should be determined experimentally. In GARField and in some cases in STRAFI, the acquisition is achieved by a Fourier analysis. The sample is excited with one pulse (limiting the total measurement area) where the resolution is determined by the acquisition time, given by ∆r ≈1/taγG. As the sample is measured using one pulse, measurements are much faster. 4.7. Conclusions This has already been shown to be valuable with confocal Raman microscopy [89] and electrochemical impedance spectroscopy [195], but could be extended towards other techniques. Author Contributions: Conceptualization, R.N., H.H., B.E. and A.O.; writing—original draft prepa- ration, R.N., H.H., B.E. and A.O.; writing—review and editing, R.N., H.H., B.E. and A.O.; supervision, H.H., B.E. and A.O.; project administration, H.H., B.E. and A.O.; funding acquisition, H.H., B.E. and A.O. All authors have read and agreed to the published version of the manuscript. Funding: This publication is part of the project PQP (Print Quality and Particles) (with project number 17099) of the research programme Collaboration program High Tech Systemen en Materialen (HTSM) 2018 TTW] which is (partly) financed by the Dutch Research Council (NWO). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 2. Anderson, A.L.; Chen, S.; Romero, L.; Top, I.; Binions, R. Thin Films for Advanced Glazing Applications. Buildings 2016, 6, 37. [CrossRef] pp g p y gy 4. Pan, Z.; Ding, J.; Hou, X.; Shi, S.; Yao, L.; Liu, J.; Li, P.; Chen, J.; Zhai, J.; Pan, H. Substantially improved energy storage capability of ferroelectric thin films for application in high-temperature capacitors. J. Mater. Chem. A 2021, 9, 9281–9290. [CrossRef] 3. Silva, J.P.B.; Sekhar, K.C.; Pan, H.; MacManus-Driscoll, J.L.; Pereira, M. Advances in Dielectric Thin Films for Energy Storage Applications, Revealing the Promise of Group IV Binary Oxides. 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Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens
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BioMed Central BioMed Central Open Ac Research article Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens Belén G Pardo1, Carlos Fernández1, Adrián Millán1, Carmen Bouza1, Araceli Vázquez-López1, Manuel Vera1, José A Alvarez-Dios2, Manuel Calaza2, Antonio Gómez-Tato3, María Vázquez4, Santiago Cabaleiro4, Beatriz Magariños5, Manuel L Lemos5, José M Leiro5 and Paulino Martínez*1 Address: 1Departamento de Genética. Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain, 2Departamento de Matemática Aplicada, Facultad de Matemáticas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain, 3Departamento de Geometría y Topología, Facultad de Matemáticas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain, 4Cluster de la Acuicultura de Galicia (CETGA), Punta de Couso s/n, 15965, Aguiño (Ribeira), A Coruña, Spain and 5Departamento de Microbiología y Parasitología. Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain Email: Belén G Pardo - mariabelen.gomez@usc.es; Carlos Fernández - carlos.fernandez.lopez@usc.es; Adrián Millán - adrian.millan@usc.es; Carmen Bouza - mcarmen.bouza@usc.es; Araceli Vázquez-López - aravazq@usc.es; Manuel Vera - manuel.vera@usc.es; José A Alvarez- Dios - joseantonio.alvarez.dios@usc.es ; Manuel Calaza - mcalaza@usc.es; Antonio Gómez-Tato - antonio.gomez.tato@usc.es; María Vázquez - ruizocenda@cetga.org; Santiago Cabaleiro - cabaleiro@cetag.org; Beatriz Magariños - beatriz.magarinos@usc.es; Manuel L Lemos - manuel.lemos@usc.es; José M Leiro - josemanuel.leiro@usc.es; Paulino Martínez* - paulino.martinez@usc.es * Corresponding author * Corresponding author Received: 23 June 2008 Accepted: 25 September 2008 Received: 23 June 2008 Accepted: 25 September 2008 This article is available from: http://www.biomedcentral.com/1746-6148/4/37 This article is available from: http://www.biomedcentr © 2008 Pardo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2008 Pardo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Veterinary Research Open Access Research article Open Ac Research article Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens Belén G Pardo1, Carlos Fernández1, Adrián Millán1, Carmen Bouza1, Araceli Vázquez-López1, Manuel Vera1, José A Alvarez-Dios2, Manuel Calaza2, Antonio Gómez-Tato3, María Vázquez4, Santiago Cabaleiro4, Beatriz Magariños5, Manuel L Lemos5, José M Leiro5 and Paulino Martínez*1 Background h b ( for the development of EST resources in Japanese flounder (8856 ESTs) and Atlantic halibut (17659 ESTs) [21-28], EST resources in turbot are scarce (3171 ESTs). Less than 800 sequences have been deposited to date in the NCBI nucleotide database, most of them from anonymous mic- rosatellite searching [29]. g The turbot (Scophthalmus maximus; Scophthalmidae; Pleu- ronectiformes) is a commercially valuable flatfish that has been intensively cultured for the last decade. Its produc- tion has steadily increased to 7120 tonnes in 2006 (80% from Spain; FEAP, 2006) and represents one of the most promising aquaculture species in Europe. However, dis- ease outbreaks in turbot have occurred frequently and losses due to infections constitute a serious problem for its culture [1,2]. The use of antibiotics, vaccines and fish health management practices has partially solved the problem, but the achievement of large-scale production in the highly competitive world market requires enhancing resistance of cultured fish to diseases. Information on the immune response of turbot is still limited, and little is known about host-pathogen interactions in fish species. The screening and identification of immune-relevant genes is essential to analyze the genetic basis for infection, immunity and resistance to pathogens of economic rele- vance in aquaculture. Expressed sequence tag (EST) anal- ysis is a powerful approach to provide a rapid and efficient method to go from expressed sequences to genes. ESTs are essential for studies of gene function [3,4], but are also useful to identify polymorphic gene markers, such as mic- rosatellites and single nucleotide polymorphisms (SNPs) [5-8]. These markers are the basis for genetic and physical mapping, and for comparative genome analysis [9-11]. From a practical perspective, maps can be applied for assisted selection programmes (MAS) and eventually for identification of genes related with quantitative traits (QTL) [12,13]. In addition, ESTs constitute the basic resources to develop microarrays for functional genomics studies [14]. With the aim of increasing the genomic resources in tur- bot and identifying relevant genes for immunity, three cDNA libraries were constructed from mRNA isolated from immunity-related tissues of turbot (liver, spleen and head kidney) at different times after infection with Aerom- onas salmonicida and the scuticociliate parasite Philasterides dicentrarchi. These pathogens are responsible for impor- tant disease outbreaks in turbot, as well as in other culture fish species [30-33]. Our main goal was to obtain the most accurate information possible to address functional genomic studies on disease resistance. Background h b ( However, the use of non-normalized cDNA libraries made it feasible to get a preliminary picture of the turbot genetic response to path- ogens through analyzing transcript distribution among infected vs. control libraries. A total of 12584 ESTs were sequenced and compared to GenBank database and a large array of defence or immune-related genes was iden- tified. Also, this large scale EST study increased the number of putative markers for mapping. A total of 191 microsatellites, of which 104 exhibited sufficient flanking sequences for primer design, and 2197 good quality SNPs were identified for the first time in turbot. The cDNA sequences generated will serve as a basis for microarray construction. This first EST study in turbot will provide the support for further research into the genetics, genomics and even proteomics of this important aquaculture spe- cies. EST sequence resources are rapidly growing in molecular databases. However, the number of ESTs in fish is gener- ally scarce, excluding some model species and Atlantic salmon among cultured fish [15-19]. The scarcity of EST resources in cultured fish limits the use of modern func- tional genomic approaches for selective breeding pur- poses [20]. Among flatfish, aquaculture production has been successfully achieved in turbot, Japanese flounder and Atlantic halibut. Compared to the very large efforts Abstract Background: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host- pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. Results: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune- related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant Page 1 of 12 (page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 http://www.biomedcentral.com/1746-6148/4/37 differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. Conclusion: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot. Page 2 of 12 (page number not for citation purposes) Gene annotation d f ESTs were identified by BLAST searches against nucleotide database at GenBank. Due to the low representation of fish genes, a protein-based homology strategy was also used in the international database searches. Protein sequences have been demonstrated to be more suitable to detect homology over long periods of evolutionary time [37]. Our EST database pprovided a graphical view of all contigs, their PROSITE/PRINTS protein patterns and a search interface by keywords, microsatellite, gene, and UniGene/GO/KEGG information. Tools to search for sequences and markers based on annotations, to perform local BLAST searches, and to select sequences for a pro- spective microarray were also included. These tools used RepeatMasker http://www.repeatmasker.org/ for masking low-complexity sequences and OligoArray 2.1 http:// berry.engin.umich.edu/oligoarray2_1/ to predict second- ary structures and potential cross-hybridization. EST projects generate a large number of redundant sequences due to the random selection of cDNAs from tis- sue libraries, especially when libraries are non-normal- ized and a high number of clones are sequenced. Clustering redundant sequences is a critical step to iden- tify genes. The program CAP3 http://seq.cs.iastate.edu./ was used to cluster EST sequences using the default parameters. As shown in Table 1, clustering yielded 3482 unique turbot sequences: 2409 singletons (69.2%) and 1073 contigs (30.8%) comprising 6847 ESTs (6.4 sequences/contig) and an average length of 527 bp. Figure 1A shows the histogram distribution of contig sizes. Although most contigs showed two (46.7%) or three-to- five (30.3%) sequences, a small number of highly As shown in Table 1, 1716 (49.3%) unique sequences dis- played no significant similarity to known sequences or ESTs in the public databases, whereas the remaining 1766 (50.7%) showed significant matches with e-values ≤ 1e-5. Among these, BLAST database searches allowed assign- ment of putative function to 816 sequences (23.4%). In spite of their lower frequency among unique sequences, contigs were annotated more frequently (59.9%) than singletons (40.1%). As in other ESTs fish studies, the lower percentage of annotated singletons suggests that these are either novel fish-specific or rapidly evolving genes [38]. Also, it is possible that bioinformatic errors could have a greater impact on singletons, since they are unique sequences whose information cannot be con- trasted with other sequences in the database. The lower annotation success regarding similar genomic projects [28,39] was probably related to the read length (around 500 bp) and specially the direction (from the 3'end) of sequencing. http://www.biomedcentral.com/1746-6148/4/37 http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 [26-28]. However, this order comprises around 600 spe- cies, many of them of great economic value both for fish- eries and farming. In this study, we have addressed the construction of an EST database for the identification of genes related with immunity and defence in turbot. expressed genes were also detected. As shown in Figure 1B, the beta-globin contig (407 ESTs) and five others con- tained more than 100 ESTs, which represents a high pro- portion (1212/9256 = 13.1%) over the total redundancy (74.0%). Most of these ESTs shared homology with genes involved in transport, protein metabolism and response to stress, all of which related to defence. At the other extreme, 77.0% of the 1073 contigs were ≤ 5 times redun- dant, indicating that most of these unique sequences rep- resent rare mRNAs and that these libraries provide a rich source of sequence information. The three cDNA libraries constructed held at least 2.5 × 106 primary recombinant clones. The directional cloning approach used for construction of cDNA libraries ensured that cDNA inserts appeared mostly in the same orienta- tion within the vector. Clones were sequenced from the 3' end with the vector primer T7 to obtain large gene-specific genomic regions for future oligo-microarray design. The libraries used were non-normalized and, as usually observed [16], substantial redundancy was obtained (around 74%). This approach allowed an analysis of the turbot response to specific pathogens by comparing the amount of transcripts across all genes or groups of genes classified in functional categories. A total of 12584 ESTs were sequenced. After trimming and vector removal 9256 high quality ESTs were obtained (Table 1), showing an average length of 409 bp. Their sequences are available in the dbEST NCBI database under numbers FE943103- FE952358. Results and Discussion cDNA libraries and ESTs EST analysis is an efficient and fast method for gene dis- covery [15,17]. In Pleuronectiformes, this approach has been recently applied in Japanese flounder (Paralichthys olivaceus; [21,22,24,25]), winter flounder (Pseudopleu- ronectes americanus; [34]), flounder (Platichthys flesus; [35,36]) and Atlantic halibut (Hippoglossus hippoglossus; Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 Sequence prevalence distribution of the identified contigs from turbot libraries Figure 1 Sequence prevalence distribution of the identified contigs from turbot libraries. (A) Absolute frequency histogram showing contig size (number of sequences) distribution. (B) Functional and BLAST hits confidence characteristics of the ten largest contigs. Only biological function according to GO terms has been included. Gene annotation d f The 3' untranslated region (UTR) is approxi- mately double the length of the 5'UTR according to Gen- Bank fish entries. So, what is gained in specificity for microarray oligo-design is lost for gene annotation. Table 1: Summary statistics of ESTs from turbot libraries Table 1: Summary statistics of ESTs from turbot libraries Number % Good-quality ESTs 9256 Redundant sequences 6847 74.0 Unique sequences 3482 36.0 Contigs 1073 30.8 Singletons 2409 69.2 Unique sequences with no BLAST hits 1716 49.3 Unique sequences with BLAST hits 1766 50.7 BLASTN 1091 61.8 BLASTX 675 38.2 Unique sequences with functional annotation 816 23.4 Contigs 489 59.9 Singletons 327 40.1 Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 A Sequence prevalence distribution of the identified contigs from turbot libraries Figure 1 Sequence prevalence distribution of the identified contigs from turbot libraries. (A) Absolute frequency histogra showing contig size (number of sequences) distribution. (B) Functional and BLAST hits confidence characteristics of the ten argest contigs. Only biological function according to GO terms has been included. B Gene Contig size e-value % Hom Biological process BLAST Hepcidin precursor 70 3e-21 83 - BLASTN Lysozyme C precursor 76 8e-42 100 Response to stress BLASTX Trypsinogen 1 precursor 86 7e-21 67 Proteolysis BLASTX Serotransferrin 88 5e-11 85 Ion transport BLASTN Trypsinogen 1 114 4e-88 69 Proteolysis BLASTX cDNA clone D. labrax 148 8e-22 82 - BLASTN Ribosomal protein S27a 155 3e-32 68 Prot. metabol. process BLASTX Ribosomal protein S2 171 0.0 93 Biosyntethic process BLASTN Hemoglobin alpha-1 chain 219 6e-32 86 Transport BLASTX Beta type globin 407 3e-127 81 Transport BLASTN Sequence prevalence distribution of the identified contigs from turbot libraries Figure 1 Sequence prevalence distribution of the identified contigs from turbot libraries. (A) Absolute frequency histo showing contig size (number of sequences) distribution. (B) Functional and BLAST hits confidence characteristics of the t largest contigs Only biological function according to GO terms has been included B Gene Contig size e-value % Hom Biological process BLAST Hepcidin precursor 70 3e-21 83 - BLASTN Lysozyme C precursor 76 8e-42 100 Response to stress BLASTX Trypsinogen 1 precursor 86 7e-21 67 Proteolysis BLASTX Serotransferrin 88 5e-11 85 Ion transport BLASTN Trypsinogen 1 114 4e-88 69 Proteolysis BLASTX cDNA clone D. labrax 148 8e-22 82 - BLASTN Ribosomal protein S27a 155 3e-32 68 Prot. http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 All unique sequences were annotated based on similarity using BLASTX or BLASTN [40] in the public databases GenBank NR and Unigene. The multiple annotations pro- vided greater assurance about gene description and fre- quency of annotation than in a single database. The use of consensus sequences allowed sequences without signifi- cant similarity regions with a known protein (e.g., 5' or 3' noncoding regions) to be annotated if they were members of an annotated contig. All hits with e-value ≤ 1e-5 and their associated alignments were stored in the database and tracked with any associated functional annotation. We also ran AutoFACT [41] on all sequences in the data- base. It is interesting to note that while AutoFACT was able to come up with more function-specific information than our online tool (provided external database hits were found), it was not able to annotate as many sequences as our online custom tool. defence/immunity were always retained given their inter- est in this study. Biological process, molecular function and cellular component categories are shown in Figures 2, 3 and 4, respectively. According to these criteria, biologi- cal processes associated with proteins (synthesis, metabo- lism and proteolysis) were the largest annotated categories, though a significant group related to transport and response to stress did appear. In accordance with this, a large proportion of sequences were classified into struc- tural constituent of ribosome and protein-related func- tions (binding, peptidase activity) regarding molecular function. A remarkably high proportion of annotated sequences were categorized as oxidoreductase activity. Finally, most gene activity was located into the ribosome, followed by the membrane, nucleus and the extracellular and cytoplasm constituents of the cell. Gene annotation d f metabol. process BLASTX Ribosomal protein S2 171 0.0 93 Biosyntethic process BLASTN Hemoglobin alpha-1 chain 219 6e-32 86 Transport BLASTX Beta type globin 407 3e-127 81 Transport BLASTN B http://www.biomedcentral.com/1746-6148/4/37 Immune genes Page 5 of 12 (page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO) Figure 3 Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO). Molecular Function 0 20 40 60 80 100 Motor activity Catalytic activity Isomerase activity Lyase activity Oxidoreductase activity Transferase activity Kinase activity Hydrolase activity Peptidase activity Enzyme regulator activity Receptor activity Transporter activity Structural molecule activity Structural constituent of ribosome Transcription regulator activity Chemokine activity Binding Protein binding Cofactor binding Heme binding Carbohydrate binding Nucleic acid binding RNA binding DNA binding Translation factor activity, nucleic acid binding Calcium ion binding Ion binding Lipid binding Nucleotide binding Oxigen binding ot unique sequences in molecular function categories following Gene Ontology (GO) rbot unique sequences in molecular function categories following Gene Ontology (GO). Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO) Figure 3 Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO). genes with defence or immunity using scientific literature information available on vertebrates. According to this analysis, 203 genes out of 816 annotated (24.9%) appeared related to defence or immunity in our work (Table 2). This observation is in agreement with the important role of liver, spleen and head kidney in the immune response of fish. though some of them have important roles in the immune response like B-cell linker, chemotaxin, comple- ment components, IgD, IgM, interferon stimulated gene 12 (b2), lipopolysaccharide (LPS)-binding protein, natu- ral killer (NK)-lysine type 1, peptidoglycan recognition protein, skin mucus lectin and tumour necrosis factor (TNF) receptor associated factor2, among others. Previ- ously, only a hepcidin [44] and a natural killer cell enhancing factor [45] had been characterized in turbot. Like in mammals, the immune system of fish is composed of non-specific and specific defence. The innate immune response is an important and highly developed defence mechanism against pathogens in fish [43]. Examples of innate immunity include anatomic barriers, mechanical removal of pathogens, bacterial antagonism, pattern-rec- ognition receptors, antigen-nonspecific defence com- pounds, complement pathways, phagocytosis, and inflammation [43]. In Table 2, a list of defence/immune- related genes that were found three or more times in our libraries is shown. Complement related genes were pre- dominant (7.9%) followed by apoptosis (4.9%) and immunoglobulin (Ig)-related (3.9%) genes. Immune genes Annotated ESTs (816) were classified into functional cat- egories according to GO terms [42] (77.0%), 367 among contigs (75.1%) and 261 among singletons (79.8%). A single sequence very often showed several GO terms in the same ontology, so we try to group them using a single more general category. Overly specific categories were also collapsed into more general terms. Any terms related to Systematic classification of annotated sequences using available bioinformatics tools (GO, KEGG) provides a useful information to analyze the functional profile of annotated genes. In our study, we were especially inter- ested in identifying immune genes or in a broader sense those genes regulated in response to specific pathogens of turbot. So, we analyzed the relationship of our annotated Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO) Figure 2 Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). Biological Process 0 20 40 60 80 10 Biological process Reproduction Pathogenesis Biological adhesion Developmental process Cell differentiation Regulation of biological process Cellular homeostasis Cellular process Cell adhesion Metabolic process Primary metabolic process Cellular metabolic process Biosyntethic process Protein metabolic process Lipid metabolic process Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process Carbohydrate metabolic process Immune system process Transport Ion trasnport Electron trasnport Protein transport Lipid transport Proteolysis Response to stress Response to stimulus Signal transduction Transcription Cytoskeleton organization & biogenesis Organelle organization & biogenesis Chromosome organization & biogenesis 0 Biological Process 0 20 Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO) Figure 2 Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). Page 5 of 12 (page number not for citation purposes) Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO) Figure 2 Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). sification of turbot unique sequences in biological processes categories following Gene Ontology (GO re 2 sification of turbot unique sequences in biological processes categories following Gene O Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO) Figure 2 Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). Page 6 of 12 (page number not for citation purposes) Immune genes Glutathione S-transferase, heat shock proteins and cytochrome P450, elastases, major histocompatibility complex (MHC) and coagulation factors involved in innate immunity were also present, as well as others like interferon, perforin, hepcidin, nephrosin or alpha-2-macroglobulin. Interest- ingly, the majority of our immune-related cDNAs (75.0%) were reported for the first time in turbot, even The availability of a large number of sequences from immune-related cDNA libraries, both from non-infected fish (controls) as well as from fish challenged with specific pathogens, suggested a comparison of transcript profiles to identify genes regulated in response to these pathogens. Four or more sequences per gene (contig) are the mini- mum necessary to get statistical power to check the null hypothesis of even distribution of sequences among the three libraries. Ninety six genes presented four or more sequences in our libraries. However, taking into account the large number of tests performed, we decided to use a more conservative set of genes (with 6 or more sequences) to avoid type I errors. This new set comprised 72 genes, whose distribution among libraries is shown in Addi- tional file 1 together with their probability of departure from the null hypothesis using a chi-square test. To use all information available and to get higher statistical power, we decided to perform an additional analysis by compar- Page 6 of 12 (page number not for citation purposes) Page 6 of 12 (page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO) Figure 4 Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO). Cellular Component 0 20 40 60 80 Extracellular region Membrane Macromolecular complex Intracellular Cytoplasm Cytosol Ribosome Cytoskeleton Mitochondrion Nucleus Chromosome 100 40 Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO) Figure 4 Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO). Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO) Figure 4 Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO). nique sequences in cellular component categories following Gene Ontology (GO) ot unique sequences in cellular component categories following Gene Ontology (GO). Table 2: Defence and immune-related annotated ESTs from turbot libraries Genes No. Markers-containing ESTs EST studies can also provide resources for the identifica- tion of polymorphic DNA markers such as microsatellites and SNPs. In our study, screening of EST sequences for short tandem repeats (2–6 bp) identified 191 microsatel- lites using a conservative criterion (≥ 6 and ≥ 8 repeats for tri/tetra/penta and dinucleotide motifs, respectively). Of these, 120 had significant hits in BLAST with e-value cut off ≤ 1e-5 and 71 were annotated. Most microsatellites were dinucleotide (128) and trinucleotide (56), while only 6 tetranucleotide and 1 pentanucleotide were found. Among these 191 ESTs sequences, 104 contained suffi- cient flanking sequences length for primer design. Fifty of these 104 microsatellites-containing ESTs were contigs, therefore the in silico comparison of the sequences included in these contigs allowed us to identify 11 puta- tive polymorphic microsatellites. However, the strongest genetic signature was observed in response to P. dicentrarchi infection. Specific genes were exclusively detected in this library and sometimes at high frequencies, like those homologous to Oryzias latipes and Paralichthys olivaceous cDNA clones and to mitochondrial ATP synthase alpha-subunit gene. Functional annotation of these unknown genes now appears relevant to under- stand the response of turbot to this pathogen. The same was reflected when GO categories were analyzed (see Additional file 2). A large number of differentially regu- lated gene categories (P = 0) appeared associated to P. dicentrarchi library according to the different GO criteria. Cytoskeleton organization and biogenesis, as well as car- bohydrate metabolic process appeared up-regulated cate- gories among biological processes. Transporter and hydrolase activities, as well as protein, ion and heme binding also were overexpressed regarding molecular function classification. Finally, cytoskeleton and cytosol appeared as the highest active cellular components. Look- ing at specific categories, a significant increase in the expression of antioxidant genes, like glutathione peroxi- dase and glutathione-S-transferase, was observed. These genes play a pivotal role to prevent cellular damage due to the increased reactive oxygen species (ROS) during infec- tion [47]. Paramá et al. [48] have recently demonstrated the increase in intracellular ROS by proteases of turbot kidney in response to this pathogen. Tissue trauma or invasion by pathogens induces changes in the quantities of several macromolecules in animal body fluids, which comprise one aspect of the acute phase response (APR). In fish, APR proteins include pentraxins, serum amyloid P, several components of the complement system, transfer- rin and thrombin. http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 general down-regulation of genes involved in proteolytic activity in response to this bacterium (chymotrypsin B precursor, trypsinogen1, chymotrypsinogen 2, trypsyno- gen-like serin protease and elastase precursor) was observed, in turn correlated to the lower presence of pepti- dase and proteolysis GO terms in this library. The possi- bility that A. salmonicida could be blocking these genes cannot be ruled out, since it would facilitate the infection process. Additionally, it was noteworthy that several iron metabolism-related genes turned out to be up-regulated after infection with A. salmonicida (haptoglobin fragment 1, globin-related proteins, hepcidin precursor). The high relevance of iron for bacteria infection could explain this observation. The up-regulation of hepcidin after bacteria infection has been recently described in turbot and gilt- head seabream [44,46]. Finally, some other immune rele- vant genes like MHC II alpha and beta antigens, complement C9, thrombin, chemotaxin, bactericidal per- meability-increasing protein, apolipoprotein A-IV3, heat shock protein 90 beta and chemotaxin showed significant up-regulation in response to A. salmonicida. transferrin was observed in response to P. dicentrarchi in our study. Transferrin up-regulation could be related to the increase in inflammation and enhanced oxidative stress typical of infections with this parasite [48]. Finally, up-regulation of other important components of the immune response such the profilin and lysozyme was observed. Profilin-like protein has been reported as a toll- like receptor (TLR) 11 ligand in some parasites [49]. TLRs are evolutionary conserved transmembrane proteins that recognize a unique pattern of molecules derived from pathogens or damaged cells, triggering robust but defined innate immune responses [49]. Immune genes unique sequences % Complement related 16 7.9 Apoptosis related 10 4.9 Immunoglobulin related 8 3.9 Glutathione S-transferase 7 3.5 Elastase 6 2.9 Cytochrome P450 6 2.9 Major histocompatibility complex 5 2.5 Coagulation factor 5 2.5 Interferon related 3 1.5 Perforin 3 1.5 Hepcidin precursor 3 1.5 Nephrosin 3 1.5 Alpha-2-macroglobulin 3 1.5 Other genes 119 58.6 Total 203 24.9 "No. unique sequences" refers to the total amount of the different annotated contigs and singletons for each gene class listed in the Table. "Total percentage" of defence/immune-related genes is referred to the number of unique annotated sequences from turbot libraries (816). Table 2: Defence and immune-related annotated ESTs from turbot libraries ing the profiles of all genes grouped according to GO cat- egories (see Additional file 2). Though this approach could be mixing genes with opposite expression patterns, it could provide significant trends associated to specific functions for complementing the "individual-level gene" analysis. Bonferroni correction for multiple tests was con- sidered in this case, because of the higher statistical power achieved with this approach. As expected, defence/immune-related genes appeared overrepresented in the set of 72 genes (37.5%) with regard to the total number of annotated genes in our libraries (24.9%). Some genes apparently responded in a similar way to both pathogens, being down- (myosin, nephrosin and several peptidases) or up-regulated (actin and lys- ozyme) regarding control. However, most genes responded to only one pathogen. The different infection profile of the pathogens used in this study, a bacterium (A. salmonicida) and a parasite (P. dicentrarchi), is expected to stimulate a specific set of genes in the host. So, protein biosynthesis was up-regulated in response to Aeromonas salmonicida, as suggested the increased amount or ribos- ome-related and elongation factor proteins, and their cor- respondent GO terms (see Additional file 2). Also, a Page 7 of 12 (page number not for citation purposes) Page 7 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 Page 8 of 12 (page number not for citation purposes) Conclusion To our knowledge, this is the first report on a large tran- scriptional analysis in turbot providing new genomic resources in this important European aquaculture species. This study describes a collection of 9256 ESTs represent- ing 3482 unique sequences obtained from three direc- tionally cloned cDNA libraries from S. maximus, all of these being novel ESTs for this species. Therefore, this is a valuable EST collection, which increases genomic resources of turbot and enhances the genomic tools avail- able for non-model fish species. The transcript profile comparison among the three libraries allowed the identi- fication of putative genes generally or specifically related with infections in turbot. In addition, a high number of putative microsatellite and SNP EST-markers are now available for turbot map and highly useful for compara- tive mapping. These ESTs will be the basis for the develop- Tissue source and challenge g Two batches of 40 individuals (20–30 g each) obtained from a mixture of heterogeneous genetic families were collected at a specialized turbot fish farm. Fish from each batch were challenged intraperitoneally with A. salmonic- ida subsp. salmonicida and P. dicentrarchi, respectively [53,54]. The dose was adjusted to obtain around 50% sur- vival (LD50). The challenges were performed at the CETGA facilities in quarantine tanks. Fish were sacrificed prior to organ extraction using a lethal dose of MS222 anesthetic. In order to obtain mRNA representative of both innate and adaptive immune systems across the infection process, liver, spleen and head kidney tissues were collected from 5 sacrificed fish at five sampling points along the infective process for A. salmonicida (12 h, 1 day, 3 days, 7 days and 21 days post-inoculation) and at four sampling points for P. dicentrarchi (1 day, 3 days, 7 days and 15 days post-inoculation). Analogous batches of control fish were injected with saline serum and sampled at the same days of challenged fish. For each sample time, equal amounts of tissue from liver, kidney and spleen of each fish were pooled and immediately frozen in liquid nitrogen, constituting 15 pools for A. salmonicida (3 tis- sues × 5 sampling points), 12 for P. dicentrarchi (3 tissues × 4 sampling points) and the same pools for their respec- tive controls. Each sample of pooled tissues was ground to a fine powder in a mortar and pestled with liquid nitrogen and stored at -80°C until being used for RNA extraction. The use of pools of individuals at each sampling point was planned, both for identifying putative SNPs, as well as for averaging individual effects in future microarray analysis on turbot immune response. Real SNPs are those which passed quality filters 1 and 2 using the pipeline QualitySNP and true SNPs are the highest quality SNPs passing the three filters (see Methods). http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 Table 3: Summary statistics of SNP identification from turbot EST resources Real SNPs True SNPs andSNPs Total sequences analysed 12584 Number of contigs 257 255 Total SNPs detected 2197 1158 SNP frequency 1.39/100 bp 0.74/100 bp Total number of transitions 749 453 C/T 556 344 A/G 193 109 Total number of transversions 974 558 A/T 161 87 A/C 352 214 T/G 251 130 C/G 210 127 Total number of indels 366 125 Tri-allelic polymorphisms 99 21 Tetra-allelic polymorphisms 9 1 Real SNPs are those which passed quality filters 1 and 2 using the pipeline QualitySNP and true SNPs are the highest quality SNPs passing the three filters (see Methods). Table 3: Summary statistics of SNP identification from turbot EST resources Table 3: Summary statistics of SNP identification from turbot EST resources ment of a turbot microarray, focused on the characterization of the transcriptional response to patho- gen exposure. Markers-containing ESTs Up-regulation of pentraxin and sero- Because of their abundance within the genome, SNPs are the most common type of genetic markers [50] for study- ing complex genetic traits and genome evolution [51]. In turbot there have no been reports on SNP identification so far. The use of non-normalized libraries and a large number of individuals in library construction made possi- ble the identification of 2197 good quality SNPs. After the three filters used in the QualitySNP pipeline [52], we finally detected 2197 real and 1158 true SNPs (Table 3), representing a rate of 1.39 and 0.74 SNPs per 100 bp, respectively. Real and true SNPs included 749 and 453 transitions, 974 and 558 transversions and 366 and 125 indels, respectively. With this pipeline, only clusters with at least 4 EST sequences were selected to minimize the detection of SNPs caused by sequencing errors. As shown in Table 4 the majority of SNPs were detected in contigs involving a larger number of sequences, which provides an additional proof of our SNP quality. The identification of microsatellite and SNPs markers within turbot ESTs will contribute to extend the turbot genetic map [10] after linkage analysis in reference families. Since these markers are linked to genes, they will be useful as Type I markers for population genomics scanning in this species and for comparative mapping and fish evolutionary studies. Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 Authors' contributions This work represents a collaboration between different institutions (Xunta de Galicia -local Government- and Universities) and turbot industry (CETGA- Centro Tec- nológico Gallego de Acuicultura-). PM and CB identified the need for this investigation, designed and supervised the project. BGP and AV constructed the cDNA libraries. BGP, CF, and MVE performed all sequencing work, and revised and compiled this information for the subsequent bioinformatics analysis. Database for editing, clustering and functional annotation of genes was developed by JAA, MC, and AG in close connection with PM, BGP, CB, CF and AM. These last two authors performed all bioinfor- matics analysis using this database. BM and MLL, for Aeromonas salmonicida, and JL, for Philasterides dicentrarchi, supplied virulent strains and performed all challenges at CETGA facilities. Maintenance, sacrifice, organ and RNA extraction from fish was done by MV and SC. The paper was written by BGP and mostly revised by PM and JAA. All authors read the manuscript and gave their approval. A bioinformatic tool was developed in order to process all data. Basecalling from chromatogram traces was per- formed by using PHRED [55,56]. Vector, poly-A tails and low-quality regions were trimmed from EST sequences using a custom Perl script, a local BLAST search engine and the trimmest utility from the EMBOSS suite. High quality ESTs (at least 100 bp and PHRED score ≥ 20 after removal of vector sequence, adapter, and poly-A tail) of both normal and infected libraries were combined and assembled to form clusters using CAP3 http:// seq.cs.iastate.edu./[57] with the overlapping identity per- centage and minimum overlapping length parameters set to > 85.0% and 50 bp, respectively, in order to obtain highly reliable contig sequences. These contigs were man- ually revised to detect possible errors all along the bioin- formatics process. ESTs that did not form contigs (singletons) and the contigs resultant of assemblage of multiple sequences were referred to as unique sequences. Singletons and consensus sequences of each contig (unique sequences) were compared against public data- bases. Unique sequences were searched by both Blastn and Blastx, and the corresponding outputs subsequently parsed using BioPerl for fish-relevant hits and significant UniGene information. GO, KEGG and COG terms were extracted from AutoFACT output, which in turn feeds from Tblastx and RPS-blast output. All results were stored in a mySQL database to be consulted and searched using a custom-designed, friendly AJAX web interface. The e- value cut off was ≤ 1e-5. Authors' contributions All annotations thus obtained RNA isolation and cDNA library construction Glycerol stocks of overnight cul- tures were prepared in 96 well plates and stored at -80°C. Bioinformatic mining of microsatellites and SNPs The set of 3482 unique sequences was searched for micro- satellites using the program SPUTNIK http://espressosoft ware.com/pages/sputnik.jsp. The minimum repeat number used for this search was 8 for dinucleotide and 6 for tri-, tetra-and pentanucleotide microsatellites. Micros- atellite-containing ESTs were identified as candidates for marker development if they presented enough flanking sequences on either side of the repeats for primer design. Detection of single nucleotide polymorphisms (SNPs) was done using the pipeline QualitySNP with default set- tings [52]. This program uses three filters for the identifi- cation of reliable SNPs: filter 1 screens for all potential SNPs; filter 2 uses a haplotype-based strategy to detect reli- able SNPs; and filter 3 screens SNPs by calculating a con- fidence score based on sequence redundancy and quality. SNPs that pass filters 1 and 2 are called real SNPs, and those that pass all of the three filters are called true SNPs. Sequencing and bioinformatics Plasmid DNA was isolated from around 4000 clones from each library using the DirectPrep® 96 Miniprep kit (QIA- GEN) and the Plasmid Miniprep96 kit (MILLIPORE) with a robotic platform (BIOMEK 3000), and checked by elec- trophoresis in 1% agarose gels. One part of the purified DNA was sequenced following the ABI Prism BigDye™ Terminator v3.1 Cycle Sequencing Kit protocol on an ABI 3100 DNA sequencer (Applied Biosystems) and the other with the DTCS kit protocol on a CEQ2000 DNA sequencer (BECKMAN COULTER). All clones were sequenced from their 3' ends using a standard T7 primer to obtain the highest specific sequences of genes for oligo- microarray design. Those clones which suffered a system- atic drop on sequencing signal after poly-A tails were sequenced from 5' end. RNA isolation and cDNA library construction y Total RNA was extracted from pooled tissues of control and infected fish using TRIZOL Reagent (Life Technolo- gies) according to manufacturer's recommendations. RNA quality was assessed in a Bioanalyzer (Bonsai Technolo- gies). RNA was quantified using gel and NanoDrop® ND- 1000 spectrophotometer (NanoDrop® Technologies Inc) estimations. Poly-A mRNA was isolated using the Dyna- beads® mRNA Purification Kit (INVITROGEN). Three cDNA libraries (from A. salmonicida and P. dicentrarchi infected fish and control) were directionally constructed (5' EcoRI, 3' XhoI), with equal amounts of RNA from each tissue at each sampling time, using the ZAP-cDNA Library Construction Kit (STRATAGENE) following manufac- turer's instructions except for size fractioning. This was performed on cDNAs prior to ligation into vector and car- ried out with the SizeSep 400 Spun Columns (GE Table 4: Real and true quality SNP distribution in contigs with 4 or more ESTs Number of contigs Real SNPs True SNPs with 4 sequences 22 58 58 with 5–10 sequences 94 295 235 with 11–20 sequences 75 437 266 with 21–30 sequences 28 322 215 with 31–50 sequences 21 289 110 with > 50 sequences 17 796 274 Total 257 2197 1158 Real SNPs are those which passed quality filters 1 and 2 using the pipeline QualitySNP and true SNPs are the highest quality SNPs passing the three filters (see Methods). Table 4: Real and true quality SNP distribution in contigs with 4 or more ESTs Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 were complemented with AutoFACT output, whenever available. HEALTHCARE). To allow characterization of the inserts in a plasmid system the library was mass excised according to the manufacturer's recommendations, converting it from plaque forming units to a phagemid in E. coli SOLR strain. Manually isolated colonies were randomly picked and arrayed into 96-well microtiter plates containing liq- uid selective medium. Glycerol stocks of overnight cul- tures were prepared in 96 well plates and stored at -80°C. HEALTHCARE). To allow characterization of the inserts in a plasmid system the library was mass excised according to the manufacturer's recommendations, converting it from plaque forming units to a phagemid in E. coli SOLR strain. Manually isolated colonies were randomly picked and arrayed into 96-well microtiter plates containing liq- uid selective medium. Additional file 2 Comparison of ESTs grouped according to GO terms among the three turbot cDNA libraries. contigs of annotated genes are shown grouped according to GO term categories (Molecular function; Biological process; Cellular component) and split into the three turbot libraries from which sequences were obtained. The expression pattern of each gene at each library is evaluated as the amount of sequences found. This pattern is com- pared among libraries and examined for its deviation from an even distri- bution among libraries by a chi-square test. Click here for file 15. Hirono I, Aoki T: Expressed sequence tags of medaka (Oryzias latipes) liver mRNA. Mol Mar Biol Biotechnol 1997, 6:345-350. p ) 16. Lo J, Lee S, Xu M, Liu F, Ruan H, Eun A, He Y, Ma W, Wang W, Wen Z, Peng J: 15,000 Unique zebrafish EST clusters and their future use in microarray for profiling gene expression pat- terns during embryogenesis. Genome Res 2003, 13:455-466. 17. Clark MS, Edwards YJ, Peterson D, Clifton SW, Thompson AJ, Sasaki M, Suzuki Y, Kikuchi K, Watabe S, Kawakami K, Sugano S, Elgar G, Johnson SL: Fugu ESTs: new resources for transcription anal- ysis and genome annotation. Genome Res 2003, 13(12):2747-2753. [http://www.biomedcentral.com/content/supplementary/1746- 6148-4-37-S2.doc] 18. Rise ML, von Schalburg KR, Brown GD, Mawer MA, Devlin RH, Kuipers N, Busby M, Beetz-Sargent M, Alberto R, Gibbs AR, Hunt P, Shukin R, Zeznik JA, Nelson C, Jones SR, Smailus DE, Jones SJ, Schein JE, Marra MA, Butterfield YS, Stott JM, Ng SH, Davidson WS, Koop BF: Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybrid- ization characteristics. Genome Res 2004, 14:478-490. 18. Rise ML, von Schalburg KR, Brown GD, Mawer MA, Devlin RH, Kuipers N, Busby M, Beetz-Sargent M, Alberto R, Gibbs AR, Hunt P, Shukin R, Zeznik JA, Nelson C, Jones SR, Smailus DE, Jones SJ, Schein JE, Marra MA, Butterfield YS, Stott JM, Ng SH, Davidson WS, Koop BF: Development and application of a salmonid EST database BF: Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybrid- ization characteristics. Genome Res 2004, 14:478-490. Additional file 1 Comparison of gene expression profiles regulated in response to turbot pathogens. The total number and distribution among libraries (Aerom- onas, Philasterides, control) of sequences from contigs with 6 or more sequences is presented. The homology with public databases (e-value), the function and the probability of departure from the null hypothesis of even distribution of sequences among libraries is shown for each contig. Click here for file [http://www.biomedcentral.com/content/supplementary/1746- 6148-4-37-S1.doc] Page 10 of 12 (page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 14. Jørgensen SM, Afanasyev S, Krasnov A: Gene expression analyses in Atlantic salmon challenged with infectious salmon anemia virus reveal differences between individuals with early, inter- mediate and late mortality. BMC Genomics 2008, 9:179. Acknowledgements This study was supported by a Consellería de Pesca e Asuntos Marítimos and the Dirección Xeral de I+D – Xunta de Galicia project (2004/CP480). Authors wish to thank to Lucía Insua, Sonia Gómez, Ana Teijido and Susana Sánchez for technical assistance. 19. 19. Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, Bouneau L, Fischer C, Ozouf-Costaz C, Bernot A, Nicaud S, Jaffe D, Fisher S, Lutfalla G, Dossat C, Segurens B, Dasilva C, Salanoub M, Levy M, Boudet N, Castellano S, Anthouard V, Jubin C, Castelli V, Katinka M, Vacherie B, Biemont C, Skalli Z, Cattolico L, Poulain J, De Berard- inis V, Cruaud C, Duprat S, Brottier P, Coutanceau JP, Gouzy J, Parra G, Lardier G, Chapple C, McKernan KJ, McEwan P, Bosak S, Kellis M, Volff JN, Guigo R, Zody MC, Mesirov J, Lindblad-Toh K, Birren B, Nusbaum C, Kahn D, Robinson-Rechav M, Laudet V, Schachter V, Quetier F, Saurin W, Scarpelli C, Wincker P, Lander ES, Weissenbach J, Roest Crollius H: Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto- karyotype. Nature 2004, 431:946-957. References 1. 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Liu ZJ, Li P, Kocabas A, Ju Z, Karsi A, Cao D, Patterson A: Micros- atellite-containing genes from the channel catfish brain: evi- dence of trinucleotide repeat expansion in the coding region of nucleotide excision repair gene RAD23B. Biochem Biophys Res Commun 2001, 289:317-324. 24. Arma NR, Hirono I, Aoki T: Genes expressed in Japanese floun- der Paralichthys olivaceus spleen: analysis of genes involved in immune function. Fisheries Sci 2005, 71:1304-1323. 7. He C, Chen L, Simmons M, Li P, Kim S, Liu ZJ: Putative SNP dis- covery in interspecific hybrids of catfish by comparative EST analysis. Anim Genet 2003, 34:445-448. 25. References Aoki T, Hirono I: Immune relevant genes of Japanese flounder Paralichthys olivaceus. Comp Biochem Phys D 2006, 1:115-121. 26 C O A SC SC 25. Aoki T, Hirono I: Immune relevant genes of Japanese flounder, Paralichthys olivaceus. Comp Biochem Phys D 2006, 1:115-121. g J p Paralichthys olivaceus. Comp Biochem Phys D 2006, 1:115-121. y p y , 26. Park KC, Osborne JA, Tsoi SCM, Brown LL, Johnson SC: Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hip- poglossus) liver, kidney and spleen tissues following vaccina- tion against Vibrio anguillarun and Aeromonas salmonicida. Fish Shellfish Immunol 2005, 18:393-415. y 8. Serapion J, Kucuktas H, Feng J, Liu Z: Bioinformatic mining of type I microsatellites from expressed sequence tags of chan- nel catfish (Ictalurus punctatus). Mar Biotechnol 2004, 6:364-377. ( p ) 9. Murakawa K, Matsubara K, Fukushima A, Yoshii J, Okubo K: Chro- mosomal assignments of 3Vdirected partial cDNA sequences representing novel genes expressed in granulocy- toid cells. Genomics 1994, 23:379-389. 27. Galloway TF, Bardal T, Kvam SN, Dahle SW, Nesse G, Randol M, Kjorsvik E, Andersen O: Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglos- sus hippoglossus L.) embryos incubated at different tempera- tures: transient asymmetric expression of MyoD. J Exp Biol 2006, 209:2432-2441. 10. Bouza C, Hermida M, Pardo BG, Fernández C, Castro J, Fortes G, Sánchez L, Presa P, Pérez M, Sanjuán A, Comesaña S, Álvarez JA, Calaza M, Cal R, Piferrer F, Martínez P: A microsatellite genetic map of the turbot (Scophthalmus maximus). Genetics 2007, 177:2457-2467. 28. Douglas SE, Knickle LC, Kimball J, Reith ME: Comprehensive EST analysis of Atlantic halibut (Hippoglossus hippoglossus), a commercially relevant aquaculture species. BMC Genomics 2007, 8:144. 11. Moen T, Hayes B, Baranski M, Berg PR, Kjøglum S, Koop BF, Davidson WS, Omholt SW, Lien S: A linkage map of the Atlantic salmon (Salmo salar) based on EST-derived SNP markers. BMC Genomics 2008, 9:223. 29. Pardo BG, Fernández C, Hermida M, Vázquez A, Pérez M, Presa P, Calaza M, Alvarez-Dios JA, Comesaña AS, Raposo-Guillán J, Bouza C, Martínez P: Development and characterization of 248 novel microsatellite markers in turbot (Scophthalmus maximus). Genome 2007, 50:329-332. 12. Poompuang S, Hallerman EM: Toward detection of quantitative trait loci and marker-assisted selection in fish. Reviews Fish Sci 1997, 5:253-277. 13. Liu ZJ, Cordes JF: DNA marker technologies and their applica- tions in aquaculture genetics. Aquaculture 2004, 238:1-37. Page 11 of 12 (page number not for citation purposes) References Page 11 of 12 (page number not for citation purposes) Page 11 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 30. Toranzo AE, Barja JL: First report of furunculosis in turbot reared in floating cages in North-West of Spain. Bull Eur Ass Fish Pathol 1992, 12:147-149. morphisms and insertions/deletions in EST data from diploid and polyploid species. BMC Bioinformatics 2006, 7:438. morphisms and insertions/deletions in EST data from diploid and polyploid species. BMC Bioinformatics 2006, 7:438. 53. 53. Magariños B, Santos Y, Romalde JL, Barja JL, Toranzo AE: Patho- genic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida. J Gen Microbiol 1992, 138:2491-2498. 31. Björnsdóttir B, Gudmundsdóttir S, Bambir SH, Gudmundsdóttir BK: Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evalua- tion of cross protection induced by a furunculosis vaccine. J Fish Dis 2005, 28:181-188. 54. Paramá A, Iglesias R, Álvarez MF, Leiro J, Aja C, Sanmartín ML: Phi- lasterides dicentrarchi (Ciliophora, Scuticociliatida): experi- mental infection and possible routes of entry in farmed turbot (Scophthalmus maximus). Aquaculture 2003, 217:73-80. 32. Iglesias R, Paramá A, Alvarez MF, Leiro J, Fernandez J, Sanmartin ML: Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scoph- thalmus maximus in Galicia (NW Spain). 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 47. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M: Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 2006, 160:1-40. 48. Paramá A, Castro R, Arranz JA, Sanmartín ML, Lamas J, Leiro J: Scu- ticociliate cysteine proteinases modulate turbot leucocyte functions. Fish Shellfish Immunol 2007, 23:945-956. 49. Ishii KJ, Uematsu S, Akira S: 'Toll' gates for future immuno- therapy. Curr Pharm Des 2006, 12:4135-4142. 50. 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Acne in late adolescence and risk of prostate cancer
International journal of cancer
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cc-by
4,536
IJC International Journal of Cancer Acne in late adolescence and risk of prostate cancer Henrik Ugge Katja Fall2,5† 1 n1, Scott Montgomery2,3,4, Sabina Davidsson1† and , Ruzan Udumyan2, Jessica Carlsson1, Ove Andre € € Department of Urology, Faculty of Medicine and Health, Orebro University, Orebro, Sweden € € Clinical Epidemiology and Biostatistics, School of Medical Sciences, Orebro University, Orebro, Sweden 3 Clinical Epidemiology Unit, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden 4 Department of Epidemiology and Public Health, University College London, London, United Kingdom 5 Department of Medical Epidemiology, Karolinska Institutet, Stockholm, Sweden 1 Cancer Epidemiology 2 Accumulating evidence suggest that Propionibacterium acnes may play a role in prostate carcinogenesis, but data are so far limited and inconclusive. The aim of this population-based cohort study was therefore to test whether presence of acne vulgaris during late adolescence is associated with an increased risk of prostate cancer later in life. We identified a large cohort of young men born in Sweden between 1952 and 1956, who underwent mandatory assessment for military conscription around the age of 18 (n 5 243,187). Test information along with health data including medical diagnoses at time of conscription was available through the Swedish Military Conscription Register and the National Patient Register. The cohort was followed through linkages to the Swedish Cancer Register to identify the occurrence of prostate cancer until December 31, 2009. We used Cox regression to calculate adjusted hazard ratios (HR) and 95% confidence intervals (95% CI) for the association between acne in adolescence and prostate cancer risk. A total of 1,633 men were diagnosed with prostate cancer during a median follow-up of 36.7 years. A diagnosis of acne was associated with a statistically significant increased risk for prostate cancer (adjusted HR: 1.43 95%; CI: 1.06– 1.92), particularly for advanced stage disease (HR: 2.37 95%; CI 1.19–4.73). A diagnosis of acne classified as severe conferred a sixfold increased risk of prostate cancer (HR: 5.70 95% CI 1.42–22.85). Data from this large prospective population-based cohort add new evidence supporting a role of P. acnes infection in prostate cancer. While prostate cancer is the most common cancer among men in the western world, its etiology is still poorly understood. Chronic inflammation is thought to play an important role in the pathogenesis of different types of cancer,1 and accumulating evidence Key words: Propionibacterium acnes, prostate cancer, acne vulgaris, inflammation, acne vulgaris Abbreviations: BMI: body mass index; CI: confidence interval; ESR: erythrocyte sedimentation rate; HR: hazard ratio; P. acnes: Propionibacterium acnes; PIA: proliferative inflammatory atrophy; PIN: prostate intraepithelial neoplasia; PH: proportional hazards Additional Supporting Information may be found in the online version of this article. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. S.D. and K.F. contributed equally and share the last authorship Grant sponsor: UK Economic and Social Research Council (ESRC) as grants to the International Centre for Life Course Studies; Grant numbers: RES-596-28-0001, ES/JO19119/1 DOI: 10.1002/ijc.31192 History: Received 10 July 2017; Accepted 17 Nov 2017; Online 4 Dec 2017 Correspondence to: Henrik Ugge, MD, Department of Urology, € € Orebro University Hospital, 701 85 Orebro, Sweden, Email: henrik. ugge@regionorebrolan.se suggests a role of inflammation also in prostate cancer.2 Both acute and chronic inflammation is often observed in prostate tumor specimens and inflammatory cells are frequently found near areas of proliferative glandular prostate epithelium with the morphological appearance of atrophy.3 This type of atrophy (PIA) is thought of as a precursor lesion that may progress to prostate cancer directly or via prostate intraepithelial neoplasia (PIN).4 Propionibacterium acnes, most commonly associated with acne vulgaris, have been identified as the most prevalent microorganisms in prostate specimens,5–8 and have been associated with presence of inflammatory foci.9 We have recently observed that P. acnes are more common in prostatic tissue from prostate cancer patients than in samples obtained from men without the disease.7 Further, a longitudinal cohort study has demonstrated that men who had received antibiotics as treatment for severe acne were at increased risk of prostate cancer later in life.10 While a few other epidemiological studies support a link between acne and prostate cancer,11 the literature is inconsistent,12–14 possibly due to the use of study designs relying on selfreported exposure information or retrospective data collection. This is to our knowledge the first prospective large-scale population-based study using clinically diagnosed acne in order to test if the condition is associated with increased prostate cancer risk. Methods This study is based on a cohort of 284,198 men born between January 1, 1952 and December 31, 1956 that were C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Int. J. Cancer: 142, 1580–1585 (2018) V Union for International Cancer Control 1581 Ugge et al. What’s new? Some evidence has suggested that acne vulgaris may increase the risk of prostate cancer, but the data have been inconsistent. The results of this prospective cohort study support a link between acne in adolescence and prostate cancer later in life, potentially implicating the bacteria Propionibacterium acnes and resulting inflammation in prostate carcinogenesis. These results also suggest that the association may be stronger for the most severe type of acne and for advanced prostate cancer. Ethical approval The study was approved by the regional Ethical Review Board, Uppsala, Sweden (decision reference 2014/324). Exposure assessment At the time of conscription examination, participants were subject to a health questionnaire and an interview performed by a medical doctor, who recorded medical conditions according to the 8th edition of the International Classification of Diseases (ICD-8). Diagnoses were recorded on a practical clinical basis, with emphasis on health problems possibly affecting the participant’s performance during military service. Acne was identified using ICD-8 codes 706.00, 706.01, 706.10, 706.11, 706.12, and 706.20 from the Swedish Military Conscription Register (n 5 4,847 in the estimation sample) and using ICD-10 codes L70 from the Swedish National Outpatient register15 (added n 5 189 to the estimation sample). Severe acne was defined using ICD-8 codes 706.00, 706.01, 706.11, 706.20 and ICD-10 codes L700C, L70.0 C, L70.1, and L70.2 (see Online Appendix). Outcome assessment Prostate cancer cases were identified using ICD-7 code 177 from the Swedish National Cancer Register, with a completeness exceeding 95% for common cancer types.16 Patients with stages T3–4, N1 or M1 were classified as advanced prostate cancer and stages T1-T2 as localized prostate cancer. Prostate cancer diagnoses registered before December 31, 2009 (end of follow-up) were included. Other measures Swedish Military Conscription Register. The Swedish Mili- Figure 1. Inclusion and exclusion flow chart. tary Conscription Register17 records extensive and standardized physical and psychological examinations by physicians and psychologists, as described in detail previously.18 Measures included height (cm) and body mass index (BMI) (kg/ m2), a physical working capacity score (0–9), a cognitive function score (1–9), a stress resilience score (1–9) and a summary disease score (0–9). The physical working capacity score was based on a standardized ergometer test, where the participant was subject to a 5 min sub maximal test, followed by a 5 min maximal test with gradually increasing load. A semistructured interview with a psychologist, covering issues such as emotional stability and social maturity, was used as an assessment of the conscript’s potential ability to cope with stress during military service and summarized in a stress resilience score. Cognitive function was obtained from standardized tests covering logical, verbal, technical and spatial abilities. The summary disease score was used to quantify the presence and severity of disease relevant to the capacity to C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Int. J. Cancer: 142, 1580–1585 (2018) V Union for International Cancer Control Cancer Epidemiology included in the Swedish Military Conscription Register. These men underwent conscription examinations in the 1970s, except for 80 who were conscripted in 1969 and 1,163 in the 1980s. Examinations were performed at ages 18 and 19 years, with a small number at later ages. At the time, assessment for military service was compulsory in Sweden. Fewer than 4% of the male population were excluded from the enlistment examination due to chronic illness or disability. In the cohort, 2,564 were excluded due to data inconsistencies such as errors in the personal identification number, female sex or uncertain vital status. Further, 182 men were excluded due to improbable measures at the conscription assessment. We also excluded individuals with ill-defined summary disease score (a standardized score based on presence and severity of health problems, n 5 35), neoplasms at the time of conscription examination (n 5 599), and those who had a malignant cancer diagnosis preceding prostate cancer diagnosis (n 5 47). Next, we excluded men with missing data for covariates used in the analysis (n 5 37,435), leaving a sample of 243,187 available for complete case analysis (Figure 1). 1582 Acne in late adolescence and risk of prostate cancer Table 1. Characteristics of the exposed and unexposed to acne among Swedish men born 1952–1956 (n 5 243,187) No acne n 5 238,151 Characteristics (%) n Acne n 5 5,036 (%) N Head of household’s occupation <0.001 Manual worker 98,525 (41.4) 2,130 (42.3) Agricultural worker 9,242 (3.9) 165 (3.3) Farm owner/manager 23,696 (9.9) 429 (8.5) Office worker 66,320 (27.8) 1,497 (29.7) Business owner/managers 25,734 (10.8) 541 (10.7) Other/unknown 14,634 (6.1) 274 (5.4) 52016 (21.8) 1,076 (21.4) Underweight (<18.5) 27,680 (11.6) 562 (11.2) Normal weight (18.5–25.0) 192,519 (80.8) 4,138 (82.2) Overweight and obese (>25.0) 17,952 (7.5) 336 (6.7) Household crowding (persons/room) 1960 census >2 people/room 0.419 Cancer Epidemiology BMI categories (kg/m2) 0.032 Height (cm) Mean (SD) <0.001 178.68 (6.43) 179.57 (6.50) 5.20 (1.97) 5.38 (1.98) 6.31 (1.81) 6.23 (1.82) 2.00 (1.00–89.00) 3.00 (1.00–48.00) Cognitive function score Mean (SD) <0.001 Physical working capacity score Mean (SD) 0.002 Erythrocyte sedimentation rate (mm/h) Median (min–max) 1 p1 <0.001 p values from v2 test (categorical), t test (continuous) or median test (ESR). undertake military duty. Erythrocyte sedimentation rate (ESR) was used as a marker of systemic inflammation, and was in the analyses adjusted for erythrocyte volume fraction (EVF). Socioeconomic and demographic characteristics. Demographic data, including information on vital status and emigration, were provided by the government organization Statistics Sweden, using the Total Population Register.19 Markers of material and social circumstances in childhood were taken from the 1960 census. Household crowding during childhood was calculated (people/habitable room) and dichotomized. Occupation of the head of household in 1960 was classified as manual, agricultural, farm owners/managers, office workers, business owners/managers and others. Region of residence was taken from the 1970 census. Statistical analysis Descriptive statistics included frequencies, proportions, means and standard deviations. We used time-dependent Coxregression to examine unadjusted and adjusted associations between acne and prostate cancer onset later in life. The functional form of the measures was explored using multivariable fractional polynomial modeling20 which indicated a linear relationship with the log hazard of the outcome for height, stress resilience score, cognitive function score, physical working capacity score and erythrocyte volume fraction. The adjusted model included the following measures: birth year, household crowding, head of household’s occupation, region of residence, BMI, summary disease score, erythrocyte sedimentation rate (modelled as categorical variables), height, stress resilience score, physical working capacity score, cognitive function score and erythrocyte volume fraction (modeled as linear measures). In sensitivity analysis, we used Coxregression to examine the associations with acne identified from conscription records only. The assumption of proportional hazards (PH) for Cox regression was evaluated by using a Grambsch–Therneau test.21 The statistical software used was Stata version 14/SE for Windows (StataCorp, College Station, Texas). Tests were two-sided and statistical significance was defined as p < 0.05 and 95% confidence intervals that do not include 1.00. Results During follow-up, 1,633 men were diagnosed with prostate cancer of which 1,317 had localized disease, 203 advanced prostate cancer, and 113 unknown stage. Median follow-up time was 36.7 years, median age at study-exit was 55 years C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Int. J. Cancer: 142, 1580–1585 (2018) V Union for International Cancer Control 1583 Ugge et al. Table 2. Hazard ratio (HR) with 95% confidence interval (CI) for the association between acne, and characteristics in late adolescence and prostate cancer among Swedish men assessed for military conscription and born 1952–1956 (n 5 243,187) N events Unadjusted HR [95% CI] Adjusted HR [95% CI]1 No acne 1,586 1.00 1.00 Acne Characteristic Acne in and after adolescence 47 1.41 [1.06,1.89] 1.43 [1.06,1.92] Nonsevere 45 1.37 [1.02,1.84] 1.38 [1.02,1.87] Severe 2 5.88 [1.47,23.55] 5.70 [1.42,22.85] 1 439 1.00 [1,00, 1.00] 1.00 [1,00, 1.00] 2–6 1,044 0.90 [0.81, 1.01] 0.93 [0.82, 1.05] 7–10 92 0.79 [0.63, 0.99] 0.83 [0.66, 1.05] 11–14 35 0.95 [0.67, 1.33] 1.02 [0.71, 1.45] 15–89 23 0.73 [0.48, 1.11] 0.80 [0.52, 1.22] - 1.04 [1.01, 1.07] 1.03 [1.00, 1.06] Underweight (<18.5) 178 0.87 [0.74, 1.02] 0.96 [0.81, 1.13] Normal weight (18.5–25.0) 1,355 1.00 [1.00, 1.00] 1.00 [1.00, 1.00] Overweight and obese (>25.0) 100 0.82 [0.67, 1.01] 0.85 [0.69, 1.04] <2 1,349 1.00 [1.00, 1.00] 1.00 [1.00, 1.00] 2 284 0.77 [0.67, 0.87] 0.86 [0.75, 0.98] Physical working capacity score (per one unit increase) 2 BMI (kg/m ) Household crowding (persons per room) 1 Adjusting for birth year, head of household’s occupation in 1960, household crowding in 1960, height, BMI, physical capacity score, summary disease score, summary cognitive score, stress resilience score, erythrocyte sedimentation rate, erythrocyte volume fraction and region of residence in 1970. The models assume proportional hazards and use 243,187 observations. and median age at prostate cancer diagnosis was 53 years. Of the 243,187 men eligible for the study, 4,847 (2%) were registered with a diagnosis of acne at time of conscription assessment, likely representing more severe cases. Another 189 men were later diagnosed with acne in the National Patient Register. A slightly higher proportion of men with acne were sons of office workers and a somewhat lower proportion to agricultural workers or farm owners. Men with acne tended to have higher cognitive function and ESR, but lower physical working capacity compared with men without acne. Men with acne further tended to be taller (Table 1). There was no difference in the prevalence of significant health problems or stress resilience in men with and without acne or in stress resilience score (data not shown). We found that men with acne had a statistically significant increased risk of prostate cancer later in life compared with men without acne (HR 5 1.43; 95% CI: 1.06–1.92) (Table 2). We further noted a higher magnitude association for men diagnosed with severe acne (adjusted HR: 5.70; 95% CI: 1.42–22.85). We found a modest but statistically significant trend of higher risk with increasing levels of physical capacity. There was no clear association of ESR levels or BMI with prostate cancer risk. When prostate cancer cases were categorized according to disease stage, we found a statistically significant association between acne and advanced prostate Table 3. Hazard ratio (HR) with 95% confidence interval (CI) for the association between acne and prostate cancer among Swedish men assessed for military conscription and born 1952–1956 (n 5 243187), by tumor stage Exposure N events Unadjusted HR [95% CI] Adjusted HR [95% CI]1 Advanced prostate cancer Acne No 194 1.00 1.00 Yes 9 2.21 [1.13,4.32] 2.37 [1.19,4.73] Localized prostate cancer Acne No 1,280 1.00 1.00 Yes 37 1.38 [0.99,1.91] 1.39 [1.00,1.94] 1 Adjusting for birth year, head of household’s occupation in 1960, household crowding in 1960, height, BMI, physical capacity score, summary disease score, summary cognitive score, stress resilience score, erythrocyte sedimentation rate, erythrocyte volume fraction and region of residence in 1970. cancer (adjusted HR: 2.37; 95% CI: 1.19–4.73). The association with localized disease was of lower magnitude and borderline statistically significant (adjusted HR: 1.39; 95% CI: 1.00–1.94) (Table 3). In a sensitivity analysis, where we C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Int. J. Cancer: 142, 1580–1585 (2018) V Union for International Cancer Control Cancer Epidemiology Erythrocyte sedimentation rate (mm/h) 1584 excluded men who were diagnosed after conscription (n 5 189), the overall association was unchanged (adjusted HR: 1.40; 95% CI: 1.06–1.92). Cancer Epidemiology Discussion In this population-based study using prospectively recorded data, we observed an association between acne in late adolescence and development of prostate cancer later in life. The data suggest that the association may be stronger for the most severe type of acne and for advanced prostate cancer. The literature is currently limited and somewhat conflicting regarding the association between acne and prostate cancer. The results from a prospective cohort study suggest that acne during young adulthood is associated with an increased risk of prostate cancer-specific death.11 Another cohort study using prospectively collected data shows an increased prostate cancer risk for men who reported treatment with tetracycline for more than four years as a marker of severe acne.10 A few case-control studies, where subjects have been asked regarding their history of acne, have on the other hand not shown any associations between acne and prostate cancer,12–14 and one case–control study reported an inverse association between acne-related facial scarring and later prostate cancer.22 In the same cohort, high serum titers of antibodies directed against P. acnes were observed to be inversely associated with prostate cancer risk.23 Studies using retrospectively collected and selfreported data are sensitive to exposure misclassification, which may explain the reported null findings.12–14,22 Reliance on selfreported information on acne has also been shown to be a less reliable measure of the disease.24 The prevalence of acne in this cohort (2%) may seem low compared with other studies, and compared with the overall incidence of acne among adolescent men. Acne vulgaris is considered to affect a majority of, if not all, adolescents to some extent, and some 20% are said to be affected by moderate to severe lesions.25,26 The latter figure corresponds roughly to the number counted as exposed in other studies examining the relationship between self-reported history of acne and prostate cancer.12–14 It is however likely that men registered with acne by a physician in the setting of conscription examination constitute a group with more severe acne. The prevalence of severe acne based on physical examinations in similar settings has earlier been estimated to 2.9–6.9 percent.27,28 While the present study may underestimate the number of exposed, it should accurately have identified clinically significant cases of acne. The other study reporting a positive association between acne and prostate cancer10 could be argued to have used a similarily strict definition of exposure since prescription of tetracycline would have required physical examination. Identification of more severe cases may thus help explain discrepancies with results of studies based on self-reported, less severe acne. Indeed, although based on very small numbers, our results suggest a stronger link with what specifically has been classified as Acne in late adolescence and risk of prostate cancer more severe forms of acne. The focus of the study is further on acne later during adolescence (age 18–19 years), and the results may not be generalizable to persons with acne earlier in life. P. acnes can induce inflammatory reactions, through complement activation and induction of proinflammatory cytokines.29,30 Induction of cytokines such as IL-6 and IL-8, partly through activation of toll-like receptors (TLRs), is thought to be involved in the pathogenesis of acne,31 and has been demonstrated to occur in both skin and prostate cells infected by P. acne.7,32,33 Although the exact mechanism of P. acnes-associated pathogenesis is not completely understood in either skin or prostate, there is evidence pointing to the importance of the host inflammatory response.32,34 P. acnes is ubiquitous on the skin, but only a smaller proportion of the population develop severe acne lesions as a consequence of colonization, which further suggests that individual variation in immune response may influence the outcome of infection.35 Immunologic phenotypes predisposing an intense dermatologic reaction could also be prone to a similar inflammatory response in the prostate. We found that the mean erythrocyte sedimentation rate (ESR), a marker of systemic inflammation, was higher among men with than without acne, but not an independent determinant of prostate cancer risk. An alternative explanation of our result may involve androgens. Hormonal activity plays a role in the pathology of acne36,37 and the central role of androgen signalling in prostate cancer and prostate cancer treatment has been known for a long time.38 The role of androgen signalling in prostate carcinogenesis is however not as clearly established.39 We were unfortunately not able to address this potentially important confounder in this study, which is a limitation. The strengths of this study include its large study population, prospective design and long, essentially complete, follow-up. Another advantage is that the exposure, the diagnosis of acne, was made as part of the physical examination during the assessment for conscription and collected independently of a later diagnosis of prostate cancer. A weakness is that the number of exposed was low and the power for detailed analyses limited. The study population is furthermore relatively young which results in a low number of prostate cancer cases, possibility leading to an underestimation of the true association. Specifically, our study could be said to have examined the association between acne in late adolescence and early prostate cancer diagnosis. Whether the association remains for prostate cancer at later ages is unknown. In conclusion, the results of this prospective cohort-study indirectly support the hypothesis that P. acnes may play a role in prostate cancer. Whether the association is explained by P. acnes, host factors associated with specific immune responses, hormonal or other factors remains to be investigated further. C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Int. J. Cancer: 142, 1580–1585 (2018) V Union for International Cancer Control 1585 Ugge et al. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7. Sfanos KS, De Marzo AM. Prostate cancer and inflammation: the evidence. Histopathology 2012; 60:199–215. De Marzo AM, Nakai Y, Nelson WG. Inflammation, atrophy, and prostate carcinogenesis. Urol Oncol 2007;25:398–400. Davidsson S, Fiorentino M, Andren O, et al. Inflammation, focal atrophic lesions, and prostatic intraepithelial neoplasia with respect to risk of lethal prostate cancer. Cancer Epidemiol Biomarkers Prevent 2011;20:2280–7. Alexeyev O, Bergh J, Marklund I, et al. Association between the presence of bacterial 16S RNA in prostate specimens taken during transurethral resection of prostate and subsequent risk of prostate cancer (Sweden). Cancer Causes Control 2006;17:1127–33. Cavarretta I, Ferrarese R, Cazzaniga W, et al. The microbiome of the prostate tumor microenvironment. Eur Urol 2017. Davidsson S, Molling P, Rider JR, et al. Frequency and typing of Propionibacterium acnes in prostate tissue obtained from men with and without prostate cancer. Infect Agents Cancer 2016;11:26 Shannon BA, Garrett KL, Cohen RJ. Links between Propionibacterium acnes and prostate cancer. Future Oncol (London, England) 2006;2: 225–32. Cohen RJ, Shannon BA, McNeal JE, et al. Propionibacterium acnes associated with inflammation in radical prostatectomy specimens: a possible link to cancer evolution? J Urol 2005;173:1969–74. Sutcliffe S, Giovannucci E, Isaacs WB, et al. Acne and risk of prostate cancer. Int J Cancer 2007; 121:2688–92. Galobardes B, Davey Smith G, Jeffreys M, et al. Acne in adolescence and cause-specific mortality: lower coronary heart disease but higher prostate cancer mortality: the Glasgow Alumni Cohort Study. Am J Epidemiol 2005;161:1094–101. Cremers RG, Aben KK, Vermeulen SH, et al. Self-reported acne is not associated with prostate cancer. Urol Oncol 2014;32:941–5. Lightfoot N, Conlon M, Kreiger N, et al. Medical history, sexual, and maturational factors and 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. prostate cancer risk. Ann Epidemiol 2004;14:655– 62. Nair-Shalliker V, Yap S, Nunez C, et al. Adult body size, sexual history and adolescent sexual development, may predict risk of developing prostate cancer: results from the New South Wales Lifestyle and Evaluation of Risk Study (CLEAR). Int J Cancer 2017;140:565–74. Socialstyrelsen. The National Patient Register. http://www.socialstyrelsen.se/register/ halsodataregister/patientregistret/inenglish Barlow L, Westergren K, Holmberg L, et al. The completeness of the Swedish Cancer Register: a sample survey for year 1998. Acta Oncol (Stockholm, Sweden) 2009;48:27–33. Whitley E, Batty GD, Gale CR, et al. Intelligence in early adulthood and subsequent risk of unintentional injury over two decades: cohort study of 1 109 475 Swedish men. J Epidemiol Community Health 2010;64:419–25. Bergh C, Udumyan R, Fall K, et al. Stress resilience in male adolescents and subsequent stroke risk: cohort study. J Neurol Neurosurg Psychiatry 2014;85:1331–6. Statistics Sweden: Statistics Sweden. https://www. scb.se/sv_/Vara-tjanster/Bestalla-mikrodata/ Royston P, Sauerbrei W. Building multivariable regression models with continuous covariates in clinical epidemiology–with an emphasis on fractional polynomials. Methods Inform Med 2005;44: 561–71. Grambsch PM, Therneau TM. Proportional hazards tests and diagnostics based on weighted residuals. Biometrika 1994;81:515–26. Giles GG, Severi G, English DR, et al. Early growth, adult body size and prostate cancer risk. Int J Cancer 2003;103:241–5. Severi G, Shannon BA, Hoang HN, et al. Plasma concentration of Propionibacterium acnes antibodies and prostate cancer risk: results from an Australian population-based case-control study. Br J Cancer 2010;103:411–5. Menon C, Gipson K, Bowe WP, et al. Validity of subject self-report for acne. Dermatology (Basel) 2008;217:164–8. Lynn DD, Umari T, Dunnick CA, et al. The epidemiology of acne vulgaris in late 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. adolescence. Adolescent Health Med Therap 2016;7:13–25. Williams HC, Dellavalle RP, Garner S. Acne vulgaris. Lancet 2012;379:361–72. Lello J, Pearl A, Arroll B, et al. Prevalence of acne vulgaris in Auckland senior high school students. N Z Med J 1995;108:287–9. Schafer T, Nienhaus A, Vieluf D, et al. Epidemiology of acne in the general population: the risk of smoking. Br J Dermatol 2001;145:100–4. Perry AL, Lambert PA. Propionibacterium acnes. Lett Appl Microbiol 2006;42:185–8. Burkhart CG, Burkhart CN, Lehmann PF. Acne: a review of immunologic and microbiologic factors. Postgrad Med J 1999;75:328–31. Das S, Reynolds RV. Recent advances in acne pathogenesis: implications for therapy. Am J Clin Dermatol 2014;15:479–88. Mak TN, Fischer N, Laube B, et al. Propionibacterium acnes host cell tropism contributes to vimentin-mediated invasion and induction of inflammation. Cell Microbiol 2012;14:1720–33. Drott JB, Alexeyev O, Bergstrom P, et al. Propionibacterium acnes infection induces upregulation of inflammatory genes and cytokine secretion in prostate epithelial cells. BMC Microbiol 2010;10: 126 Szabo K, Kemeny L. Studying the genetic predisposing factors in the pathogenesis of acne vulgaris. Hum Immunol 2011;72:766–73. Webster GF. Inflammation in acne vulgaris. J Am Acad Dermatol 1995;33:247–53. Lolis MS, Bowe WP, Shalita AR. Acne and systemic disease. Med Clin North Am 2009;93:1161–81. Thiboutot D, Harris G, Iles V, et al. Activity of the type 1 5 alpha-reductase exhibits regional differences in isolated sebaceous glands and whole skin. J Invest Dermatol 1995;105:209–14. Huggins C, Hodges CV. Studies on prostatic cancer. I. The effect of castration, of estrogen and androgen injection on serum phosphatases in metastatic carcinoma of the prostate. CA Cancer J Clin 1972;22:232–40. Klap J, Schmid M, Loughlin KR. The relationship between total testosterone levels and prostate cancer: a review of the continuing controversy. J Urol 2015;193:403–13. C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Int. J. Cancer: 142, 1580–1585 (2018) V Union for International Cancer Control Cancer Epidemiology References
https://openalex.org/W4311220768
https://jurnal.uns.ac.id/jmme/article/download/64421/38258
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Analysis of Distance Learning Activities to Teach Collaboration Ability of Undergraduate Students of Mathematics Education Study Program
Journal of Mathematics and Mathematics Education
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ERROR: type should be string, got "https://jurnal.uns.ac.id/jmme https://jurnal.uns.ac.id/jmme https://jurnal.uns.ac.id/jmme p-ISSN 2089-8878; e-ISSN 2715-8276 \nVol. 12, No. 02, December 2022 * Correspondence purposes, email: dewiisabella5@gmail.com * Correspondence purposes, email: dewiisabella5@gmail.com Abstrak: Pembelajaran jarak jauh adalah pembelajaran yang dilakukan secara terpisah antara \npeserta didik dengan pendidik, pembelajaran yang dilakukan didesain menggunakan alat \nkomunikasi atau media manajemen belajar untuk mendukung pembelajaran. Pada abad ke – 21 \nini terdapat kemampuan-kemampuan yang harus dikuasai oleh seseorang yang disebut \nKompetensi Abad 21 yang salah satunya adalah kemampuan kolaborasi. Tujuan dari penelitian \nini adalah mendeskripsikan aktivitas pembelajaran jarak jauh untuk membelajarkan kemampuan \nkolaborasi mahasiswa dan mendeskripsikan pengaruh aktivitas pembelajaran pada pembelajaran \njarak jauh dalam mengembangkan kemampuan kolaborasi. Jenis penelitian ini adalah deskriptif \nkualitatif dengan metode kajian literatur yang dilengkapi data lapangan. Hasil penelitian \nmenunjukkan ada beberapa hal yang perlu diperhatikan dalamn merancang aktivitas pembelajaran \njarak jauh untuk membelajarkan kemampuan kolaborasi mahasiswa, yaitu 1) membagi \nmahasiswa menjadi beberapa kelompok, 2) digunakan media yang mendukung mahasiswa untuk \nmemberikan tanggapan, gagasan, komentar dan sebagainya baik berupa forum video conference \nmaupun LMS, 3) instruksi tentang aktivitas umpan balik, dan 4) budaya yang dibangun dalam \naktivitas pembelajaran. Diperoleh juga indikator kemampuan kolaborasi yaitu 1) kerjasama dan \nsaling ketergantungan untuk mencapai tujuan bersama seperti kerjasama dalam kelompok, \nberdiskusi, berkontribusi aktif, dan fleksibel, 2) bertanggung jawab atas dasar belajar dan perilaku \nmasing-masing seperti bertanggung jawab dalam pekerjaan dan bekerja secara produktif, dan 3) \nterjadinya pelaksanaan suatu aktivitas kerja yang kohesif seperti dapat beradaptasi, \nberkomunikasi dengan baik, dan menghargai sesama. Selain itu, terdapat kelebihan dan \nkekurangan dari rancangan aktivitas pembelajaran jarak jauh ini. Accepted: July 01, 202x \nApproved: July 24, 2022 \nPublished: Dece\nDOI: 10.20961/jmme.v12i2.64421 \n(*)This study was presented at ICMME 2022 on 24-26 July 2022 \n[ 61 ] ANALYSIS OF DISTANCE LEARNING ACTIVITIES TO \nTEACH COLLABORATION ABILITY OF \nUNDERGRADUATE STUDENTS OF MATHEMATICS \nEDUCATION STUDY PROGRAM(*) Dewi Isabella Palma1*, Elina Nadiasari1 \n1Sanata Dharma University, Yogjakarta, Indonesia Kata kunci : Pembelajaran jarak jauh, Kompetensi abad 21, Kolaborasi Abstract: Distance learning is learning that is carried out separately between students and \neducators, the learning carried out is designed using communication tools or learning management \nmedia to support learning. In the 21st century, there are abilities that must be mastered by someone \ncalled 21st Century Competencies, one of which is the ability to collaborate. The purpose of this \nstudy is to describe distance learning activities to teach student collaboration skills and describe \nthe influence of learning activities on distance learning in developing collaboration skills. This \ntype of research is descriptive qualitative with a literature review method equipped with field data. The results showed that there are several things that need to be considered in designing distance \nlearning activities to teach students' collaboration skills, namely 1) dividing students into groups, Published: December 01, 2022 Accepted: July 01, 202x \nApproved: July 24, 2022 \nDOI: 10.20961/jmme.v12i2.64421 \n(*)This study was presented at ICMME 2022 on 24-26 July 2022 \n[ 61 ] 62 62 p-ISSN 2089-8878; e-ISSN 2715-8276 p-ISSN 2089-8878; e-ISSN 2715-8276 62 2) using media that supports students to provide responses, ideas, comments and so on in the form \nof video conference forums and LMS, 3) instructions on feedback activities, and 4) the culture \nbuilt in learning activities. Indicators of collaboration ability are also obtained, namely 1) \ncooperation and interdependence to achieve common goals such as cooperation in groups, \ndiscussion, active contribution, and flexibility, 2) being responsible for the basis of learning and \nbehavior of each such as being responsible for work and working productively, and 3) the \nimplementation of a cohesive work activity such as being able to adapt, communicate well, and \nrespect for others. In addition, there are advantages and disadvantages of this design of distance \nlearning activities. Keywords: Distance Learning, 21st Century Competencies, Collaboration INTRODUCTION Distance learning is an institution-based formal education where study groups are separated and \ninteractive telecommunications systems are used to connect learners, resources and instructors \n(Simonson et al., 2019). According to Abidin (Abidin et al., 2020), distance learning is a learner who is \nplanned in another place or outside his place of teaching and when the learning process does not occur \nface-to-face directly between the teacher and learner, so that learning emphasizes independent learning \nand uses special techniques in designing materials and special methodologies for communication \nthrough media such as computers, telephones, television and so on. Then, according to Law No. 20 of \n2003 concerning the National Education System Article 1 (Yuangga & Sunarsi, 2020) states that distance \neducation is an education whose students are separated from educators and learning is carried out using \nvarious learning resources through telecommunications, information and other media. Based on some of \nthe opinions above, it can be concluded that distance learning is learning that is carried out separately \nbetween students and educators (not face-to-face in person), where learning is carried out independently \nby students and the learning carried out is designed using communication tools or learning management \nmedia such as Whatsapp, Zoom, Moodle, and so on to support learning both synchronously and \nasynchronously. Based on this, the concept of distance learning has begun to be developed before the \nCOVID-19 pandemic which began at the tertiary level. This is stated in Law No. 12 of 2012 with the \naim of providing Higher Education services to groups of people who cannot attend regular face-to-face \neducation and expanding access and facilitating Higher Education and learning services. On the other hand, along with the times, in the 21st century there are competencies that must be \nmastered by students from an early age. P21 (Partnership for 21st Century Learning) develops a learning \nframework in the 21st century that requires students to have skills, knowledge and abilities in the fields \nof technology, media and information, learning skills and innovation as well as life and career skills \n(Wijaya et al., 2016). Within the framework, there are four abilities commonly referred to as 21st century Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… \n \n63 63 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 63 competencies, which students must master in learning, namely critical thinking, communication, \ncollaboration, and creativity. INTRODUCTION Triling and Fadel (Wijaya et al., 2016)describe that critical thinking and \novercoming problems are students able to use reason for various situations, use system thinking methods, \nmake decisions and overcome problems; communication and collaboration, namely students are able to \ncommunicate clearly and collaborate with other group members; creativity and innovation, namely \nstudents are able to think creatively, work creatively and create new innovations. Even though distance \nlearning is growing and exists at every level of education, educators still have to make sure students \nmaster these four abilities. The importance of mastering and developing these four abilities, so that \nstudents can become superior and qualified human beings. One of the important 21st century competencies is collaboration. P21 (Roekel, 2010) defines \ncollaboration as the ability to work effectively and respectfully with diverse teams, exercise flexibility \nand willingness to assist in making the necessary compromises to achieve common goals, and \nresponsibility for joint work and respect for the individual contributions made by each member of the \ngroup. Roekel (2010) explains that collaboration is important because a large group of diverse \nindividuals will produce better estimates and collaboration can create knowledge for many people. Based \non this, learning that supports collaboration will have a good impact on student learning, because through \ncollaboration, students will get a variety of knowledge from the results of collaboration. There are several \nstudies that examine the application of collaborative learning models to distance learning. In a study \nconducted by Farah (Fauziah, 2021) found that collaborative learning carried out in the experimental \ngroup was in the category of quite effective, namely 63.7%. Then research conducted by Thompson and \nKu (Thompson & Ku, 2006), found that there was ineffective communication, conflicts between group \nmembers, and negative attitudes that posed challenges in collaborating online, but more collaborative \ngroups produced better quality projects and had a more positive attitude towards online learning. Based \non this, it can be seen that collaboration is a challenge in distance learning. Based on the importance of the current ability of collaboration and development of distance \nlearning, this study will discuss distance learning activities to teach student collaboration skills and \ndescribe the influence of learning activities on distance learning in developing collaboration skills. JMME Halaman 61-71 RESEARCH METHOD This research is a type of qualitative descriptive research. Qualitative descriptive is a type of \nresearch that gives an idea of an object that is observed on the basis of qualitatively visible facts as they \nare. The main source of data in the study is words. In addition, the source of this research data is the \nresult of a literature review supported by interviews with lecturers and surveys with students of the JMME Halaman 61-71 64 \n \n \nMathematics Education Study Program, one of the private universities in Yogyakarta who are the \nsubjects of this study. p-ISSN 2089-8878; e-ISSN 2715-8276 64 p-ISSN 2089-8878; e-ISSN 2715-8276 Mathematics Education Study Program, one of the private universities in Yogyakarta who are the \nsubjects of this study. Mathematics Education Study Program, one of the private universities in Yogyakarta who are the \nsubjects of this study. The data collection methods in this study are literature review, direct interview methods and \nsurvey methods. Interviews were conducted to explore information related to distance learning activities \nto improve student collaboration skills. Then a survey was conducted to collect information from \nstudents regarding the response and effects of distance learning activities to improve student \ncollaboration skills. The instruments used in this study are interview guidelines and questionnaires where \nthe instruments have been declared valid by validators. RESULTS AND DISCUSSION According to (Simonson et al., 2015) there are four characteristics of distance learning. First, \ndistance learning is carried out by accredited institutions equivalent to institutions that offer traditional \nface-to-face learning and not independent learning or learning in a nonacademic learning environment. Secondly, learners and teachers are separated geographically and perhaps even time so accessibility and \nconvenience are essential in distance learning. Third, interactive telecommunications connects students \nwith each other and also connects students with teachers. Whatever the medium, interaction is also very \nimportant in distance learning as learning in general, the relationship between students and teachers no \nlonger depends on the physical but on the sophistication of communication technology. Fourth, like other \neducation, distance learning also creates a learning community consisting of students, teachers, and \nlearning resources such as books, modules, videos, and others. Garrison and Shale (Simonson et al., \n2015) stated the criteria in determining the characteristics of the distance learning process. First, distance \nlearning implicitly suggests that the educational communication created between teachers and students \ndoes not occur in physical proximity. Second, distance learning should involve two-way communication \nbetween teachers and students in order to support and facilitate the learning process. Third, distance \nlearning uses media to mediate the need for two-way communication. In teaching collaboration skills, it is necessary to design the right activities. according to \n(Nurhayati et al., 2019) learning is carried out in groups with the aim of training cooperation between \nstudents in solving a problem. In addition, collaboration skills according to (Zubaidah, 2018)include \ngiving and receiving feedback from colleagues or fellow group members to perform the same task, so in \ndesigning learning activities it is necessary to facilitate students to provide feedback to fellow friends, \nespecially group members. Media is also needed in teaching collaboration skills in distance learning, \nthis is in accordance with the characteristics of Distance Learning according to (Simonson et al., 2015) \nabove. Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… \n \n65 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 65 Indicators of collaboration ability according to Suryani (Sunardi et al., 2017) are 1) each member \nworks together to achieve common goals and interdependence, 2) individuals are responsible for each \nother's learning and behavior, 3) Classes or groups are encouraged towards the implementation of \ncohesive group work activities. RESULTS AND DISCUSSION In addition, Trilling and Fadel (Mariamah et al., n.d.), there are five \nindicators of collaboration ability, namely cooperation in groups, adjusting to group friends, being \nresponsible for group work, discussing in making decisions, and communicating well in groups. . Then \nGreenstein (Hidayat et al., 2021) also revealed that the indicators that indicate the ability of collaboration \nare to actively contribute, work productively, show flexibility and compromise, show responsibility and \nshow respect. Based on these three opinions, it can be concluded that the indicators of collaboration \nability are 1) Cooperation and interdependence to achieve common goals such as cooperation in groups, \ndiscussion, active contribution, and flexibility, 2) being responsible for each other's learning and \nbehaviors such as being responsible for work and working productively, and 3) implementing cohesive \nwork activities such as being able to adapt, communicate well, and value others. To support a literature review of distance learning activities in teaching collaboration skills, we \nanalyzed distance learning activities designed by lecturers and their impact on students' collaboration \nability based on indicators of collaboration ability. Based on the results of interviews with lecturers at \none of the private universities, it was found that learning activities designed online require collaboration \nskills from students to carry out some learning activities or assignments given. The learning media used \ndepends on the type of activity and task being designed. For example, each small group provided a forum \non ZOOM such as a breakout room to facilitate student discussions. In addition, you can use the Learning \nManagement System (LMS) to provide a place for students to share work, share ideas, comments, \ncriticisms and suggestions with each other as well as a place to collect assignments. The encouragement \nof cooperation between students in groups leads students to use media that help collaboration such as \ngoogle documents that can be accessed simultaneously. Then, the learning activities that are designed \nrequire some attention so that the learning activities can run well. First, the activities carried out are to \ndivide students into small groups and it is also offered that in each small group there is a group leader to \norganize activities and tasks in the group to solve problems or projects. Furthermore there is an emphasis \ngiven to students verbally that the contribution between students is equal or evenly distributed. Then the \nsecond is peer-to-peer feedback activity. RESULTS AND DISCUSSION This feedback activity facilitates students to be able to convey \nideas, correct, observe, make comments, and assess fellow students. Research conducted by Ching & \nHsu (Purwadi, 2020) revealed that peer feedback has a positive impact on student learning and can \nincrease active participation in distance learning because of the freedom to express ideas and ideas they \nhave. Purwadi (2020) found that accustoming students to dare to think critically can develop cognitive JMME Halaman 61-71 66 p-ISSN 2089-8878; e-ISSN 2715-8276 66 skills to be able to think at a higher level. From these two explicit instructions for collaborating, it slowly \ndisappears so that students do not rely on instructions from lecturers. The culture applied in distance \nlearning is to support the development of collaboration, that is, a culture of learning and a culture of \nmutual respect. skills to be able to think at a higher level. From these two explicit instructions for collaborating, it slowly \ndisappears so that students do not rely on instructions from lecturers. The culture applied in distance \nlearning is to support the development of collaboration, that is, a culture of learning and a culture of \nmutual respect. 1. A learning culture is a learning culture that invites feedback givers to realize that the results \nare still learning outcomes. Likewise, for recipients of input, they are invited to realize that \nthe comments, values, suggestions or criticisms given are the result of learning from judging \nor correcting things that are lacking. 2. A culture of mutual respect is a culture of respect that invites feedback givers to realize that \nthe work done requires a long process and requires thought, time and effort. Similarly, the \nrecipient of the feedback is invited to realize that the comments, values, suggestions and \ncriticisms given are the efforts of the feedback giver to correct, observe, comment and assess \nthe shortcomings contained in his work. In addition to the learning activities carried out, the interview also explained the assessment \ncarried out by the lecturer in assessing learning activities that require cooperation. First, the assessment \nof contributions between peers. This assessment is delivered at the beginning of the lesson with the aim \nthat students are motivated to contribute well in each group. This assessment contains a range of scores \n(1 to 5) to rate each friend in a group. RESULTS AND DISCUSSION In the interview it was also mentioned that there are collaborative activities that are easier to do \nonline than offline. For example, when students present their answers online, it will be easier because \nstudents can directly share their answers through the screen sharing feature. In terms of time, sharing \nanswers is more efficient online than offline. In this activity, it was also found that one of the learning \nactivities that can be done offline and online is the presentation of student answers. Then, there are \nlearning activities that cannot be done online, but can be done offline, which is to monitor the \ncontribution of each student in a small group. Not all students can contribute for reasons that are often \ngiven by students with signal constraints, lecturers cannot be sure of the truth of these reasons. What \nlecturers can do is observe the consistency of student reasons. On the other hand, offline monitoring can \nbe easily done as it is enough to go around visiting each group and checking the answers. if the same or \nsimilar errors are found in each group, the lecturer can easily give a classical explanation for the mistakes \nmade. Furthermore, to find out the effectiveness of the lecture activities designed, a questionnaire was \ndistributed to students who took courses taught by the lecturer. There were 34 students who were \nrespondents. From the common questions regarding online learning that students do, several things were \nfound. The most widely used media is WhatsApp which is 94.1% and after that uses Moodle with 64.7% \nstatement; some of the other media students mentioned were zoom, google classroom, and desmos. In \naccessing online lectures, as many as 94.1% of students stated that they used laptops, all students stated \nthat they also accessed online lectures using mobile phones. And in accessing online lectures, 11.4% of \nstudents stated that they experienced difficulties due to network constraints. In lectures, students are \ndivided into groups which usually consist of 2-5 students. In large groups sometimes consist of 6-8 \nstudents. Students also stated that after being divided into groups, the lecturer will visit each group. Some of the difficulties experienced by students, the first is difficulty in communicating, this is \ncharacterized by the presence of group friends who are passive or do not respond when group discussions \nare running either via Whatsapp or via zoom. RESULTS AND DISCUSSION For example, in one group there are three students A, B, and C, \nthen A will assess B and C, as well as students B and C. Through this kind of assessment, it will be seen \nhow much contribution each student has in each group. Although there are some cases where students \nare reported to lecturers for not making any contribution to the group. The effort that can be made by \nthe lecturer is to ask the student directly concerned about the reason for not making any contribution in \nthe group and this is a consideration for the lecturer to the members of the group concerned. Second, \nassessment in the form of frequency in classical discussions. At the beginning of learning, lecturers will \nprovide multiple-choice questions that are conceptual and tricky. This trap question is given so that \nstudents do not lean towards one of the correct answers. So that several groups of students are formed \nwho answer different questions. Then the activity carried out is that the lecturer asks one of the student's \nanswer representatives to give an idea, then other students who have different answers are welcome to \nrespond or comment on the answer. Activities like this assess the frequency of student participation in a \ngiven activity. Third, the assessment of the analysis of social networks. Social network analysis is a way \nof assessing activities by visualizing the interactions that occur, for example student A comments on \nstudent B, student B comments on student C, this interaction gets a link or network. If this is visualized \nto all students, a picture will be obtained in the form of webs that visualize the relationship. Through this Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 67 visualization, you will see a circle that represents a student, the larger the circle, the student is the most \nactive student. The results of this analysis are also presented and explained to students which ones give \nthe most responses, receive responses and so on. This will give an indication that it is the student who \ngets the most responses, so that the topic or discussion given attracts the attention of the student. As for \nstudents who responded a lot, it was indicated that the student was active in discussion activities. RESULTS AND DISCUSSION Second, students experience difficulties in the internet \nnetwork which is sometimes constrained. Third, students sometimes find it more difficult to understand \nthe material being studied so that they have difficulty in finding solutions in groups. Some students also \nfeel that when studying offline, students can be more free to chat and joke and can coordinate in groups JMME Halaman 61-71 p-ISSN 2089-8878; e-ISSN 2715-8276 68 better. However, most students also stated that the advantage of online lectures is that online lectures are \nmore effective in terms of time and place. Students feel that online lectures become less tense and dare \nto express ideas in class. Students also state that they can learn new technologies through online lectures. In addition, online lectures are considered effective because they require students to be active, activities \nin online lectures are also considered to still make it easier for students to discuss, students can study in \ngroups or through other groups. The specific questions are based on the three indicators of collaboration capabilities used. The \nresults obtained show that all three indicators of collaboration ability are met. The first indicators are \ncooperation and interdependence to achieve common goals such as cooperation in groups; Discuss; \nactively contribute; and flexibility is met, this is shown from the percentage of students' ability to work \ntogether and express opinions in groups is 100%, but 8.8% of students stated that they did not work in \ngroups and felt that they could only work with a few groups. member. In decision making, all students \nstated that decisions are taken jointly and not unilaterally with a 100% percentage of answers. The second \nindicator is responsibility on the basis of learning and behavior of each such as being responsible for \nworking and working productively. All students stated offering to divide assignments into groups with \na 100% statement percentage, but 5.2% of students said they did not work on assigned assignments in \ngroups. However, most students work on assignments that have been shared in groups. In presenting \nideas in groups, most students stated that they conveyed the idea of completing the task, which was \n97.1%. The third indicator is the implementation of cohesive work activities such as being able to adapt, \ncommunicate well, and respect others. The fulfillment of these indicators is shown by the results of the \nquestionnaire which states that all students state that they accept every opinion submitted. RESULTS AND DISCUSSION All students \nalso stated that they could accept and appreciate the work of each group member. Most students also felt \nadaptable to group members although 5.9% of students stated they were unable to adapt. Based on the above results, it was found that distance learning can develop student collaboration \nskills. Distance learning activities should be designed by requiring cooperation from students, namely \n1) dividing students into groups either through direct instruction delivered or formed from the given \nanswer choices, 2) using media that supports students to provide responses, ideas, comments and so on. both in the form of video conference forums and LMS, 3) instructions for feedback activities, and 4) \nculture built in learning activities such as learning culture and mutual respect culture. It was also \nconveyed by students that they were divided into groups with members between 2-5 people for small \ngroups or 6-8 people for large groups, and in lectures adequate technology was used to support learning \nactivities such as using Whatsapp, Moodle, Google classes, and Desmos. Vol. 12, No. 02, December 2022 69 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 69 The assessment of cooperation that can be carried out also depends on the activities or tasks given, \nnamely the assessment of contributions between colleagues, the assessment of the frequency of classical \ndiscussions, and the assessment of social network analysis. In assessing the contribution of peers, it can \nhelp lecturers to know that there are unbalanced student contributions. This is also conveyed by students, \nthat students experience some communication problems because there are group members who are \npassive or do not respond when discussing. The opinion of this student is in accordance with what is \nconveyed by the lecturer regarding peer assessment related to the contribution of his group of friends. Advantages and disadvantages of distance learning activities that require cooperation in learning \nactivities and assignments. The advantage of this activity is the efficiency of learning time. If learning \nactivities can run well, learning time will be more efficient than offline. the shortcomings experienced \nare aspects of supervision of the contributions of students in the group. According to the results of \nlecturer interviews and student questionnaires, it was found that signal problems are one of the most \nfrequently conveyed reasons and it is difficult to ascertain the truth. This causes the contribution of \nstudents to be uneven. RESULTS AND DISCUSSION It is also conveyed by students that they are experiencing internet network \ndifficulties. JMME Halaman 61-71 CONCLUSIONS AND SUGGESTIONS Based on the results and discussion above, it can be concluded that there are several things that \nneed to be considered in designing learning activities to teach student collaboration skills, namely 1) \ndividing students into groups either through instructions directly delivered or formed from the selection \nof answers given, 2) media that supports students to provide responses, ideas, comments and so on both \nin the form of video conference forums and so on. LMS, 3) instruction on feedback activities, and 4) \ncultures built into learning activities such as culture of learning and culture of mutual respect. These four \nthings are found to be able to help teach students collaborative abilities as measured by three indicators, \nnamely, 1) cooperation and interdependence to achieve common goals such as cooperation in groups, \ndiscussion, active contribution, and flexibility, 2) being responsible for the basis of learning and behavior \nof each such as being responsible for work and working productively, and 3) the implementation of a \ncohesive work activity as can adapt, communicate well, and value others. In addition, the assessment \nthat can be done depends on the activity or task given, some assessments that can be given are the \nassessment of contributions between peers, an assessment in the form of frequency in classical \ndiscussions, and an assessment of social network analysis. The effectiveness of learning activities to teach student collaboration skills can be seen from the \nadvantages and disadvantages shown. The advantage of this learning activity is that the time and place JMME Halaman 61-71 70 p-ISSN 2089-8878; e-ISSN 2715-8276 of learning becomes more efficient. The disadvantage of this activity is the lack of supervision aspects \nthat lecturers can carry out regarding the correctness of the reasons for network constraints. ACKNOWLEDGMENTS The researcher would like to thank all parties who helped carry out research activities. This \ngratitude is addressed to the research subject who volunteered to be the subject of this study, Dr. M. Andy Rudhito, S.Pd who has provided guidance in conducting this research and Sanata Dharma \nUniversity who has contributed to the researcher. Zubaidah, S. (2018). Mengenal 4C: Learning and Innovation Skills untuk Menghadapi Era Revolusi 4.0. \n2nd Science Education National Conference, 13(April), 1–18. JMME Halaman 61-71 REFERENCES Abidin, Z., Hudaya, A., & Anjani, D. (2020). Efektivitas Pembelajaran Jarak Jauh Pada Masa Pandemi \nCovid-19. Research \nand \nDevelopment \nJournal \nof \nEducation, \n1(1), \n131. https://doi.org/10.30998/rdje.v1i1.7659 Fauziah, F. (2021). Implementasi Pembelajaran Kolaboratif Menggunakan Breakout Room Zoom \nMeeting Pada Pembelajaran Jarak Jauh. Edutech : Jurnal Inovasi Pendidikan Berbantuan \nTeknologi, 1. https://doi.org/https://doi.org/10.51878/edutech.v1i3.683 Hidayat, A. K., Yulianti, D., & Herpratiwi. (2021). Penggunaan Google Sites Dalam Membangun \nKolaborasi Pada Materi Korosi Ditinjau Dari Kemandirian Belajar Siswa. Jurnal Edukasi \nMatematika Dan Sains), 9(2), 440–451. https://doi.org/10.25273/jems.v9i2.10997 Mariamah, S., Bachtiar, M. Y., Pgpaud, P. P. G., & Makassar, U. N. (n.d.). Penerapan Project Based \nLearning Untuk Meningkatkan Kemampuan Kolaborasi Anak Usia Dini. 125–130. Nurhayati, D. I., Yulianti, D., & Mindyarto, B. N. (2019). Bahan Ajar Berbasis Problem Based Learning \npada Materi Gerak Lurus untuk Meningkatkan Kemampuan Komunikasi dan Kolaborasi Siswa. Unnes Physics Education Journal, 8(2), 218. Purwadi, I. M. A. (2020). Pengaruh Pemberian Umpan Balik Dari Teman Sejawat Terhadap \nKemampuan Kemampuan Berpikir Kritis Dan Komunikasi Matematis Siswa Smp Laboratorium \nUndiksha \nSingaraja. Jurnal \nPendidikan \nMatematika \nUndiksha, \n11(1), \n63–72. https://ejournal.undiksha.ac.id/index.php/JJPM/article/view/24382 Roekel, D. Van. (2010). Preparing 21st Century Students for a Global Society: An Educator’s Guide to \nthe \n“Four \nCs.” \nNational \nEducation \nAssociation. https://www.nea.org/professional-\nexcellence/student-engagement/tools-tips/being-21st-century-educator Simonson, M., Smaldino, S., & Zvacek, S. (2015). Teaching and Learning at a Distance (Sixth Edition). Information Age Publishing, Inc. Simonson, M., Smaldino, S., & Zvacek, S. (2019). Teaching and Learning at a Distance: Foundations of \nDistance Education (7th ed.). Information Age Publishing. Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… \n71 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 71 Sunardi, Kurniati, D., Sugiarti, T., Yudianto, E., & Nurmaharani, R. (2017). Pengembangan Indikator \n4c’s Yang Selaras Dengan Kurikulum 2013 Pada Mata Pelajaran Matematika Sma/Ma Kelas X \nSemester 1. Jurnal Ilmiah AdMathEdu, 7. Thompson, L., & Ku, H.-Y. (2006). A Case Study Of Online Collaborative Learning. Quarterly Review \nof Distance Education, 7(4). Wijaya, E. Y., Sudjimat, D. A., & Nyoto, A. (2016). Transformasi Pendidikan Abad 21 Sebagai Tuntutan \nPengembangan Sumber Daya Manusia di era Global. Jurnal Pendidikan, 1, 263–278. Yuangga, K. D., & Sunarsi, D. (2020). Pengembangan Media Dan Strategi Pembelajaran Untuk \nMengatasi Permasalahan Pembelajaran Jarak Jauh Di Pandemi Covid- 19. Jurnal Guru Kita, 4(3), \n51–58. Zubaidah, S. (2018). Mengenal 4C: Learning and Innovation Skills untuk Menghadapi Era Revolusi 4.0. 2nd Science Education National Conference, 13(April), 1–18. JMME Halaman 61-71"
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‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools in Canada
Journal of religious education
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cc-by
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Journal of Religious Education (2022) 70:327–340 https://doi.org/10.1007/s40839-022-00182-w Journal of Religious Education (2022) 70:327–340 https://doi.org/10.1007/s40839-022-00182-w Abstract Pope Francis met representatives of the Indigenous peoples of Canada in Rome in April 2022 and in Canada in July 2022. At these meetings he offered sincere apologies for the ways in which the Catholic Church had colluded with the strategy of cultural assimila­ tion of the Indigenous people in Canada. This was especially abhorrent in the residential schools, operated by the Catholic Church (and other Christian churches), that aimed to ‘evangelise’ and ‘civilise’ the Indigenous children. He emphasised that this was a ‘disas­ trous error’ and the ‘overall effects of the policies linked to the residential schools were catastrophic’. He was very clear at both meetings that this abuse was ‘incompatible with the Gospel of Jesus Christ’. These Catholic residential schools and the impact of these schools on the Indigenous people are discussed in this article. Keywords  Catholic residential schools · Canada · Pope Francis Keywords  Catholic residential schools · Canada · Pope Francis ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools in Canada Stephen J. McKinney1 Accepted: 15 September 2022 / Published online: 29 October 2022 © The Author(s) 2022 Stephen J. McKinney Stephen.mckinney@glasgow.ac.uk 1 School of Education, University of Glasgow, Glasgow, Scotland Stephen J. McKinney Stephen.mckinney@glasgow.ac.uk 1 School of Education, University of Glasgow, Glasgow, Scotland 2  Forgotten histories and missing voices There are a number of ways to frame or contextualise the history of the Catholic residen­ tial schools in Canada and other parts of the world. They can be framed as a (misguided) opportunity for evangelisation, for spreading the word of God, or as destructive products of a rapacious colonial system and as instruments of power and social control (Aquash, 2013; Johnson, 2018). These are, of course, highly relevant frames and will be referred to in this article. There are other ways to frame this history using the lenses of forgotten histories and missing voices. Forgotten histories and missing voices are very closely related, but there is a distinction to be drawn between them. All remembering is selective, and this can induce selective forgetting (Stone et al., 2017). Remembering is often used to support the sense of the ‘self-concept’ for an individual per­ son. The self-concept includes the following dimensions but is not synonymous with any of them: self-esteem; self-worth; self-image; ideal self; identities or roles and personal traits and qualities (Ackerman, 2018). A person acts and interacts with others based on their own self-concept and, if others accept this self-concept in interactions, they help to affirm the self-concept for that person. The remembering associated with the self-concept can be a combination of positive and negative memories, but an emphasis on more positive memo­ ries can preclude more negative memories. By extension and adaptation, this can be applied to the concept of an institution that can be held by those in leadership positions or positions of power – and this enables a deeper understanding of the internal conception and external projection of the institution. The insti­ tution can act and interact based on the held concept and this can be affirmed in interactions. The held concept and projection of an institution, for example, can be premised on the view that the institution is socially just, and the remembering and recall will be about the positive times that demonstrate this social justice. This then can lead to a hesitancy or a failure to recall times when social justice was not demonstrated – which would obviously contradict the held concept of the institution. This leads to a forgetting or supressing of histories or parts of histories. Forgotten histories refers to unacknowledged, overlooked, obscured or hidden histories that have not been recalled or remembered. 1  Introduction The visit of Pope Francis to Canada in July 2022 has assumed significant historical impor­ tance. During the visit, the Pope apologised to the Indigenous people for the role of the Catholic Church and religious communities in the cultural and religious abuses they have experienced. These abuses were experienced by many children who had been placed in the Indigenous residential schools. This article discusses the context for these apologies and the apologies themselves in three parts. First, the article will use forgotten histories and missing voices as a framework. This framework will enable a deeper understanding of the institu­ tional significance of the revelations about the treatment of the Indigenous children in the residential schools. Second, the rationale for the residential schools and the treatment of the Indigenous children will be scrutinised, with a focus on religious abuse. Third, the apologies 1 3 3 3 328 S. J. McKinney of Pope Francis will be examined, and some concluding remarks are offered about why it is important to remember this history. of Pope Francis will be examined, and some concluding remarks are offered about why it is important to remember this history. 1 3 2  Forgotten histories and missing voices They may have been forgotten because they were about persons or groups of persons who were not deemed to be important or relevant in the ways in which the narratives of the history of the institution have been configured and been projected. For example, the stories of lower placed members of the institution or women may have been forgotten because other narratives were more prominent, such as hierarchical and male dominated narratives. Or they were forgotten because the history was supressed as it exposed uncomfortable or shameful attitudes, actions or events that disrupted the held concept and projection of the institution. There is a danger that the held conception becomes a form of self-deception. These forgotten histories will include the missing voices from the past which were not considered to be important or were deliberately excluded. It would be a mistake to consign missing voices solely to past periods of time, to periods of forgotten history. Missing voices can refer to the present and voices that are missing 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 329 in the contemporary world. Within the Catholic Church, missing voices still refers to the voices of women, including Catholic women religious who are scrutinised, at times, and excluded by male hierarchies (Brigham, 2015). There are signs that Pope Francis is attempt­ ing to address this, and good recent examples have included the appointment of four women (three religious and a lay woman) as consultors to the secretariat of the Synod of Bishops and, at the time of writing, his intention to appoint two women to the Dicastery that helps him select bishops (Lamb, 2019, Vatican news, 2022a). Missing voices still refers to the voices of children and groups of children who are not heard. Very recently it has referred to the voices of children who identify as LGBTQ in Catholic schools (Huchting & Fisher, 2019). A focus on forgotten histories and missing voices can have a positive outcome in the sense that some extraordinary histories can be uncovered, and missing voices heard and highlighted. The life of Saint Katherine Drexel, foundress of the sisters of the Blessed Sac­ rament in America is a good example of a Catholic educator who began to listen to the missing voices. She divided opinion during her lifetime and her legacy continues to divide opinion (Lipperini, 2013; Morton, 2014, 2022). 2  Forgotten histories and missing voices For some she brought the message of Christ to the unchurched black masses, to others she ‘imposed the U.S. Catholic Church’s Jim Crow policies on Black education in the South’. Morton (2022, 19–20) argues for a move away from binary understandings and a more nuanced vision of the process of her life work: In the Afro-Catholic Diaspora, Mother Katherine is neither hero nor villain; she is a beloved witness to this self-determined Black Catholic education…Mother Katherine experienced a shift from being a missionary to unchurched black souls to becoming an accomplice to the holistic survival of Black people – mind, body, and spirit. Within this analysis of the work of Saint Katherine one of the greatest parts of the devel­ opment of her vocation was that she began to listen to the missing voices of the African Americans. Within this analysis of the work of Saint Katherine one of the greatest parts of the devel­ opment of her vocation was that she began to listen to the missing voices of the African Americans. While it is helpful to examine the more positive outcomes of a nuanced approach to for­ gotten histories, it is clear that forgotten histories and missing voices often refer to darker histories that have been deliberately forgotten or suppressed. Forgotten histories and abuses of power are being uncovered in the sex abuse scandals that have emerged in many Chris­ tian churches. The sex abuse has taken many forms and the impact of the scandals, certainly in the Catholic Church, has been exacerbated by the attempts to cover up or to deny, and even to bury the scandals (Dale & Alpert, 2007; Formicola, 2016) In other words trying to ensure they remain forgotten histories and that the missing voices of the victims of abuse are suppressed. This is beginning to be researched from different sociological, legal and theological perspectives (Moran, 2019; Formicola, 2020). 3  Indigenous children in Catholic schools – Australia and the USA Forgotten histories and missing voices are revealed in the contemporary histories of the treatment of the children of Indigenous peoples by the Catholic Church in various locations including Canada, USA and Australia. In all three countries, the aim was to assimilate, not integrate, the Indigenous children into state languages, cultures and even accepted forms of 1 3 330 S. J. McKinney religion in the 19th and 20th centuries. This was partly a legacy of the Doctrine of Discovery and a series of papal bulls that had been used from the 15th century to legally justify the colonisation by the European powers (Miller, 2019). religion in the 19th and 20th centuries. This was partly a legacy of the Doctrine of Discovery and a series of papal bulls that had been used from the 15th century to legally justify the colonisation by the European powers (Miller, 2019). This proposed assimilation was of a limited nature, in that it did not provide equal oppor­ tunities for the Indigenous children. It usually meant assimilation into the lowest socio- economic status and conditions: the boys were prepared for manual labour or farming, and the girls were prepared for some form of domestic service (Smith, 2009). It is useful to note that the assimilation in the early days did not extend to citizenship as this was only granted to all Indians in the United States in 1924 and in Canada in 1960 (Dawson, 2012). In Aus­ tralia the 1967 Referendum granted rights and equality to the Aboriginal people and Torres Strait Islanders – citizenship was granted in different states at different times prior to this (Gordon, 2018). Zucker (2008) notes that the assimilation policy in Australia did not recognise the Aboriginal culture. The Aborigines were considered to have no culture, no history and to be without spirituality. The Indigenous people would be improved by adopting the superior white way of life and the Christian religion. Indigenous children were separated from their parents to be educated into Christianity and in many places by Catholic missionaries. While different stories have emerged, and some suggest that the children were often treated better in the homes run by the Catholic Church than government institutions, the children were still separated from their families and for a specific purpose – evangelisation and civilisation. 3  Indigenous children in Catholic schools – Australia and the USA A similar story unfolds in the United States of America where a recent investigation has confirmed that the United States was responsible for supporting 408 boarding schools across 37 states between 1819 and 1969 (United States Department of the Interior, 2022). The Catholic Church was funded to run some of the Federal Indian boarding schools, as were other Christian denominations. Smith (2009, 5–6) argues that this was conceived as part of a deliberate choice of cultural genocide rather than physical genocide because, in the context of the United States of America, cultural genocide was perceived to be more economically efficient. Cultural genocide or ‘ethnocide’ does not involve the mass death of physical geno­ cide but the attempt to ‘destroy culture, language and religion’ and steal land and outlaw customs (MacDonald & Hudson, 2012, 442). The rejection of the spirituality and religious practices of the children and the imposition of Catholic Christianity were components of the cultural genocide that had tragic consequences. The recent investigation has prompted the United States bishops to seek a greater understanding of this history, promising to share their findings and create a greater culture of inclusion (Ruff, 2022). 4  Indigenous children in Catholic schools – Canada In Canada the strategies to ‘educate’ the Indigenous children (First Nations, Métis, and Inuit children) began before the opening of the residential schools in the late nineteenth century. There was an unsuccessful attempt at a Catholic boarding school for Aboriginal boys in the early 17th century (The Truth and Reconciliation Commission of Canada, 2015a). Sir Pere­ grine Maitland, the lieutenant Governor of Upper Canada proposed a plan in the early 1820s to ‘civilise’ the Indians that would focus on the young as it was expected that this would be more successful than focussing on adults (Hutchings, 2016). Maitland’s chief advisor, John Strachan, who would become Toronto’s first Anglican bishop, believed that Aboriginal 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 331 children should be separated from their families and raised by pious white people. A number of small boarding schools, operated by different denominations, were set up in the early to mid 19th century. children should be separated from their families and raised by pious white people. A number of small boarding schools, operated by different denominations, were set up in the early to mid 19th century. In the latter part of the 19th century, Nicholas Davin was commissioned by Sir John A. Macdonald Canada’s first prime minister, to write a report on Industrial Schools for Indi­ ans and Half-Breeds (published in 1879) (Indian Residential School History and Dialogue Centre Collections, 2022). Davin drew on the example of the Industrial schools in America which were deemed to be a ‘principal feature of the policy of aggressive civilisation’ (Davin, 1979). The evidence from America was that the day school did not work ‘because the influ­ ence of the wigwam was stronger than the influence of the school’. Christian churches were, in the opinion of Davin, best placed to supplant Aboriginal spirituality with a better one (Johnson, 2018). Macdonald justified the residential schools in 1883 as follows: When the school is on the reserve the child lives with its parents, who are savages; he is surrounded by savages, and though he may learn to read and write his habits, and training and mode of thought are Indian. He is simply a savage who can read and write. 4  Indigenous children in Catholic schools – Canada It has been strongly pressed on myself, as the head of the Department, that Indian children should be withdrawn as much as possible from the parental influence, and the only way to do that would be to put them in central training industrial schools where they will acquire the habits and modes of thought of white men. The first industrial residential school was opened by the Canadian government in Battlefield in 1883 and the strategy to ‘Christianise’ and ‘civilise’ would be put into operation in many more Government funded residential schools, the majority of which were operated by the Christian churches until 1969 (White & Peters, 2009; Morcom, 2017). Over 150,000 chil­ dren attended these schools between the late nineteenth century and the late 1990s when the remaining federally supported schools were finally closed. These schools were founded on the principles of the inherent superiority of whites over the uncivilised and savage Indians and attempted to implement the policy of the ‘aggressive civilisation’ of the children, as described by Davin in his 1879 report (Bombay et al., 2014; Hanson et al., 2020). Similar to America, this was an attempt at cultural genocide. Part of the impetus for the cultural genocide was to ensure the acquisition of the lands occupied by the Indigenous peoples and the natural resources on these lands (Rose, 2018). The Indian Act of 1920 allowed the government to ‘compel any First Nations child to attend residential school’, but it was never compulsory for the children to attend residential schools and some children attended day schools (The Truth and Reconciliation Commission of Canada, 2015a, 62). These government funded residential schools were run on behalf of the government by the Catholic Church, the Anglican Church, the Presbyterian Church, the Methodist Church and later the United Church and other denominations. The Catholic Church was responsible for approximately 60% of the residential schools, the Anglican Church 30% and the remaining 10% was between the other denominations (Feir, 2016). The schools provided religious instruction and basic academic and industrial skills. On arrival the children were given a new name (Euro-Canadian) to replace their Aborigi­ nal name and were given a number (The Truth and Reconciliation Commission of Canada, 2015a). It is reported that in many schools the numbers were used daily rather than names. Their hair was cut, and they swapped their traditional clothing for uniform. 4  Indigenous children in Catholic schools – Canada In many of the 1 3 3 332 S. J. McKinney schools, children were punished if they practiced their own customs/culture or used their own language. Most of the children spoke an Aboriginal language and had scant or no understanding of French or English, the languages used in the schools. This meant that the arrival at the school was a bewildering and frightening experience for the children as they could not communicate and could not understand what was being said to them. Over time, some children lost fluency in their own language or abandoned their own tongue. There were exceptions and in one of the Alberta schools, Fr Mullen translated the Bible and hymns into Cree for the children. In some schools the children were free to converse in their own languages in the playground. Brothers and sisters were separated in the schools. There was a high death rate due to maltreatment, poor sanitation, under nourishment and inadequate health care (Mosby & Galloway, 2017; Wilk et al., 2017). Clearly the basic needs were not met for many of the children in terms of care, nourishment and health. Many of the survivors of the residential schools would suffer from long term physical and mental health problems. 5  Truth and Reconciliation Commission of Canada The Truth and Reconciliation Commission of Canada was set up in 2008 to reveal the ‘complex history and on-going legacy of the church-run residential schools to the Canadian people’ and ‘guide and inspire’ a process that would lead to reconciliation. The Commission travelled throughout Canada for six years to hear the voices of the Aboriginal people who had been educated in the residential schools and produced a lengthy report in 2015 with ninety-four Calls to Action (The Truth and Reconciliation Commission of Canada, 2015a). A previous Commission had produced the Report of the Royal Commission on Aboriginal Peoples in 1996 that urged for a reconciliation process in Canada. This 400-page report and the 440 recommendations initially received some media attention but was largely ignored by the government (Hurley & Wherrett, 1999). Nevertheless, this report did begin the process of raising wider awareness of the treatment of the Aboriginal people (McGregor, 2018). The Truth and Reconciliation Commission of Canada provided a second opportunity to examine and understand how the Aboriginal people had been treated and seek reconciliation. The Truth and Reconciliation Commission of Canada (2015a, 1) used the term cultural genocide and distinguished cultural genocide from physical and biological genocide: Physical genocide is the mass killing of the members of a targeted group, and biologi­ cal genocide is the destruction of the group’s reproductive capacity. Cultural genocide is the destruction of those structures and practices that allow the group to continue as a group. States that engage in cultural genocide set out to destroy the political and social institutions of the targeted group. Land is seized, and populations are forcibly transferred, and their movement is restricted. Languages are banned. Spiritual lead­ ers are persecuted, spiritual practices are forbidden, and objects of spiritual value are confiscated and destroyed. And, most significantly to the issue at hand, families are disrupted to prevent the transmission of cultural values and identity from one genera­ tion to the next. 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 333 The Commission concluded that this separation of children from their families meant that ‘the Canadian Government essentially declared the Aboriginal people to be unfit parents’ (The Truth and Reconciliation Commission of Canada, 2015a 4). The Commission heard over 6,000 testimonies from former students of the residential schools, family members, community members and interested participants. 5  Truth and Reconciliation Commission of Canada Some of the testimonies were from former staff of the residential schools. The missing voices began to be heard in the testimonies of the adults who had been forced to attend the residential schools as children (Sedehi, 2019). The testimonies speak about physical, sexual, mental, cultural and religious abuse. 6  Religious abuse This section draws on the testimonies of survivors of Catholic residential schools as recounted to The Truth and Reconciliation Commission of Canada (The Truth and Recon­ ciliation Commission of Canada, 2015b). Some former students appreciated the Christian upbringing they received through religious education and the daily rituals of prayer. How­ ever, there is also evidence of religious abuse that took a number of forms. First, the children were not allowed to practice their own religion and spirituality, and some were threatened with punishment if they participated in their own rituals. In some cases, this even applied to participation in rituals that were practiced outside school term time (Johnson, 2018). Sarah McLeod recalls returning to Kamloops school with a small totem pole, a birthday present. This was taken from her by one of the nuns and discarded; the nun described it as being ‘all devil’. Second, the imposition of a strict regime of daily Christian prayer was common: morn­ ing and evening prayers, before and after meals and before classes. Antonette White who had attended the Catholic school at Kuper Island near Vancouver Island, British Columbia commented: I think the worst thing, is the praying. It’s, it’s like you pray, pray, pray, and yet there’s still no peace in that prayer of what they made you do. Noel Starblanket attended the Lebret (Qu’Appelle, St. Paul’s, Whitecalf) Industrial School. This was operated by the Missionary Oblates of Mary Immaculate and the Grey Nuns from 1884 to 1973 (University of Regina, 2022). He recalls a very similar strict regime, as does Victoria Boucher-Grant who attended Fort William (St. Joseph’s) school, Ontario: I learned how to pray. I learned how to, this became a way of life, kneeling on my knees, and praying to, to some, some God that made me feel guilty because I was, I was not a very clean person. Third, fear of the judgment of God and the images of the devil and hell were used to coerce children in a number of ways. Joseph Martin Larocque at the Qu’Appelle school remembers being scared of images of the devil with a pitchfork in religious education classes. Father Lacombe’s instructional ladder, a pictorial catechism, was used in a number of the Catholic schools. This represented stairs leading to heaven and Jesus and the angels. At the bottom of 1 3 334 S. J. 6  Religious abuse McKinney the stairs was fire, and according to Fred Bass, Indian people. Bass stated that he was told at the Roman Catholic school, Kamsack, Sastatchewan that if he and the other children did not change their ways, they would end up in the fire. Strict prohibition of the children’s spiri­ tuality and the denial of their right to practice their rituals and the imposition of Catholic religious instruction and practice, then, were used as weapons to evangelise and catechise. 7  The Christian churches and Canada apologise The apologies by the Christian churches were for the collusion with the assimilationist poli­ cies of colonialism and for the work of the churches in the residential schools. These apolo­ gies began with the United Church of Canada in 1986 apologising for being an agent of colonialism (Bush, 2015). At this point, there was no explicit apology for the collusion with the Residential school system. Later apologies included contrition for involvement in the residential schools. Two Anglican bishops apologised in 1991 and 1993 and the Presbyte­ rian Church in 1994. In the Catholic Church the apologies were issued, most notably, by the Missionary Oblates of Mary Immaculate and different dioceses. On 24 July, 1991 Fr Doug Crosby, then President of the Oblates apologised on behalf of the members of the order for the damage caused by the schools they operated (Robinson, 2019). He apologised for three things. In the first instance, he apologised for the existence of the schools that separated chil­ dren from their families. In the second instance, he apologised for the physical and sexual abuse. In the third instance, he apologised for dismissing the Indigenous religious tradition and the attempts at assimilation. On June 11, 2008 Stephen Harper, the Prime Minister of Canada, apologised on behalf of Canada for the role of the residential schools in the policy of assimilation. 1 3 9  Pope Francis and the apologies to the Indigenous people in Canada The Truth and Reconciliation Commission of Canada (2015a, 221) noted that that the Roman Catholic Church did not have a single spokesperson and the apologies had been issued by different diocese and the religious organisations. On April 29, 2009, Pope Benedict XVI met with Mr Phil Fontaine, the Grand Chief of the Assembly of First Nations of Canada and The Most Reverend James Weisbgerber, President of the Canadian Conference of Bishops (Vatican, 2009). In the short communique that was released, the Pope expressed his ‘sorrow at the anguish caused’ by the church involvement in the residential schools. These words of regret were not an official apology and he did not use the word apology. The Commission contrasted this with the pastoral letter of Benedict issued in Ireland in 2010 as a response to the child abuse in the church in Ireland. In this letter, the Pope stated: ‘You have suffered grievously and I am truly sorry’ (Pope Benedict, 2010, Sect. 6). This led the Commission to develop Call to Action number 58 which called upon the Pope to issue an apology to survivors and their families and communities for the ‘spiritual, cultural, emotional, physi­ cal and sexual abuse’ of the children in the Catholic residential schools. They called for an apology similar to the pastoral letter of 2010 and that the apology should be delivered by the Pope one year after he received the report of the Commission (The Truth and Reconciliation Commission of Canada, 2015a, 223, 330). On July 24 to 30, 2022, Pope Francis visited Canada on an apostolic journey. On July 25, he met with the Indigenous people (First Nation, Métis and Inuit) of Canada and described his visit as a penitential pilgrimage (Pope Francis, 2022a). The Pope had previously met some representatives of the Indigenous people in Rome in March/April 2022. At that meet­ ing in Rome, the Pope apologised for the ‘suffering, hardship, discrimination’ and abuse experienced by the people and he drew attention to the role of the residential schools in the attempt to rob the Indigenous people of their cultural identity. Pope Francis stated that all of this had made him feel indignation and shame very strongly (Pope Francis, 2022b). He was emphatic that when confronted with historical memory we must have a commitment to learn from past mistakes. 8  The unmarked graves The report of the discovery of unmarked graves of 215 Indigenous children in 2021 at the site of Kamloops Indian Residential School in South Central British Columbia, and later at the sites of other former residential schools, once again highlighted the tragic consequences of the injustices of the residential schools (Thorne & Moss, 2022). Kamloops was run by the Catholic Church. The exact number of children who died may never be known because of the practice of poor record keeping (Supernant, 2022). There are parallels in other parts of the world. In Scotland, the unmarked grave of possi­ bly as many as 400 children is in the cemetery of St. Mary’s Parish Church Lanark (Gamble, 2018). The children had been residents at the Smyllum Park School run by the Daughters of Charity as an orphanage and for poor children (1864–1981) and they were buried in the ‘Smyllum plot’ in the graveyard. While the children appeared to have died from natural causes and economic cost may explain the lack of headstones, this is within the context of the grim evidence of emotional, physical and sexual abuse towards children in the school that has been uncovered in the Scottish Child Abuse Inquiry Case Study no. 1 (Scottish Child Abuse Inquiry, 2018). The unmarked graves in Canada and Scotland are chilling indicators that the children were not to be remembered and would be part of forgotten history. This recovered history 1 3 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 335 of the unmarked graves and the abuse experienced by many of the Indigenous children in the residential homes in Canada demonstrates how far removed the care for the children was from the conception of social justice that is espoused by Catholic Social Teaching. The inherent God-given human dignity of all individuals as revealed in sacred scripture which underpins contemporary Catholic Social Teaching was not recognised (Genesis 1:26–27; Pontifical Council for Justice and Peace, 2004). Christian ‘dignity’ was imposed with a European cultural form of Catholic Christianity with terrible consequences. 9  Pope Francis and the apologies to the Indigenous people in Canada The Pope was very clear that the abuses the Indigenous people had experienced were ‘contrary to the Gospel of Jesus Christ’. In his address at Maskwacis in Canada on July 25, 2022, the Pope apologised again for the devastation of the Indigenous families and their culture caused by the assimilationist policies and the residential schools. He drew attention to the role of Catholic Church and the religious communities in the cultural destruction and the physical, verbal, psychological and spiritual abuse of the children in their care. Pope Francis emphasised that the ‘overall effects of the policies linked to the residential schools were catastrophic’ and that this was a ‘disastrous error’. Once again, he was very clear that this abuse was ‘incompatible with the 3 3 336 S. J. McKinney Gospel of Jesus Christ’. Pope Francis recognised the importance of memory and recovering the forgotten histories (Pope Francis, 2022a). The Pope acknowledged that the public apol­ ogy is only a first step in a process that must seek to ensure there is no repetition of the dam­ aging culture and practices that have caused such harm for a vulnerable group of people. In the second address on July 25, 2022, at a meeting with Indigenous peoples and mem­ bers of the parish community of Sacred Heart Edmonton, the Pope again recalled the role of the residential schools in ‘robbing communities and individuals of their cultural and spiri­ tual identity’ (Pope Francis, 2022c). This was part of the ‘violation of dignity, the experience of evil, the betrayal of trust’ and shameful for believers. He does not offer a simple solution but urges that the way forward is to be reconciled in the crucified Christ, and he used this powerful image to stress the betrayal that took place in the residential schools: This is the way forward: to look together to Christ, to love betrayed and crucified for our sake; to look to Christ, crucified in the many students of the residential schools. If we truly want to be reconciled with one another and with ourselves, to be reconciled with the past, with wrongs endured and memories wounded, with traumatic experi­ ences that no human consolation can ever heal, our eyes must be lifted to the crucified Jesus; peace must be attained at the altar of his cross. 9  Pope Francis and the apologies to the Indigenous people in Canada Pope Francis, despite limited physical mobility, undertook the penitential pilgrimage to deliver frequent apologies to the Indigenous Peoples for the harm inflicted on them and damage caused by the cultural suppression in the residential schools. The repeated apologies emphasised the severity of the harm and damage. His final public statements were delivered in a Homily at Vespers with Bishops, Priests, Deacons, Consecrated Persons, Seminarians, and Pastoral Workers in the Cathedral of Notre Dame in Quebec on July 28, 2022 (Pope Francis, 2022d). He discussed the cultural coercion of the Indigenous peoples and also asked forgiveness of the victims of sexual abuse. 10  Concluding remarks This article has examined the history of the residential schools in Canada, the meetings of Pope Francis with the Indigenous people of Canada and his apologies for the cultural and religious injustices imposed on the Indigenous people and the grievous harm caused by the residential schools. This has been discussed within the frameworks of forgotten histories and the missing voices of the survivors of the Catholic residential schools. The Pope has listened to the voices and urges that the history is investigated, and a healing process can begin. The Pope did not refer to the term cultural genocide during the encounters with Indig­ enous people in Rome and Canada, despite this being the term used by the Truth and Recon­ ciliation Commission of Canada. However, he did use this term in his response to a reporter on the flight back to Rome from Canada. Pope Francis said the following (Vatican News, 2022b): …I didn’t use the word because it didn’t come to my mind, but I described the geno­ cide and asked for forgiveness, pardon for this activity that is genocidal. For example, 1 3 337 …and yet there’s still no peace’ Catholic Indigenous Residential Schools… I condemned this too: taking away children, changing culture, changing mentality, changing traditions, changing a race, let’s put it that way, an entire culture. Yes, geno­ cide is a technical word. I didn’t use it because it didn’t come to my mind, but I described it… It’s true, yes, yes, it’s genocide. It is vitally important that the forgotten history of the Indigenous Residential schools is being recovered and the missing voices are heard. Education is needed to ensure that people, especially non-Indigenous people in Canada and beyond, are aware of this history (Neufeld et al., 2022). This history impacts not only on the Canadian people but also on the associ­ ated Christian churches and, for the purposes of this study, especially the Catholic Church. Education in this history is crucial for current and future generations – that they avoid the deeply harmful mistakes of the past (Kuhl, 2017).i Significant numbers of the children did not receive an adequate education in the harsh environment of many of the residential schools. 10  Concluding remarks The education in the residential schools, for the most part, did not cohere with the vision of education described by Pope Francis in his address to the parish community of Sacred Heart Edmonton (Pope Francis, 2022c): Education must always start from respect and the promotion of talents already present in individuals. It is not, nor can it ever be, something pre-packaged and imposed. For education is an adventure, in which we explore and discover together the mystery of life. Further, the Catholic culture and religious education in the residential schools did not cohere with the vision of Catholic education based on the love of Christ that is outlined in the series of contemporary church documents on Catholic education. This series begins with Gravissi­ mum Educationis (1965) through The Catholic School (1977) to the Identity of the Catholic School for a Culture of Dialogue (2022) (Pope Paul VI, 1965; The Sacred Congregation for Catholic Education, 1977; Congregation for Catholic Education 2022).f These documents offer a vision of Catholic education that is free of coercion; an educa­ tion that is not imposed to support a strategy of cultural genocide. This education respects the inherent God-given dignity of every person and aims to ensure that all people have opportunities to develop their talents and gifts, enabling them to contribute to the com­ mon good of society. Often, the children in the residential schools were not provided with sufficient opportunities to develop their talents and gifts - opportunities which would have enabled them to fulfil their potential and make a meaningful contribution to the common good of society (Brady, 2010). Funding  The author did not receive support from any organisation for the submitted work. Declarations Conflict of interest  The author declares that he has no conflict of interest. The author has no relevant financial or non-financial interests to disclose. 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Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. irshdc.ubc.ca/index.php/Detail/objects/9427 (2017). Residential schools and the effects on Indigenous health and well-being in Canada—a scoping review. Public Health Reviews, 38(8), 1–23. https://doi.org/10.1186/ s40985-017-0055-6 Zucker, M. (2008). Open hearts: the Catholic church and the stolen generation in the Kimberley. Journal of the Australian Catholic Historical Society, 29, 23–37. https://link.gale.com/apps/doc/A190243415/ AONE?u=glasuni&sid=googleScholar&xid=eca28645 Publisher’s Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1
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SECRET THINKER SOMETIMES LISTENING ALOUD: SOCIAL COMMITMENT IN DAVID BOWIE’S LYRICS
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https://openalex.org/W2801736036
https://lipidworld.biomedcentral.com/track/pdf/10.1186/s12944-018-0734-4
English
null
Handgrip strength is positively related to blood pressure and hypertension risk: results from the National Health and nutrition examination survey
Lipids in health and disease
2,018
cc-by
6,581
RESEARCH Open Access © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ji et al. Lipids in Health and Disease (2018) 17:86 https://doi.org/10.1186/s12944-018-0734-4 Ji et al. Lipids in Health and Disease (2018) 17:86 https://doi.org/10.1186/s12944-018-0734-4 Handgrip strength is positively related to blood pressure and hypertension risk: results from the National Health and nutrition examination survey Chao Ji1†, Liqiang Zheng1†, Rui Zhang2, Qijun Wu1 and Yuhong Zhao1* Chao Ji1†, Liqiang Zheng1†, Rui Zhang2, Qijun Wu1 and Yuhong Zhao1* Abstract Background: Isometric handgrip resistance exercise, a nonpharmacological lifestyle modification, has been recommended as a first-line treatment for hypertension. This study aimed to examine the relationship of handgrip strength to blood pressure and the risk of hypertension. Methods: The responses and examination of 4597 participants in the National Health and Nutrition Examination Survey (NHANES) were analyzed in this study. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were transformed to age- and sex-specific z-scores. Handgrip strength was adjusted by weight (kg) and converted to an age- and sex-specific z-score. The relationships of SBP and DBP to handgrip strength were analyzed by Pearson correlation test and multivariable linear regression. Binary logistic regression was used to analyze the association between handgrip strength and prevalence of hypertension. Results: Handgrip strength was positively related to higher DBP in men and women. In men, logistic regression models revealed that increased handgrip strength was associated with higher risk of hypertension after adjusting for age, BMI, smoking and drinking status; OR was 1.24 (95%CI: 1.04–1.48). After stratifying on BMI, handgrip strength was significantly associated with higher risk of hypertensions after adjusting for age, BMI, smoking and drinking status in overweight and obese men; OR was 1.31 (95%CI: 1.05–1.63). No significant associations were observed in women. Conclusions: Increased handgrip strength is associated with higher DBP in men and women. In men, especially Conclusions: Increased handgrip strength is associated with higher DBP in men and women. In men, especially overweight and obese men, strong handgrip strength may be associated with higher risk of hypertension. rds: Systolic blood pressure, Diastolic blood pressure, Handgrip strength, Hypertension, Body mass ind Handgrip strength Handgrip strength, as an indicator of muscle strength, was evaluated using a handgrip strength test according to the recommendations of the Institute of Medicine [28]. Takei Digital Grip Strength Dynamometer, Model T.K.K.5401 (Takei Scientific Instruments Co., Niigata, Japan) was used for all muscle strength assessments. Each hand was tested three times, alternating hands with a 60-s rest between measurements on the same hand. Before measurement of handgrip strength, the dyna- mometer was adjusted to participants’ hands size, until the second joint of the index finger was at a 90-degree angle to the handle. The placements of the handle were marked for each hand at the same time. Participants stood straight with the feet hip with apart and arms fully extended along side of thigh with palms facing the thigh. The hand, which was in line with the wrist and forearm, performed a quick, maximally hard squeeze of the han- dle. The complete handgrip strength testing protocol is described in NHANES muscle strength procedures manual. Participants and samples The NHANES is a cross-sectional study that examines the health and nutrition of children and adults in the United States. It collects data about demographics, health and health behaviors. NHANES uses a complex stratified, multistage, probability cluster sample designed to represent the U.S. population with respect to age, sex and race [24, 25]. Handgrip dynamometer assessment was recently in- cluded in NHANES. Handgrip strength data is available in the 2011–2012 and 2013–2014 survey cycles. Hand- grip strength data from the participants aged ≥18 years old who participated in these survey cycles were ana- lyzed in this study. Written informed consent was ob- tained from all the participants. The NHANES protocol was reviewed and approved by the National Center for Health Statistics Research Ethics Review Board. Background which has become a global public health problem [2]. Risk factors increased the prevalence of hypertension include population growth, increased age and behavioral risk factors, such as unhealthy diet, tobacco use, con- sumption of alcohol, excess weight, exposure to per- sistent stress, high cholesterol, diabetes mellitus, and lack of physical activity. Strategies that have been im- plemented to prevent and manage hypertension in- clude reducing exposure to behavioral risk factors and early detection and treatment of hypertension [3]. Despite these strategies, the mortality trend for hyper- tension continues to increase [4]. Hypertension, also known as high or raised blood pressure (BP), is a condition in which the pressure in the arteries is persistently elevated. It contributes to the burden of heart disease, stroke, kidney failure, premature mortality and disability. Cardiovascular disease accounts for approxi- mately 17 million deaths a year, which is nearly 1/3 of an- nual global deaths [1]. Every year, worldwide, 9.4 million people deaths die from complications of hypertension, * Correspondence: zhaoyh@sj-hospital.org †Equal contributors 1Department of Clinical Epidemiology, Shengjing Hospital of China Medical University, No. 36, San Hao Street, Shenyang 110004, Liaoning, China Full list of author information is available at the end of the article Page 2 of 7 Page 2 of 7 Ji et al. Lipids in Health and Disease (2018) 17:86 Ji et al. Lipids in Health and Disease (2018) 17:86 Ji et al. Lipids in Health and Disease (2018) 17:86 Current national and international guidelines for pre- vention of hypertension recommend that adults perform muscle strengthening activities 2 or more days a week to obtain at least 150 min/week of moderate-intensity or perform 75 min/week of vigorous-intensity aerobic physical activity [5–8]. Evidence indicates that isometric resistance training is an effective exercise modality to lower resting BP in both normotensive and hyperten- sive populations [9–12]. The handgrip strength test, a quick and inexpensive way to measure an individ- ual’s muscle strength, is verified to be a satisfactory indicator of resistance training in an epidemiological study [13–15]. Blood pressure BP measurements were taken during the NHANES one- examination visit. All participants sat all the way to the back of the chair to keep the spine straight and rested quietly for 5 min prior to BP measurement. The arm was bare and unrestricted by clothing with the palm of the hand turned upward. The elbow slightly flexed and positioned so that the midpoint of the upper arm was at the level of the heart. Three consecutive BP readings were obtained by the auscultatory or oscillatory method. If a BP measurement was interrupted or incomplete, a fourth measurement was performed. All BP determina- tions (systolic and diastolic) were performed in the NHANES mobile examination center. Hypertension was defined as resting BP persistently at or above 140/ 90 mmHg according to the international standard [27]. Although most evidence indicates that greater hand- grip strength is associated with lower BP [16–19], some of them are limited to small sample size studies [19]. And other studies did not find this association [20, 21]. In contrast, in the oldest old persons, higher BP was found to be associated with greater handgrip strength [22]. Dong [14] and Diez-Fernandez [23] suggested that the effect of strong handgrip strength was medi- ated by body mass index (BMI). However, after adjust- ment for BMI, strong handgrip strength was still associated with higher, not lower, adolescent BP [14]. Analysis of the association between handgrip strength and BP adjusted for BMI in a large-scale population is relatively scarce. Therefore, using a representative sample from the database of the National Health and Nutrition Examin- ation Survey (NHANES) 2011–2012 and 2013–2014 sur- vey participants aged ≥18 years old, we examined the association between handgrip strength and BP in adults after stratifying by, or adjusting for, age, sex and BMI. This study may have important implications for hypertension intervention measures. Anthropometry Height was measured to the nearest 0.1 cm using a digital stadiometer connected to an acrylic headpiece that interfaced directly with the Integrated Survey Infor- mation System (ISIS) anthropometry software applica- tion. Weight was measured in kilograms used a digital weight scale. BMI was calculated as weight in kilograms divided by height in meters squared (kg/m2), which was categorized as underweight, normal, overweight and obese according the NIH criteria [26]. Statistical analysis Descriptive characteristics were expressed as mean ± standard deviation (SD) for continuous variables and as frequency with percentage for categorical variables. Page 3 of 7 Ji et al. Lipids in Health and Disease (2018) 17:86 Page 3 of 7 Baseline characteristics between men and women were compared using t test and chi-square test. (r = −0.084, P < 0.01), DBP (r = −0.034, P < 0.05) and BMI (r = −0.691, P < 0.01). BMI was positively correlated with SBP and DBP (both, P < 0.01). The similar results were observed in women. In men, higher BMI was also correlated to higher SBP and DBP (both, P < 0.01); how- ever, handgrip strength SBP and DBP were not signifi- cantly correlated with each other. (r = −0.084, P < 0.01), DBP (r = −0.034, P < 0.05) and BMI (r = −0.691, P < 0.01). BMI was positively correlated with SBP and DBP (both, P < 0.01). The similar results were observed in women. In men, higher BMI was also correlated to higher SBP and DBP (both, P < 0.01); how- ever, handgrip strength SBP and DBP were not signifi- cantly correlated with each other. To avoid the potential bias effect of body weight on the estimation of handgrip strength, relative handgrip strength [handgrip (kg)/body weight (kg)] was calculated to assess the handgrip strength. This adjustment was based on standard assumptions about morphological ef- fects [13]. Because distribution of handgrip strength, SBP and DBP varied by age and sex, these parameters were transformed into sex and age specific SD scores (z-scores) in the analysis of the association between handgrip strength and risk of hypertension by binary logistic regres- sion (z-score = (value −mean)/SD) [29]. Pearson correl- ation test, partial correlation and linear regression analysis were used to analyze the relationships between handgrip strength, BMI, SBP and DBP. Binary logistic regression was used to analyze the association between handgrip strength and prevalence of hypertension. While controlling for BMI in partial correlation tests, handgrip strength increased with SBP (r = 0.033, P < 0. 05) and DBP (r = 0.094, P < 0.01) in all subjects. The similar correlations were observed in men. However, in women handgrip strength was only positively correlated to DBP. The associations between BP and handgrip strength, based on linear regression models, are shown in Additional file 1: Table S1 and Table S2. Statistical analysis SBP was positively associated with handgrip strength (1 SD higher increase in handgrip strength was related to 0.065 SD higher SBP, P < 0.01) in men after adjusting for age and BMI (model 2). After also adjusting for smoking and drinking status (model 3), the regres- sion coefficient changed minimally; however, the as- sociation was no longer significant (P = 0.062). Significant association was also observed between SBP and unadjusted handgrip strength in women (model 1). However, in women, no significant associ- ations were founded between handgrip strength and SBP in adjusted models 2 and 3. Higher handgrip strength was significantly associated with higher DBP in men: One SD increase in handgrip strength was related Data are presented as mean ± SD or count (%) Data are presented as mean ± SD or count (%) aDifference between men and women was significant using t- test bDifference between men and women was significant using chi-square test BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure Data are presented as mean ± SD or count (%) aDifference between men and women was significant using t- test bDifference between men and women was significant using chi-square test BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure p ifference between men and women was significant using t- test Discussion The present study revealed that handgrip strength was positively and significantly associated with DBP in men and women. There were no significant associations be- tween handgrip strength and SBP in women. Handgrip strength appears to increase the risk of hypertension in men especially for those who were overweight and obese (BMI ≥25). However, the same results were not observed in women. Table 2 shows the association between handgrip strength and hypertension, stratified by gender; binary logistic regression was used to estimate the odds ratio (OR) of handgrip strength for the risk of hypertension. Unadjusted handgrip strength was not associated with risk of hypertension in men (model 1). After adjusting for age and BMI (model 2) and smoking and drinking status addition (model 3), significant associations were observed: ORs were 1.229 (95%CI: 1.036–1.458) and 1. 241 (95%CI: 1.038–1.484), respectively. No significant associations between handgrip strength and hyperten- sion were observed in women. Emerging evidence has demonstrated the effect of iso- metric handgrip exercise on reducing BP in normoten- sive and hypertensive populations [30]. Meta-analyses of randomized controlled trials revealed that isometric handgrip training could reduce resting BP [19, 31]. Re- cent randomized trials also support the same conclusion [16, 17]. Possible mechanisms for these effects include improvements in conduit and resistance due to vessel endothelium dependent dilation, oxidative stress and autonomic regulation of heart rate and BP [30]. How- ever, most of these studies included relatively few sub- jects, and the exercise trainings were not uniform. Recent studies report variable findings about the associ- ation between handgrip strength and BP. A recent ran- domized controlled trial found that isometric handgrip exercise did not decrease BP [20]. In 2010, Dong et al. [14] conduct a survey of 89,655 adolescents in China aged 13–17 years; their results indicated that strong handgrip strength was associated with increased adoles- cent BP. Another longitudinal cohort study conducted by Taekema et al. reported that higher BP is associated with greater handgrip strength in the oldest old, aged over 85 years [22]. In their middle-age subjects, BP and handgrip strength were not significantly associated. Results Baseline characteristics of the study participants are shown in Table 1. Our study included 4597 participants; mean age was 47.4 ± 18.3 years. Compared with women, men had higher height, weight, BMI, handgrip strength, SBP and DBP. However, the gender-related difference in hypertension prevalence was not significant. The relationships of handgrip strength with BMI, SBP and DBP were analyzed using a separate Pearson correl- ation test for each pair of variables. In all participants, higher handgrip strength was correlated with lower SBP Table 1 Baseline characteristics of study subjects by gender All (4597) Men (2184) Women (2413) t/χ2 P Age (yrs) 47.4 ± 18.3 47.1 ± 18.3 47.8 ± 18.3 1.322 0.186 Height (cm) 167.1 ± 10.1 174.3 ± 7.8 160.5 ± 7.1 61.562a < 0.01 Weight (kg) 81.5 ± 22.4 86.9 ± 21.8 76.5 ± 21.7 15.776a < 0.01 BMI (kg/m−2) 29.1 ± 7.3 28.5 ± 6.5 29.6 ± 7.9 5.360a < 0.01 BMI Category Underweight 84 (1.9) 38 (1.8) 46 (2.0) 63.541b < 0.01 Normal 1278 (29.0) 594 (28.3) 684 (29.6) Overweight 1368 (31.1) 767 (36.6) 601 (26.1) Obese 1674 (38.0) 698 (33.3) 976 (42.3) Handgrip strength (kg) 33.9 ± 10.4 41.5 ± 8.8 26.7 ± 5.7 61.850a < 0.01 SBP (mm Hg) 122.8 ± 17.7 124.5 ± 16.3 121.2 ± 18.8 6.197a < 0.01 DBP (mm Hg) 69.7 ± 11.4 70.8 ± 12.0 68.8 ± 10.8 5.562a < 0.01 Smoking 1944 (42.4) 1096 (50.4) 848 (35.2) 107.976b < 0.01 Drinking 2855 (70.0) 1579 (80.5) 1276 (60.2) 199.285b < 0.01 Hypertension 715 (16.7) 355 (17.3) 360 (16.1) 0.985 0.321 Data are presented as mean ± SD or count (%) Table 1 Baseline characteristics of study subjects by gender Ji et al. Lipids in Health and Disease (2018) 17:86 Page 4 of 7 Page 4 of 7 Table 2, no significant associations were found be- tween handgrip strength and hypertension risk (Table 4). to 0.165 SD higher in DBP in men after adjusting for age and BMI, P < 0.01 (model 2). In men, after also adjusting for smoking and drinking, one SD increase in handgrip strength was related to 0.155 SD higher DBP (model 3). In contrast to the lack of association between handgrip strength and SBP in women, DBP was significantly associ- ation with handgrip strength in women. Unadjusted hand- grip strength was negatively associated with DBP (model 1). Results After adjusting for age and BMI, one SD increase in handgrip strength in women was related to 0.096 SD higher DBP (model 2) and 0.081 SD higher in DBP after also adjusting for smoking and drinking status (model 3). Discussion A higher percen of body fat generally translates to a higher rate of pearance of free fatty acids in plasma, which could induce higher blood pressure [44]. Thus, considering Table 4 Association in women between handgrip strength Underweight & β S.E. Model Model 1: unadjusted −0.163 0.154 Model 2: adjusted for age and BMI −0.133 0.211 Model 3: as Model 2 and smoking and drinking −0.123 0.215 BMI body mass index. All participants were categorized into two strata ac ≥25). Handgrip was transformed into age and sex specific SD scores (z-sc This study had several strengths for studying the asso- ciation between handgrip strength and BP and risk of hypertension. The National Health and Nutrition Exam- ination Survey (NHANES) is a program of studies de- signed to assess the health and nutritional status of adults and children in the United States, which examines a nationally representative sample and can be general- ized to western populations. Secondly, in this study, handgrip strength was adjusted for age, BMI, and smok- ing and drinking status. Few studies of handgrip strength are adjusted for body size and composition, although that was suggested in previous studies [45]. A weakness of this study was that simply finding an association be- tween handgrip strength and BP and risk of hyperten- sion does not support a causal relationship because this study used a cross-sectional design. Longitudinal data and clinical trials are needed to clarify whether the rela- tionship between muscle strength and BP and risk of hypertension is causal. Another limitation is that find- ings based on the NHANES database may not be generalizable to non-western populations; this study needs to be replicated in other populations to better understand the external validity of our findings. y Several mechanisms could explain the positive associa- tions between handgrip strength and BP. Peripheral vas- cular resistance increases with chronological age due to reduced sympatholysis, which results in an elevated sym- pathetic tone [35, 36], or vascular resistance increased with morphological changes in the arteriolar network [37]. Another possible explanation is that BP is associ- ated with the age-associated loss of lean mass [38, 39]. In addition, handgrip strength is probably associated with other risk factors for hypertensions, such as meta- bolic syndrome or cardiovascular disease biomarkers, in- cluding triglyceride, HDL, LDL, glycohemoglobin (HbA1c), uric acid, insulin resistance and serum adipo- nectin levels [40–44]. Discussion Although participants were first categorized by the international classification of adult underweight, normal, overweight, and obesity according to BMI value, to per- form BMI-stratified analysis of the association between handgrip strength and hypertension these four weight categories were combined into Underweight & Normal (BMI < 25) and Overweight & Obese (BMI ≥25) groups to categorize all participants into two BMI strata. Table 3 shows the association in men between handgrip strength and hypertension risk stratified by these weight groups. No significant associations were observed in the normal/ underweight group. In the overweight/obese group, un- adjusted handgrip strength was not associated with hypertension risk (model 1). After adjusting for age and BMI, the association between handgrip strength and hypertension was significant (P < 0.05, model 2); the OR was 1.288 (95%CI: 1.041, 1.594). After adjusting also for smoking and alcohol status, this association did not changed (P < 0.05, model 3); the OR was 1.311 (95%CI: 1.053, 1.631). In women, similar to the results in The association between BP and handgrip strength in adolescents may be confounded by BMI. Few of these studies adjusted for BMI; such adjustment was suggested, particularly in samples of younger adults [23, 32–34]. The present study revealed that handgrip strength was positively related to BP, the inverse of this relationship before adjusting for BMI. Moreover, Table 2 Association between handgrip strength and hypertension stratified by gender Table 2 Association between handgrip strength and hypertension stratified by gender Men (N = 1587) Women (N = 1960) β S.E. OR (95% CI) P β S.E. OR (95% CI) P Model Model 1: unadjusted −0.083 0.058 0.921 (0.822, 1.032) 0.154 −0.098 0.071 0.907 (0.789, 1.043) 0.169 Model 2: adjusted for age and BMI 0.206 0.087 1.229 (1.036, 1.458) 0.018 0.007 0.117 1.007 (0.800, 1.268) 0.950 Model 3: as Model 2 and smoking and drinking 0.216 0.091 1.241 (1.038, 1.484) 0.018 −0.009 0.122 0.991 (0.781, 1.257) 0.939 BMI body mass index. Handgrip was transformed into age and sex specific SD scores (z-score) Ji et al. Lipids in Health and Disease (2018) 17:86 Page 5 of 7 Table 3 Association in men between handgrip strength and hypertension stratified by two BMI categories Underweight & Normal (N = 563) Overweight & Obese (N = 1292) β S.E. OR (95%CI) P β S.E. Discussion Although the exact mechanisms of handgrip strength induced lipids changes and interaction with hypertension remain unclear, several potential hy- potheses have been proposed. Adiponectin directly stim- ulates the activation of muscle autophagy, which could affect the type and size of muscle cells [41]. The associ- ation between handgrip strength and metabolic syn- drome seemed to be mediated by increasing insulin resistance [42]. Insulin resistance stimulates muscle glu- cose uptake and increases body fat. A higher percentage of body fat generally translates to a higher rate of ap- pearance of free fatty acids in plasma, which could also induce higher blood pressure [44]. Thus, considering the Discussion OR (95%CI) P Model Model 1: unadjusted −0.106 0.127 0.899 (0.701, 1.153) 0.402 −0.047 0.079 0.954 (0.817, 1.115) 0.557 Model 2: adjusted for age and BMI 0.127 0.158 1.136 (0.833, 1.549) 0.421 0.253 0.109 1.288 (1.041, 1.594) < 0.05* Model 3: as Model 2 and smoking and drinking 0.030 0.171 1.030 (0.737, 1.440) 0.863 0.270 0.112 1.311 (1.053, 1.631) < 0.05* BMI body mass index. All participants were categorized into two strata according to BMI value: Underweight & Normal (BMI < 25) and Overweight & Obese (BMI ≥25). Handgrip was transformed into age and sex specific SD scores (z-score). * P < 0.05 numerous factors associated with BP and handgrip strength, the mechanism of the relationship between BP and handgrip strip strength has not yet been fully ex- plained; clarification requires further study. that association did not change after controlling for smoking and drinking status. Similar results were also seen in the association between handgrip strength and risk of hypertension. These findings in- dicated that handgrip strength was positively associ- ated with BP and the risk of hypertension. g g also seen in the association between hand strength and risk of hypertension. These findings dicated that handgrip strength was positively ass ated with BP and the risk of hypertension. Several mechanisms could explain the positive asso tions between handgrip strength and BP. Peripheral cular resistance increases with chronological age du reduced sympatholysis, which results in an elevated s pathetic tone [35, 36], or vascular resistance incre with morphological changes in the arteriolar netw [37]. Another possible explanation is that BP is ass ated with the age-associated loss of lean mass [38, In addition, handgrip strength is probably associ with other risk factors for hypertensions, such as m bolic syndrome or cardiovascular disease biomarkers cluding triglyceride, HDL, LDL, glycohemogl (HbA1c), uric acid, insulin resistance and serum ad nectin levels [40–44]. Although the exact mechanism handgrip strength induced lipids changes and interac with hypertension remain unclear, several potential potheses have been proposed. Adiponectin directly s ulates the activation of muscle autophagy, which c affect the type and size of muscle cells [41]. The ass ation between handgrip strength and metabolic drome seemed to be mediated by increasing ins resistance [42]. Insulin resistance stimulates muscle cose uptake and increases body fat. Conclusion The current study shows that handgrip strength is sig- nificantly associated with lower BP. In contrast to most previous reports, this study found that strong handgrip Table 4 Association in women between handgrip strength and hypertension stratified by two BMI categories Underweight & Normal (N = 636) Overweight & Obese (N = 1323) β S.E. OR (95%CI) P β S.E. OR (95%CI) P Model Model 1: unadjusted −0.163 0.154 0.849 (0.628, 1.150) 0.291 −0.043 0.104 0.958 (0.782, 1.174) 0.679 Model 2: adjusted for age and BMI −0.133 0.211 0.876 (0.579, 1.326) 0.531 0.022 0.152 1.022 (0.759, 1.377) 0.885 Model 3: as Model 2 and smoking and drinking −0.123 0.215 0.884 (0.580, 1.348) 0.568 0.008 0.158 1.008 (0.740, 1.373) 0.961 BMI body mass index. All participants were categorized into two strata according to BMI value: Underweight & Normal (BMI < 25) and Overweight & Obese (BMI ≥25). Handgrip was transformed into age and sex specific SD scores (z-score) Ji et al. Lipids in Health and Disease (2018) 17:86 Page 6 of 7 Page 6 of 7 Page 6 of 7 Received: 24 December 2017 Accepted: 3 April 2018 strength was related to increased DBP, and greater hand- grip strength was associated with greater the risk of hypertension after adjusting for age, BMI, smoking and drinking status, which were closely related to BP. Fur- ther clinical trials and cohort studies from large scale samples are needed to verify these results and explain the possible mechanisms of this relationship. This study may have important relevance for hypertension intervention. Received: 24 December 2017 Accepted: 3 April 2018 Competing interests Competing interests Competing interests The authors declare that they have no competing interests. 15. 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YZ approved the manuscript. YZ is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript. 12. Millar PJ, Levy AS, McGowan CL, McCartney N, MacDonald MJ. Isometric handgrip training lowers blood pressure and increases heart rate complexity in medicated hypertensive patients. Scand J Med Sci Sports. 2013;23:620–6. 12. Millar PJ, Levy AS, McGowan CL, McCartney N, MacDonald MJ. Isometric handgrip training lowers blood pressure and increases heart rate complexity in medicated hypertensive patients. Scand J Med Sci Sports. 2013;23:620–6. 13. Artero EG, Lee DC, Lavie CJ, Espana-Romero V, Sui X, Church TS, Blair SN. Effects of muscular strength on cardiovascular risk factors and prognosis. J Cardiopulm Rehabil Prev. 2012;32:351–8. 13. Artero EG, Lee DC, Lavie CJ, Espana-Romero V, Sui X, Church TS, Blair SN. Effects of muscular strength on cardiovascular risk factors and prognosis. J Cardiopulm Rehabil Prev. 2012;32:351–8. Acknowledgements 7. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, Jones DW, Materson BJ, Oparil S, Wright JT Jr, Roccella EJ. The seventh report of the joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure: the JNC 7 report. JAMA. 2003;289: 2560–72. We thank NHANES for providing the data openly. Thank you Liqiang Zheng, the co-first author, for helping me to design the research and edit the manu- script. We thank the National Key Research and Development Program of China (No. 2017YFC0907400 for Yu-Hong Zhao) for supporting this research. 8. US Department of health and Human service. 2008 physical activity guidelines for Americans; be active, healthy, and happy. Washington, DC: Department of Health and Human Services; 2008. https://stacks.cdc.gov/ view/cdc/23099. Accessed 11 Dec 2008. Funding Thi k This work was supported by the National Key Research and Development Program of China (No. 2017YFC0907400 for Yu-Hong Zhao). 9. Wiley RL, Dunn CL, Cox RH, Hueppchen NA, Scott MS. Isometric exercise training lowers resting blood pressure. Med Sci Sports Exerc. 1992;24: 749–54. Availability of data and materials 10. Badrov MB, Freeman SR, Zokvic MA, Millar PJ, McGowan CL. Isometric exercise training lowers resting blood pressure and improves local brachial artery flow-mediated dilation equally in men and women. Eur J Appl Physiol. 2016;116:1289–96. The datasets used and/or analyzed during the current study are available from the NHANES website. Additional file Additional file 1: Table S1. Association between handgrip strength and SBP adjusted for age and BMI stratified by gender. Table S2. Association between handgrip strength and DBP stratified by gender. (DOCX 15 kb) 4. World Health Organization. The global burden of disease: 2004 update. Geneva. http://www.who.int/healthinfo/global_burden_disease/2004_ report_update/en/. Accessed 1 Jan 2008. 4. World Health Organization. The global burden of disease: 2004 update. Geneva. http://www.who.int/healthinfo/global_burden_disease/2004_ report_update/en/. Accessed 1 Jan 2008. 5. Mancia G, De Backer G, Dominiczak A, Cifkova R, Fagard R, Germano G, Grassi G, Heagerty AM, Kjeldsen SE, Laurent S, et al. 2007 ESH-ESC practice guidelines for the Management of Arterial Hypertension: ESH-ESC task force on the Management of Arterial Hypertension. J Hypertens. 2007;25:1751–62. Publisher’s Note 16. Hess NC, Carlson DJ, Inder JD, Jesulola E, McFarlane JR, Smart NA. Clinically meaningful blood pressure reductions with low intensity isometric handgrip exercise. A randomized trial. Physiol Res. 2016;65:461–8. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 17. Carlson DJ, Inder J, Palanisamy SK, McFarlane JR, Dieberg G, Smart NA. The efficacy of isometric resistance training utilizing handgrip exercise for blood pressure management: a randomized trial. Medicine. 2016;95:e5791. Ethics approval and consent to participate The protocol used in the study was approved by the National Center for Health Statistics (NCHS) according to the Helsinki Declaration. 14. Dong B, Wang Z, Arnold L, Song Y, Wang HJ, Ma J. The association between blood pressure and grip strength in adolescents: does body mass index matter? Hypertens Res. 2016;39:919–25. Author details 1 f 1Department of Clinical Epidemiology, Shengjing Hospital of China Medical University, No. 36, San Hao Street, Shenyang 110004, Liaoning, China. 2Institute of Chronic Disease, Liaoning Provincial Center for Disease Control and Prevention, Shenyang, China. 18. Sayer AA, Syddall HE, Dennison EM, Martin HJ, Phillips DI, Cooper C, Byrne CD. Grip strength and the metabolic syndrome: findings from the Hertfordshire cohort study. QJM. 2007;100:707–13. Page 7 of 7 Ji et al. Lipids in Health and Disease (2018) 17:86 Page 7 of 7 19. Kelley GA, Kelley KS. Isometric handgrip exercise and resting blood pressure: a meta-analysis of randomized controlled trials. J Hypertens. 2010;28:411–8. 20. Ash GI, Taylor BA, Thompson PD, MacDonald HV, Lamberti L, Chen MH, Farinatti P, Kraemer WJ, Panza GA, Zaleski AL, et al. The antihypertensive effects of aerobic versus isometric handgrip resistance exercise. J Hypertens. 2017;35:291–9. 21. Goessler K, Buys R, Cornelissen VA. Low-intensity isometric handgrip exercise has no transient effect on blood pressure in patients with coronary artery disease. J Am Soc Hypertens. 2016;10:633–9. y y 22. Taekema DG, Maier AB, Westendorp RG, de Craen AJ. Higher blood pressure is associated with higher handgrip strength in the oldest old. Am J Hypertens. 2011;24:83–9. 23. Diez-Fernandez A, Sanchez-Lopez M, Gulias-Gonzalez R, Notario-Pacheco B, Canete Garcia-Prieto J, Arias-Palencia N, Martinez-Vizcaino V. BMI as a mediator of the relationship between muscular fitness and cardiometabolic risk in children: a mediation analysis. PLoS One. 2015;10:e0116506. 24. Johnson CL, Dohrmann SM, Burt VL, Mohadjer LK. National health and nutrition examination survey: sample design, 2011-2014. Vital Health Stat 2. 2014;162:1–33. 25. Marquez B, Murillo R. Racial/ethnic differences in weight-loss strategies among US adults: National Health and nutrition examination survey 2007- 2012. J Acad Nutr Diet. 2017;117:923–8. 26. National Institutes of Health, National Heart, Lung, and Blood Institute in cooperation with the National Institute of Diabetes and Digestive and Kidney Diseases. Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults: The Evidence Report. Bethesda (MD): National Heart, Lung, and Blood Institute; 1998. http://www.nhlbi.nih.gov/ guidelines/obesity/ob_gdlns.pdf. 27. Poulter NR, Prabhakaran D, Caulfield M. Hypertension. Lancet. 2015;386:801–12. 28. Pate RR, Daniels S. Institute of Medicine report on fitness measures and health outcomes in youth. JAMA Pediatr. 2013;167:221–2. 29. Sedgwick P. Standardising outcome measures using z scores. BMJ. 2014; 349:g5878. 30. Millar PJ, McGowan CL, Cornelissen VA, Araujo CG, Swaine IL. 42. Kawamoto R, Ninomiya D, Kasai Y, Kusunoki T, Ohtsuka N, Kumagi T, Abe M. Handgrip strength is associated with metabolic syndrome among middle- aged and elderly community-dwelling persons. Clin Exp Hypertens. 2016;38: 245–51. 45. Perna FM, Coa K, Troiano RP, Lawman HG, Wang CY, Li Y, Moser RP, Ciccolo JT, Comstock BA, Kraemer WJ. Muscular grip strength estimates of the U.S. population from the National Health and nutrition examination survey 2011-2012. J Strength Cond Res. 2016;30:867–74. 43. Lee WJ, Peng LN, Chiou ST, Chen LK. Relative handgrip strength is a simple indicator of cardiometabolic risk among middle-aged and older people: a nationwide population-based study in Taiwan. PLoS One. 2016;11:e0160876. Ji et al. Lipids in Health and Disease (2018) 17:86 42. Kawamoto R, Ninomiya D, Kasai Y, Kusunoki T, Ohtsuka N, Kumagi T, Abe M. Handgrip strength is associated with metabolic syndrome among middle- aged and elderly community-dwelling persons. Clin Exp Hypertens. 2016;38: 245–51. 43. Lee WJ, Peng LN, Chiou ST, Chen LK. Relative handgrip strength is a simple indicator of cardiometabolic risk among middle-aged and older people: a nationwide population-based study in Taiwan. PLoS One. 2016;11:e0160876. 44. Wolfe RR. The underappreciated role of muscle in health and disease. Am J Clin Nutr. 2006;84:475–82. 45. Perna FM, Coa K, Troiano RP, Lawman HG, Wang CY, Li Y, Moser RP, Ciccolo JT, Comstock BA, Kraemer WJ. Muscular grip strength estimates of the U.S. population from the National Health and nutrition examination survey 2011-2012. J Strength Cond Res. 2016;30:867–74. 44. Wolfe RR. The underappreciated role of muscle in health and disease. Am J Clin Nutr. 2006;84:475–82. 43. Lee WJ, Peng LN, Chiou ST, Chen LK. Relative handgrip strength is a simple indicator of cardiometabolic risk among middle-aged and older people: a nationwide population-based study in Taiwan. PLoS One. 2016;11:e0160876. 44. Wolfe RR. The underappreciated role of muscle in health and disease. Am J Clin Nutr. 2006;84:475–82. Author details 1 f Evidence for the role of isometric exercise training in reducing blood pressure: potential mechanisms and future directions. Sports Med. 2014;44:345–56. 31. Jin YZ, Yan S, Yuan WX. Effect of isometric handgrip training on resting blood pressure in adults: an updated meta-analysis of randomized controlled trials. J Sports Med Phys Fitness. 2015;57:154-60. 32. Atlantis E, Martin SA, Haren MT, Taylor AW, Wittert GA. Inverse associations between muscle mass, strength, and the metabolic syndrome. Metabolism. 2009;58:1013–22. 33. Stenholm S, Sallinen J, Koster A, Rantanen T, Sainio P, Heliovaara M, Koskinen S. Association between obesity history and hand grip strength in older adults–exploring the roles of inflammation and insulin resistance as mediating factors. J Gerontol A Biol Sci Med Sci. 2011;66:341–8. 34. Peterson MD, Saltarelli WA, Visich PS, Gordon PM. Strength capacity and cardiometabolic risk clustering in adolescents. Pediatrics. 2014;133:e896–903. 35. Hansen J, Sander M, Thomas GD. Metabolic modulation of sympathetic vasoconstriction in exercising skeletal muscle. Acta Physiol Scand. 2000;168: 489–503. 36. Parker BA, Smithmyer SL, Jarvis SS, Ridout SJ, Pawelczyk JA, Proctor DN. Evidence for reduced sympatholysis in leg resistance vasculature of healthy older women. Am J Physiol Heart Circ Physiol. 2007;292:H1148–56. 37. Bearden SE. Effect of aging on the structure and function of skeletal muscle microvascular networks. Microcirculation. 2006;13:279–88. 38. Frontera WR, Hughes VA, Fielding RA, Fiatarone MA, Evans WJ, Roubenoff R. Aging of skeletal muscle: a 12-yr longitudinal study. J Appl Physiol. 2000;88:1321–6. 39. Corish CA, Kennedy NP. Anthropometric measurements from a cross- sectional survey of Irish free-living elderly subjects with smoothed centile curves. Br J Nutr. 2003;89:137–45. 40. Wang Y, Xu D. Effects of aerobic exercise on lipids and lipoproteins. Lipids Health Dis. 2017;16:132. 41. Agostinis-Sobrinho C, Santos R, Moreira C, Abreu S, Lopes L, Oliveira-Santos J, Rosario R, Povoas S, Mota J. Association between serum adiponectin levels and muscular fitness in Portuguese adolescents: LabMed physical activity study. Nutr Metab Cardiovasc Dis. 2016;26:517–24.
https://openalex.org/W1989999381
https://zenodo.org/records/1976390/files/article.pdf
English
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MEDICAL EXAMINERS TO INSURANCE COMPANIES.
Lancet
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public-domain
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S!RS,&mdash;I note with surprise in your leading article on the giving of scholarships for research on the effects of alcohol by the National Temperance League that you heartily commend the movement. What value such a research, con- ducted under the auspices of a strongly biased syndicate and superintended by those who are well known to be prejudiced, can possibly have I for one entirely fail to see. The teetotal attitude towards science in the past has been very much like that of the Caledonian Sabbatarian who, when he was reminded that Christ regarded the Sabbath from a less narrow point of view than he did, replied that he knew it and did not think any the better of Him for so doing. Assyrians I am, Sirs, yours faithfully, Cambridge, Oct. 5th, 1907. W. H. S. JONES. W. H. S. JONES. Assyrians I am, Sirs, yours faithfully, Cambridge, Oct. 5th, 1907. W. H. S. JONES. W. H. S. JONES. 111 such evidence has been given before a committee or an agreed arbitrator, each medical witness shall sign the state- ment of his evidence and may add any necessary explanation or correction," and that statement seems to be used generally for a report or statement made in writing. The question is one of interest and will, no doubt, be raised and decided authoritatively before very long.-ED. L. microscope, and the action of quinine. The ancient doctor- used perforce his eyes and his fingers, which accordingly became so sensitive that symptoms were noticed which seem to have escaped modern observers. A study of these- symptoms might throw light upon some problems of tropical and sub-tropical disease, for a physician like Galen had the- accumulated experience of centuries to help him in forming his conclusions. such evidence has been given before a committee or an agreed arbitrator, each medical witness shall sign the state- ment of his evidence and may add any necessary explanation or correction," and that statement seems to be used generally for a report or statement made in writing. The question is one of interest and will, no doubt, be raised and decided authoritatively before very long.-ED. L. A large portion, perhaps some hundreds of pages, of the- ancient medical writers describe fevers having symptoms suggestive now of typhoid, now of malaria. Of these fevers some may be Malta fever, many are doubtless remittent malaria, but most appear to be cases of typhoid in patients previously infected with the malaria parasite. Instances of this must have been extremely common in highly malarious districts like parts of ancient Greece and Italy. Further study of the Hippocratic treatises, which of course imply much experience previous to Hippocrates, convinces me that- the Greeks of the East were familiar with malaria long before it became endemic in Attica. Will anyone try to discover whether malaria influenced the history of the Assyrians and the Persians ? 7 To the Editors of THE LANCET. SiBS.&mdash;Complaints still continue to reach us from medical men who have been induced to take out policies in insurance companies by promises of appointments as district medical examiners to the companies. The fees they are led to expect do not accrue. Will you, therefore, kindly allow me through your columns to warn medical practitioners against all insurance companies which offer the position of district medical examiner in return for a policy ? policy ? I am, Sirs, yours faithfully, am, Sirs, yours faithfully, HUGH WOODS, s faithfully, HUGH WOODS, am, Sirs, yours faithfully, HUGH WOODS, s faithfully, HUGH WOODS, You lay stress upon the fact that only men who have the confidence of the profession are to be chosen, but the names published are those of well-known teetotalers. As well might one expect to arrive at an impartial judgment regarding, let us say, the question of vivisection by studying the results of a research carried out by the a,ntivivisectionists. These investigators are to test the effects of alcohol upon a man’s capacity for work, but the capacity for work (unless the man’s whole life’s work be reviewed) is not a determining factor in the case for over-activity and an apparently increased power for work may be as much due to an abnormal state as the opposite condition. Reaction-time experiments upon which so much stress has been laid give us no real informa- tion as to the ultimate benefit to be derived from the use of alcohol which in certain quantities temporarily slows or hinders the output of energy, but whether this is good or bad for the individual must be determined by experience alone. I can remember a food faddist who tried to live almost entirely on nuts, boasting that he could walk 40 miles without undue fatigue, but he died young after an illness due to the effects of this regime and no doubt his extreme energy was due to an altogether abnormal physiological condition. , General Secretary, London and Counties Medical Craven-street, Strand, W.C. To the Editors of THE LANCET. SiRs,-I was recently consulted by a patient about what appears to be a new form of occupation or craft palsy due to. driving a motor car. The patient’s complaint was that on waking in the morning his left hand was firmly closed. He could extend the first two fingers with some difficulty but the last two had to be rubbed and forcibly extended before the spasm was overcome. There seems to be some dis- comfort at all times not amounting to pain. The only explanation which is forthcoming is that he has driven his car himself for the past year or two, and on driving with him I noticed that he maintained a constant grip with his left hand, all the levers being operated by the right. I have not come across this condition before, but perhaps some of your readers may have seen it and may be able from experience to say whether there is any cure for it short of giving up driving. It is conceivable that, just as occurs in writer’s palsy, the voluntary control of the muscles might be abrogated without warning, the consequence of which might be a serious accident.&mdash;I am Sirs, yours faithfully, In a recent work under similar auspices, entitled " Alcohol and the Human Body," it is never made clear to the reader whether the use or the abuse of alcohol is under discussion ; it is to be hoped that in this investigation they will at least not confound the man " saturated with alcohol " with the temperate user of the same. ; am, Sirs, yours faithfully, Putney, Oct. 12th, 1907. DONALD F. SHEARER. DONALD F. SHEARER. rs faithfully, DONALD F. SHEARER. DONALD F. SHEARER. a , S s, yours a t u y, Putney, Oct. 12th, 1907. DONALD F. SHEARER. DONALD F. SHEARER. temperate I am, Sirs, yours faithfully, A DISCLAIMER. , , yours faithfully, Old Burlington-street, W., Oct. 13th, 1907. A. W. FULLER. A. W. FULLER. , y y, Old Burlington-street, W., Oct. 13th, 1907. A. W. FULLER. A. W. FULLER. To the Editors of THE LANCET. MALARIA IN ANCIENT GREECE AND ROME. SIRS,-I wrote to you yesterday that through no fault of mine a sensational account of a lecture by me has appeared in the general press. I see to-day extracts from it put together as if there had been an interview. I cannot de scribe how profoundly sorry I am that this discreditable sensation has occurred and wish to tender my humblest- apologies through your medium to all members of my pro- ftssion. I may hold singular individual views but I have no desire to ventilate medical matters through the lay press. I Sirs respectfully MEDICAL EXAMINERS TO INSURANCE COMPANIES. g When science has appeared to support total abstinence well and good, but if not then so much the worse for science. A hundred fallacies are apt to creep into such an attempt as this to reduce to the limits of an exact science what is essentially an inexact one. It has been stated that frog and pigeon physiology has been the bane of clinical medicine, so likewise are these pseudo-scientific investigations instituted not really with a view of arriving at an impartial judgment but to bolster upaparticularfdd, and At the outset this over- whelming difficulty presents itself that that which is harmful to one person may be indifferent in its effects upon another or even beneficial to him." , y y, Old Burlington-street, W., Oct. 13th, 1907. A. W. FULLER. A. W. FULLER. 1115 1115 To the Editors of THE LANCET. SIRS,-The little book which has just appeared on the above subject is necessarily limited in scope, as ’its true object is to show how malaria affects character and some- times even influences the destiny of nations. But there are a few other points which are, I think, worthy of attention at tbehands of medical men. A modern physician, in diagnos- es a case ot malaria, relies upon his thermometer, his respectfully, BERNARD HOLLANDER, BERNARD HOLLANDER. , , you s respectfully, Welbeck-street, W., Oct. 10th, 1907. BERNARD HOLLANDER, BERNARD HOLLANDER. , , you s respectfully, Welbeck-street, W., Oct. 10th, 1907. BERNARD HOLLANDER, BERNARD HOLLANDER.
https://openalex.org/W4285088178
https://scipost.org/10.21468/SciPostPhysProc.8.077/pdf
English
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Can $\overline{MS}$ parton distributions be negative?
SciPost physics proceedings
2,022
cc-by
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⋆felix.hekhorn@unimi.it Proceedings for the XXVIII International Workshop on Deep-Inelastic Scattering and Related Subjects, Stony Brook University, New York, USA, 12-16 April 2021 doi:10.21468/SciPostPhysProc.8 Abstract We review our recent paper, where we proved Parton distributions functions (PDFs) to be positive at NLO in the MS factorization scheme. We present some additional material that was useful in completing the steps for the actual proof, but we won’t attempt to report on the full proof, but referring the reader to the paper itself. Copyright A. Candido et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 25-08-2021 Accepted 22-03-2022 Published 12-07-2022 Check for updates doi:10.21468/SciPostPhysProc.8.077 1 Introduction In our recent paper [1] we tackled the long-standing problem of positive parton distribution functions (PDFs) head-on and finally gave a concise proof that, indeed, PDFs are perturbatively positive at NLO. This has some immediate consequences for the actual fitting procedure as it affects the parametrizations of the PDFs. The rest of the paper is structured as follows. We add some material about the proof and a sketch of it (Section 2), and we show the impact on an actual fit (Section 3). Alessandro Candido, Stefano Forte and Felix Hekhorn⋆ Tif Lab, Dipartimento di Fisica, Università di Milano and INFN, Sezione di Milano, Via Celoria 16, I-20133 Milano, Italy Tif Lab, Dipartimento di Fisica, Università di Milano and INFN, Sezione di Milano, Via Celoria 16, I-20133 Milano, Italy SciPost Phys. Proc. 8, 077 (2022) 2 About the Proof In the following we quickly highlight the necessary step in order to prove the NLO statement. First, we give a review of relevant coefficient functions of DIS and hadronic processes (as prototypes of one and two-partons-involved processes) identifying the collinear substraction as the source of negativity. Next, we present a possible physical scheme, i.e. a factorization scheme in which the coefficient function for some candidate processes are reabsorbed at all orders in the PDFs (like DIS scheme), in which PDFs are granted to be positive because they coincide with actual observables. Then, we construct a still positive scheme POS, resembling MS, just modifying the MS coefficient functions to make them positive as well. We use the 077.1 SciPost Phys. Proc. 8, 077 (2022) explicit positivity of coefficient functions, and exploit perturbativity (and taking care of the large x logarithms) to prove that at NLO PDFs remain positive. Finally, we compare the MS scheme to the constructed positive scheme POS, in order to prove the former to be positive as well. explicit positivity of coefficient functions, and exploit perturbativity (and taking care of the large x logarithms) to prove that at NLO PDFs remain positive. Finally, we compare the MS scheme to the constructed positive scheme POS, in order to prove the former to be positive as well. Remark We would like to explicitly comment here that we are not interested in fitting any of the intermediate schemes (e.g. POS), they are just artifacts used as intermediate steps to prove MS to be positive. 1There is a lower bound on a, in order to get that positive domain in N-space, such that it goes to 1 as b → It is easy to compute but not relevant for the argument. 2.1 Positivity in N-space and x-space In [1, Section 3.1] we have shown that the positivity of the coefficient functions is a necessar and (perturbatively) sufficient condition for PDF positivity. During this process, we started working in Mellin space (or N-space) since convolutions are mapped onto ordinary products. The transformation also turns distributions into regular functions. We wanted to exploit the fact that we can control the sign of a multiplication and hence just use the inverse transformation to prove positivity. We were mainly interested in the large x region (x →1), since it is the only region were MS coefficients are actually negative, and it is well known that large-N region asymptotically corresponds to the large x region. Nevertheless, we found remarkably that this is not enough for positivity: a sufficiently positive function in N-space might map onto a function which is negative in the large x. Explicit example An explicit example can be shown by making use of a simple polynomial in x-space, and performing the Mellin transform analytically: f (x) = x(1 −x)  x −7 8 ‹ x −3 4 ‹ (1) F(N) = M[f (x)](N) = N 2 −17N + 108 32(N + 1)(N + 2)(N + 3)(N + 4) . (2) (1) (2) In this case the N-space function is positive in the range: In this case the N-space function is positive in the range: S+ = (−∞,−4) ∪(−3,−2) ∪(−1,∞) (3) (3) and thus all N > 1. Nevertheless, the x-space function is not positive in the full interval [0,1], but is only positive in:  ‹  ‹  0, 3 4 ‹ ∪ 7 8,1 ‹ . (4) (4) The former example is a specific case of the following functional form: f (x) = x(1 −x)(x −a)(x −b) (5) (5) that of course is negative in the interval (a, b). Moreover ∀b > 5 −2 p 6 ∼0.101 ∃a : ∀N ∈S+(a, b) : F(N) > 0, (6) (6) where S+(a, b) a set defined to S+ 1. Than the function might be negative at arbitrarily large x and still be positive for large N. where S+(a, b) a set defined to S+ 1. Than the function might be negative at arbitrarily large x and still be positive for large N. This required us to develop the whole argument directly in x-space, taking care of distri- butions and convolutions directly. 2.1 Positivity in N-space and x-space 1There is a lower bound on a, in order to get that positive domain in N-space, such that it goes to 1 as b →1. It is easy to compute but not relevant for the argument. 077.2 SciPost Phys. Proc. 8, 077 (2022) 0.2 0.4 0.6 0.8 1.0 x 0.01 0.02 0.03 0.04 0.05 0.06 f(x) 10 100 1000 N 10- 7 10- 6 10- 5 10- 4 0.001 0.010 F(N) 0.80 0.90 x 0.0010 f(x) Figure 1: Plotting our example function in x-space (left-hand side) and N-space (right-hand side) SciPost Phys. Proc. 8, 077 (2022) 10 100 1000 N 7 6 5 4 1 0 F(N) 0.2 0.4 0.6 0.8 1.0 x 0.01 0.02 0.03 0.04 0.05 0.06 f(x) 0 0 0.80 0.90 x 0.0010 f(x) Figure 1: Plotting our example function in x-space (left-hand side) and N-space (right-hand side) 2.2 Considering the MS Scheme Following [1, Section 3.2], we consider the scheme change between the POS scheme and the actual MS scheme. In Mellin space we can denote the scheme change by a linear transforma- tion: 1 Following [1, Section 3.2], we consider the scheme change between the POS scheme and the actual MS scheme. In Mellin space we can denote the scheme change by a linear transforma- tion: f MS(Q2) = h I + αs 2πKPOSi−1 f POS(Q2), (7) (7) here the PDFs f (Q2) are understood as a vector dimension over flavor space and the actual heme transformation KPOS as a matrix in flavor space. While it was not clear to us from the beginning that the MS scheme actually is a positive scheme, we were trying to construct a scheme change that explicitly enforces or breaks pos- itivity. To that end we came up with the idea of a graphical representation for the scheme change as shown in Fig. 2. As an explicit example of such a plot, we plot here the third singlet moment against the third gluon moment and indicate by the arrows the effect that the scheme change has. For reference we show the actual configuration that is realized by the central member of the NNPDF3.1 PDF set [2] by the red square. As can be seen, acutally all arrows are pointing into the first quadrant which means the scheme change from the POS to the MS scheme will make any configuration more positive. 3 Impact onto NNPDF 4.0 The strict positivity of PDFs in the flavor basis is implemented in the forthcoming NNPDF4.0 release [3] by the use of Lagrange mulipliers. In Fig. 3 we demonstrate the impact of impos- ing this constraint by two exemplary PDFs. Indeed, as expected, PDFs can and will become negative in the large x region and with the newly imposed constraint we avoid this unphysical region. Furthermore, we even see a reduced error band when imposing positivity. 4 Conclusion We observe that posivitiy in momentum fraction space space (x-space) and Mellin moment space (N-space) do not coincide. Furthermore, we give a graphical representation of the scheme change that is used in the actual proof and which demonstrates the actual positiv- ity in a more visual sense. 077.3 077.3 SciPost Phys. Proc. 8, 077 (2022) 0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10 g N = 3.000 POS scheme with NNPDF31_nlo_as_0118 at Q² = 100.0 GeV² Figure 2: Graphical representation of the scheme change between the POS and MS scheme: we plot the singlet moment against the gluon moment at N = 3. The ar- rows indicate the shift of moments that the scheme change imposes. The red square denotes the reference value for the NNPDF3.1 PDF set [2] 0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10 g N = 3.000 POS scheme with NNPDF31_nlo_as_0118 at Q² = 100.0 GeV² POS scheme with NNPDF31_nlo_as_0118 at Q² = 100.0 GeV² POS scheme with NNPDF31_nlo_as_0118 at Q² = 100.0 GeV² Figure 2: Graphical representation of the scheme change between the POS and MS scheme: we plot the singlet moment against the gluon moment at N = 3. The ar- rows indicate the shift of moments that the scheme change imposes. The red square denotes the reference value for the NNPDF3.1 PDF set [2] (b) ¯s(x) distribution (a) ¯d(x) distribution (b) ¯s(x) distribution Figure 3: Impact of imposing positivity on two selected PDF distributions at µ = 100GeV (a) ¯d(x) distribution Figure 3: Impact of imposing positivity on two selected PDF distributions at µ = 100GeV The positivity of MS PDFs leads to an improved fitting methodology due to the reduced function space that has to be explored. This has a sizable impact on the fitting procedure, but it remains unclear for the moment whether there exists a yet stricter condition that can be imposed to enforce positive, physical cross sections. However, we want to point out again that positivity is neither a necessary nor a sufficient condition for obtaining physical PDFs: it is not necessary because the convolution a partially negative PDF with a sufficiently good coefficient functions might still yield positive, physical cross sections. Yet it is not a sufficient condition because due to numerical or methodological inefficientcies (e.g. the use of perturbation theory itself) also the convolution of strictly positive PDFs with sufficiently bad coefficient functions might yield negative, unphysical cross sections. Acknowledgements We acknowledge the members of the NNPDF and N3PDF collaborations for careful reading and discussions. 077.4 077.4 SciPost Phys. Proc. 8, 077 (2022) SciPost Phys. Proc. 8, 077 (2022) SciPost Phys. Proc. 8, 077 (2022) Funding information This work is supported by the European Research Council under the European Union’s Horizon 2020 research and innovation Programme (grant agreement n.740006). References [1] A. Candido, S. Forte and F. Hekhorn, Can MS parton distributions be negative?, J. High Energy Phys. 11, 129 (2020), doi:10.1007/JHEP11(2020)129. [2] R. D. Ball et al., Parton distributions from high-precision collider data, Eur. Phys. J. C 77, 663 (2017), doi:10.1140/epjc/s10052-017-5199-5. [3] E. R. Nocera, The NNPDF4.0 global PDF determination and related studies, this proceed- ings. [3] E. R. Nocera, The NNPDF4.0 global PDF determination and related studies, this proceed- ings. 077.5
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String periods in the order-preserving model
Information and computation
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String Periods in the Order-Preserving Model Garance Gourdel ENS Paris-Saclay, Cachan, France garance.gourdel@ens-paris-saclay.fr Tomasz Kociumaka University of Warsaw, Warsaw, Poland kociumaka@mimuw.edu.pl Jakub Radoszewski University of Warsaw, Warsaw, Poland jrad@mimuw.edu.pl Jakub Radoszewski University of Warsaw, Warsaw, Poland jrad@mimuw.edu.pl Wojciech Rytter University of Warsaw, Warsaw, Poland rytter@mimuw.edu.pl Wojciech Rytter University of Warsaw, Warsaw, Poland rytter@mimuw.edu.pl Arseny Shur Ural Federal University, Ekaterinburg, Russia arseny.shur@urfu.ru Arseny Shur Ural Federal University, Ekaterinburg, Russia arseny.shur@urfu.ru Tomasz Waleń University of Warsaw, Warsaw, Poland walen@mimuw.edu.pl © Garance Gourdel, Tomasz Kociumaka, Jakub Radoszewski, Wojciech Rytter, Arseny Shur, and Tomasz Waleń; licensed under Creative Commons License CC-BY 35th Symposium on Theoretical Aspects of Computer Science (STACS 2018). Editors: Rolf Niedermeier and Brigitte Vallée; Article No. 38; pp. 38:1–38:16 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany ▶Example 1. 5 2 7 5 1 3 10 3 5 ≈6 4 7 6 3 5 9 5 6. ▶Example 1. 5 2 7 5 1 3 10 3 5 ≈6 4 7 6 3 5 9 5 6. Order-equivalence is a special case of a substring consistent equivalence relation (SCER) that was defined in [37]. For a string S of length n, we can create a new string X of length n such that X[i] is equal to the number of distinct symbols in S that are not greater than S[i]. The string X is called the shape of S and is denoted by shape(S). It is easy to observe that two strings S, T are order-equivalent if and only if they have the same shape. ▶Example 2. shape(5 2 7 5 1 3 10 3 5) = shape(6 4 7 6 3 5 9 5 6) = 4 2 5 4 1 3 6 3 4. ▶Example 2. shape(5 2 7 5 1 3 10 3 5) = shape(6 4 7 6 3 5 9 5 6) = 4 2 5 4 1 3 6 3 4. ▶Example 2. shape(5 2 7 5 1 3 10 3 5) = shape(6 4 7 6 3 5 9 5 6) = 4 2 5 4 1 3 6 3 4. Periods in the op-model. We consider several notions of periodicity in the op-model, illustrated by Fig. 1. We say that a string S has a (general) op-period p with shift s ∈J0..p−1K if and only if p < |S| and S is a factor of a string V1V2 · · · Vk such that: |V1| = · · · = |Vk| = p, V1 ≈· · · ≈Vk, and S[s + 1..|S|] is a prefix of V2 · · · Vk. The shape of the op-period is shape(V1). One op-period p can have several shifts; to avoid ambiguity, we sometimes denote the op-period as (p, s). We define Shiftsp as the set of all shifts of the op-period p. An op-period p is called initial if 0 ∈Shiftsp, full if it is initial and p divides |S|, and sliding if Shiftsp = J0..p−1K. Initial and sliding op-periods are particular cases of block-based and sliding-window-based periods for SCER, both of which were introduced in [37]. Models of periodicity. In the standard model, a string S of length n has a period p iff S[i] = S[i + p] for all i = 1, . . . , n −p. String Periods in the Order-Preserving Model String Periods in the Order-Preserving Model Abstract The order-preserving model (op-model, in short) was introduced quite recently but has already attracted significant attention because of its applications in data analysis. We introduce several types of periods in this setting (op-periods). Then we give algorithms to compute these periods in time O(n), O(n log log n), O(n log2 log n/ log log log n), O(n log n) depending on the type of periodicity. In the most general variant the number of different periods can be as big as Ω(n2), and a compact representation is needed. Our algorithms require novel combinatorial insight into the properties of such periods. 2012 ACM Subject Classification Theory of computation →Pattern matching Keywords and phrases Order-preserving Pattern Matching, Period, Efficient Algorithm Keywords and phrases Order-preserving Pattern Matching, Period, Efficient Algorithm Digital Object Identifier 10.4230/LIPIcs.STACS.2018.38 Related Version A full version of the paper is available at https://arxiv.org/abs/1801.01404, [29]. Funding A part of this work was done during Garance Gourdel’s internship at University of Warsaw, Poland. Tomasz Kociumaka, Jakub Radoszewski, Wojciech Rytter and Tomasz Waleń were supported by the Polish National Science Center, grant no. 2014/13/B/ST6/00770. Acknowledgements A part of this work was done during the workshop “StringMasters in Warsaw 2017” that was sponsored by the Warsaw Center of Mathematics and Computer Science. The authors thank the participants of the workshop, especially Hideo Bannai and Shunsuke Inenaga, for helpful discussions. © Garance Gourdel, Tomasz Kociumaka, Jakub Radoszewski, Wojciech Rytter, Arseny Shur, and Tomasz Waleń; licensed under Creative Commons License CC-BY 35th Symposium on Theoretical Aspects of Computer Science (STACS 2018). Editors: Rolf Niedermeier and Brigitte Vallée; Article No. 38; pp. 38:1–38:16 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany 38:2 1 Introduction Study of strings in the order-preserving model (op-model, in short) is a part of the so-called non-standard stringology. It is focused on pattern matching and repetition discovery problems in the shapes of number sequences. Here the shape of a sequence is given by the relative order of its elements. The applications of the op-model include finding trends in time series which appear naturally when considering e.g. the stock market or melody matching of two musical scores; see [32]. In such problems periodicity plays a crucial role. One of motivations is given by the following scenario. Consider a sequence D of numbers that models a time series which is known to repeat the same shape every fixed period of time. For example, this could be certain stock market data or statistics data from a social network that is strongly dependent on the day of the week, i.e., repeats the same shape every consecutive week. Our goal is, given a fragment S of the sequence D, to discover such repeating shapes, called here op-periods, in S. We also consider some special cases of this setting. If the beginning of the sequence S is synchronized with the beginning of the repeating shape in D, we refer to the repeating shape as to an initial op-period. If the synchronization takes place also at the end of the sequence, we call the shape a full op-period. Finally, we also consider sliding op-periods that describe the case when every factor of the sequence D repeats the same shape every fixed period of time. Order-preserving model. Let Ja..bK denote the set {a, . . . , b}. We say that two strings X = X[1] . . . X[n] and Y = Y [1] . . . Y [n] over an integer alphabet are order-equivalent (equivalent in short), written X ≈Y , iff∀i,j∈J1..nK X[i] < X[j] ⇔Y [i] < Y [j]. Order-preserving model. Let Ja..bK denote the set {a, . . . , b}. We say that two strings X = X[1] . . . X[n] and Y = Y [1] . . . Y [n] over an integer alphabet are order-equivalent (equivalent in short), written X ≈Y , iff∀i,j∈J1..nK X[i] < X[j] ⇔Y [i] < Y [j]. ▶Example 1. 5 2 7 5 1 3 10 3 5 ≈6 4 7 6 3 5 9 5 6. ▶Example 1. 5 2 7 5 1 3 10 3 5 ≈6 4 7 6 3 5 9 5 6. The famous periodicity lemma of Fine and Wilf [26] states that a “long enough” string with periods p and q has also the period gcd(p, q). The 38:3 G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń y 0 0 3 2 1 1 3 2 1 1 4 3 1 1 2 5 1 1 3 4 1 1 2 4 Figure 1 The string to the left has op-period 4 with three shifts: Shifts4 = J0..0K ∪J2..3K. Due to the shift 0, the string has an initial—therefore, a full—op-period 4. The string to the right has op-period 4 with all four shifts: Shifts4 = J0..3K. In particular, 4 is a sliding op-period of the string. Notice that both strings (of length n = 12) have (general, sliding) periods 4, but none of them has the order-border (in the sense of [36]) of length n −4. Figure 1 The string to the left has op-period 4 with three shifts: Shifts4 = J0..0K ∪J2..3K. Due to the shift 0, the string has an initial—therefore, a full—op-period 4. The string to the right has op-period 4 with all four shifts: Shifts4 = J0..3K. In particular, 4 is a sliding op-period of the string. Notice that both strings (of length n = 12) have (general, sliding) periods 4, but none of them has the order-border (in the sense of [36]) of length n −4. exact bound of being “long enough” is p + q −gcd(p, q). This result was generalized to arbitrary number of periods [9, 31, 40]. Periods were also considered in a number of non-standard models. Partial words, which are strings with don’t care symbols, possess quite interesting Fine–Wilf type properties, including probabilistic ones; see [4, 5, 6, 38, 39, 30]. In Section 2, we make use of periodicity graphs introduced in [38, 39]. In the abelian (jumbled) model, a version of the periodicity lemma was shown in [15] and extended in [7]. Also, algorithms for computing three types of periods analogous to full, initial, and general op-periods were designed [19, 24, 25, 33, 34, 35]. In the computation of full and initial op-periods we use some number-theoretic tools initially developed in [33, 34]. Remarkably, the fastest known algorithm for computing general periods in the abelian model has essentially quadratic time complexity [19, 35], whereas for the general op-periods we design a much more efficient solution. String Periods in the Order-Preserving Model length m in optimal, O(m) time (or O(m+Occ) time if we are to report all Occ occurrences). The index can be constructed in O(n log log n) expected time or O(n log2 log n/ log log log n) worst-case time. We use the index itself and some of its applications from [18]. Other developments in this area include a multiple-pattern matching algorithm for the op-model [32], an approximate version of op-pattern matching [28], compressed index constructions [12, 21], a small-space index for op-pattern matching that supports only short queries [27], and a number of practical approaches [8, 10, 11, 13, 23]. Our results. We give algorithms to compute: Our results. We give algorithms to compute: all full op-periods in O(n) time; the smallest non-trivial initial op-period in O(n) time; all initial op-periods in O(n log log n) time; all sliding op-periods in O(n log log n) expected time or O(n log2 log n/ log log log n) worst- case time (and linear space); all sliding op-periods in O(n log log n) expected time or O(n log2 log n/ log log log n) worst- case time (and linear space); all general op-periods with all their shifts (compactly represented) in O(n log n) time and space. The output is the family of sets Shiftsp represented as unions of disjoint intervals. The total number of inter als o er all p is O(n log n) all general op-periods with all their shifts (compactly represented) in O(n log n) time and space. The output is the family of sets Shiftsp represented as unions of disjoint intervals. The total number of intervals, over all p, is O(n log n). In the combinatorial part, we characterize the Fine–Wilf periodicity property (aka interaction property) in the op-model in the case of coprime periods. This result is at the core of the linear-time algorithm for the smallest initial op-period. Structure of the paper. Combinatorial foundations of our study are given in Section 2. Then in Section 3 we recall known algorithms and data structures for the op-model and develop further algorithmic tools. The remaining sections are devoted to computation of the respective types of op-periods: full and initial op-periods in Section 4, the smallest non-trivial initial op-period in Section 5, all (general) op-periods in Section 6, and sliding op-periods in Section 7. String Periods in the Order-Preserving Model Some proofs have been omitted due to space constraints; they can be found in the preprint [29]. 2 Fine–Wilf Property for Op-Periods The following result was shown as Theorem 2 in [37]. Note that if p and q are coprime, then the conclusion is void, as every string has the op-period 1. ▶Theorem 3 ([37]). Let p > q > 1 and d = gcd(p, q). If a string S of length n ≥p + q −d has initial op-periods p and q, it has initial op-period d. Moreover, if S has length n ≥p+q−1 and sliding op-periods p and q, it has sliding op-period d. ▶Theorem 3 ([37]). Let p > q > 1 and d = gcd(p, q). If a string S of length n ≥p + q −d has initial op-periods p and q, it has initial op-period d. Moreover, if S has length n ≥p+q−1 and sliding op-periods p and q, it has sliding op-period d. The aim of this section is to show a periodicity lemma in the case that gcd(p, q) = 1. ▶Example 1. 5 2 7 5 1 3 10 3 5 ≈6 4 7 6 3 5 9 5 6. A version of the periodicity lemma for the parameterized model was proposed in [2]. Op-periods were first considered in [37] where initial and sliding op-periods were introduced and direct generalizations of the Fine–Wilf property to these kinds of op-periods were developed. A few distinctions between the op-periods and periods in other models should be mentioned. First, “to have a period 1” becomes a trivial property in the op-model. Second, all standard periods of a string have the “sliding” property; the first string in Fig. 1 demonstrates that this is not true for op-periods. The last distinction concerns borders. A standard period p in a string S of length n corresponds to a border of S of length n −p, which is both a prefix and a suffix of S. In the order-preserving setting, an analogue of a border is an op-border, that is, a prefix that is equivalent to the suffix of the same length. Op-borders have properties similar to standard borders and can be computed in O(n) time [36]. However, it is no longer the case that a (general, initial, full, or sliding) op-period must correspond to an op-border; see [37]. Previous algorithmic study of the op-model. The notion of order-equivalence was intro- duced in [32, 36]. (However, note the related combinatorial studies, originated in [22], on containment/avoidance of shapes in permutations.) Both [32, 36] studied pattern matching in the op-model (op-pattern matching) that consists in identifying all consecutive factors of a text that are order-equivalent to a given pattern. We assume that the alphabet is integer and, as usual, that it is polynomially bounded with respect to the length of the string, which means that a string can be sorted in linear time (cf. [16]). Under this assumption, for a text of length n and a pattern of length m, [32] solve the op-pattern matching problem in O(n + m log m) time and [36] solve it in O(n + m) time. Other op-pattern matching algorithms were presented in [3, 14]. An index for op-pattern matching based on the suffix tree was developed in [18]. For a text of length n it uses O(n) space and answers op-pattern matching queries for a pattern of STACS 2018 38:4 String Periods in the Order-Preserving Model 2.1 Preliminary Notation For a string S of length n, by S[i] (for 1 ≤i ≤n) we denote the ith letter of S and by S[i..j] we denote a factor of S equal to S[i] . . . S[j]. If i > j, S[i..j] denotes the empty string ε. A string which is strictly increasing, strictly decreasing, or constant, is called strictly monotone. A strictly monotone op-period of S is an op-period with a strictly monotone shape. Such an op-period is called increasing (decreasing, constant) if so is its shape. Clearly, any divisor of a strictly monotone op-period is a strictly monotone op-period as well. A string S is 2-monotone if S = S1S2, where S1, S2 are strictly monotone in the same direction. 38:5 38:5 G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń S = q q q p p p i j k Figure 2 Op-periods (p, i) and (q, j) synchronized at position k. Figure 2 Op-periods (p, i) and (q, j) synchronized at position k. Below we assume that n > p > q > 1. Let a string S = S[1..n] have op-periods (p, i) and (q, j). If there exists a number k ∈J1..n −1K such that k mod p = i and k mod q = j, we say that these op-periods are synchronized and k is a synchronization point (see Fig. 2). ▶Remark. The proof of Theorem 3 can be easily adapted to prove the following. ▶Theorem 4. Let p > q > 1 and d = gcd(p, q). If op-periods p and q of a string S of length n ≥p + q −1 are synchronized, then S has op-period d, synchronized with them. ▶Theorem 4. Let p > q > 1 and d = gcd(p, q). If op-periods p and q of a string S of length n ≥p + q −1 are synchronized, then S has op-period d, synchronized with them. ▶Observation 5. ▶Observation 5. (1) A string is strictly monotone iffits trace is a unary string. (2) If S has an op-period p with shift i, then trace(S) “almost” has a period p, namely, trace(S)[j] = trace(S)[k] for any j, k ∈J1..n −1K such that j = k (mod p) and j ̸= i (mod p). (This is because both trace(S)[j] and trace(S)[k] equal the sign of the difference between the same positions of the shape of the op-period of S.) (2) If S has an op-period p with shift i, then trace(S) “almost” has a period p, namely, trace(S)[j] = trace(S)[k] for any j, k ∈J1..n −1K such that j = k (mod p) and j ̸= i (mod p). (This is because both trace(S)[j] and trace(S)[k] equal the sign of the difference between the same positions of the shape of the op-period of S.) ▶Example 6. Consider the string 7 5 8 1 4 6 2 4 5. It has an op-period (3, 1) with shape 2 3 1. The trace of this string is: - + - + + - + + The positions giving the remainder 1 modulo 3 are shown in gray; the sequence of the remaining positions is periodic. It turns out that the existence of two coprime op-periods makes a string “almost” strictly monotone. One can use periodicity graphs [38, 39] to show the following result. ▶Theorem 7. Let S be a string of length n that has coprime op-periods p and q with shifts i and j, respectively, such that n > p > q > 1. Then: ▶Theorem 7. Let S be a string of length n that has coprime op-periods p and q with shifts i and j, respectively, such that n > p > q > 1. 2.2 Periodicity Theorem For Coprime Periods For a string S, by trace(S) we denote a string X of length |S| −1 over the alphabet {+, 0, -} such that: X[i] =    + if S[i] < S[i + 1] 0 if S[i] = S[i + 1] - if S[i] > S[i + 1]. X[i] =    + if S[i] < S[i + 1] 0 if S[i] = S[i + 1] - if S[i] > S[i + 1]. X[i] =    + if S[i] < S[i + 1] 0 if S[i] = S[i + 1] - if S[i] > S[i + 1]. 3 Algorithmic Toolbox for Op-Model For a string S of length n, we introduce a table op-PREF[1..n] such that op-PREF[i] is the length of the longest prefix of S[i..n] that is equivalent to a prefix of S. It is a direct analogue of the PREF array used in standard string matching (see [20]) and can be computed similarly in O(n) time using one of the standard encodings for the op-model that were used in [14, 18, 36]. ▶Lemma 8. For a string of length n, the op-PREF table can be computed in O(n) time. ▶Lemma 8. For a string of length n, the op-PREF table can be computed in O(n) tim Let us mention an application of the op-PREF table that is used further in the algorithms. We denote by op-LPPp(S) (“longest op-periodic prefix”) the length of the longest prefix of a string S having p as an initial op-period. ▶Lemma 9. For a string S of length n, op-LPPp(S) for a given p can be computed in O(op-LPPp(S)/p + 1) time after O(n)-time preprocessing. ▶Lemma 9. For a string S of length n, op-LPPp(S) for a given p can be computed in O(op-LPPp(S)/p + 1) time after O(n)-time preprocessing. Proof. We start by computing the op-PREF table for S in O(n) time. We assume that op-PREF[n + 1] = 0. To compute op-LPPp(S), we iterate over positions i = p + 1, 2p + 1, . . . and for each of them check if op-PREF[i] ≥p. If i0 is the first position for which this condition is not satisfied (possibly because i0 > n−p+1), we have op-LPPp(S) = i0 +op-PREF[i0]−1. Clearly, this procedure works in the desired time complexity. ◀ For a string S, we define a longest common extension query op-LCP(i, j) in the order- preserving model as the maximum k ≥0 such that S[i..i+k−1] ≈S[j..j+k−1]. Symmetrically, op-LCS(i, j) is the maximum k ≥0 such that S[i −k + 1..i] ≈S[j −k + 1..j]. Similarly as in the standard model [17], LCP-queries in the op-model can be answered using lowest common ancestor (LCA) queries in the op-suffix tree; see the following lemma. ▶Lemma 10. For a string of length n, after preprocessing in O(n log log n) expected time or in O(n log2 log n/ log log log n) worst-case time one can answer op-LCP-queries in O(1) time. 3 Algorithmic Toolbox for Op-Model The factor S[i..i+2p−1] is called an order-preserving square (op-square) iffS[i..i+p−1] ≈ S[i + p..i + 2p −1]. For a string S of length n, we define the set op-Squaresp = {i ∈J1..n −2p + 1K : S[i..i + 2p −1] is an op-square}. Op-squares were first defined in [18] where an algorithm computing all the sets op-Squaresp for a string of length n in O(n log n + P p |op-Squaresp|) time was shown. p We say that an op-square S[i..i + 2p −1] is right shiftable if S[i + 1..i + 2p] is an op-square and right non-shiftable otherwise. Similarly, we say that the op-square is left shiftable if S[i −1..i + 2p −2] is an op-square and left non-shiftable otherwise. Using the approach of [18], one can show the following lemma. ▶Lemma 11. All the (left and right) non-shiftable op-squares in a string of length n can be computed in O(n log n) time. ▶Observation 5. Then: (a) if n > pq, then S has a strictly monotone op-period pq; (b) if 2p < n ≤pq and the op-periods are synchronized, then S is 2-monotone; (c) if p+q < n ≤2p and the op-periods are synchronized, then (q, j) is a strictly monotone op-period of S; (d) if n > max{2p, p+2q} and the op-periods are not synchronized, then S is strictly mono- tone; (e) if n > 2p, the op-periods are not synchronized, and p is initial, then S is strictly monotone; (f) if p+q < n ≤2p and p is initial, then (q, j) is a strictly monotone op-period of S. STACS 2018 38:6 String Periods in the Order-Preserving Model 4 Computing All Full and Initial Op-Periods For a string S of length n, we define op-PREF′[i] for i = 0, . . . , n as: if op-PREF[i + 1] = n −i if op-PREF[i + 1] = n −i op-PREF′[i] =  n if op-PREF[i + 1] = n − op-PREF[i + 1] otherwise. 38:7 G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń Algorithm 1: Computing All Initial Op-Periods of S. 1 T := op-PREF′; 2 for j := n down to 2 do 3 foreach prime divisor q of j do 4 P[j/q] := min(P[j/q], P[j]); 5 for p := 1 to n do 6 if P[p] ≥p then p is an initial op-period; Algorithm 1: Computing All Initial Op-Periods of S. 1 T := op-PREF′; 2 for j := n down to 2 do 3 foreach prime divisor q of j do 4 P[j/q] := min(P[j/q], P[j]); 5 for p := 1 to n do 6 if P[p] ≥p then p is an initial op-period; Algorithm 1: Computing All Initial Op-Periods of S. Here we assume that op-PREF[n + 1] = 0. In the computation of full and initial op-periods we heavily rely on this table according to the following obvious observation. ▶Observation 12. p is an initial op-period of a string S of length n if and only if op-PREF′[ip] ≥p for all i = 1, . . . , ⌊n/p⌋. ▶Theorem 14. All initial op-periods of a string of length n can be computed in O(n log log n) time. ▶Theorem 14. All initial op-periods of a string of length n can be computed in O(n log log n) time. Proof. By Lemma 8, the op-PREF table for the string—hence, the op-PREF′ table—can be computed in O(n) time. Then we use Algorithm 1. Each prime number q ≤n has at most n q multiples below n. Therefore, the complexity of Eratosthenes’s sieve and the number of updates on the table T in the algorithm is P q∈Primes,q≤n n q = O(n log log n); see [1]. ◀ 4.1 Computing Initial Op-Periods Let us introduce an auxiliary array P[0..n] such that: P[p] = min{op-PREF′[ip] : i = 1, . . . , ⌊n/p⌋}. Straight from Observation 12 we have: P[p] = min{op-PREF′[ip] : i = 1, . . . , ⌊n/p⌋}. Straight from Observation 12 we have: ▶Observation 13. p is an initial period of S if and only if P[p] ≥p. The table T could be computed straight from definition in O(n log n) time. We improve this complexity to O(n log log n) by employing Eratosthenes’s sieve. The sieve computes, in particular, for each j = 1, . . . , n a list of all distinct prime divisors of j. We use these divisors to compute the table via dynamic programming in a right-to-left scan, as shown in Algorithm 1. ▶Theorem 16. All full op-periods of a string of length n can be computed in O(n) time. Proof. We apply Algorithm 2. The complexity of the first for-loop is O(n) by Fact 15. The second for-loop works in O(n) time as the sizes of the sets Div(n), Div(i) are O(√n) and the elements of these sets can be enumerated in O(√n) time as well. ◀ Proof. We apply Algorithm 2. The complexity of the first for-loop is O(n) by Fact 15. The second for-loop works in O(n) time as the sizes of the sets Div(n), Div(i) are O(√n) and the elements of these sets can be enumerated in O(√n) time as well. ◀ 4.2 Computing Full Op-Periods Let us recall the following auxiliary data structure for efficient gcd-computations that was developed in [34]. We will only need a special case of this data structure to answer queries for gcd(x, n). ▶Fact 15 (Theorem 4 in [34]). After O(n)-time preprocessing, given any x, y ∈{1, . . . , n}, the value gcd(x, y) can be computed in constant time. Let Div(i) denote the set of all positive divisors of i. In the case of full op-periods we only need to compute P[p] for p ∈Div(n). As in Algorithm 1, we start with T = op-PREF′. Then we perform a preprocessing phase that shifts the information stored in the array from STACS 2018 38:8 String Periods in the Order-Preserving Model String Periods in the Order-Preserving Model Algorithm 2: Computing All Full Op-Periods of S. 1 T := op-PREF′; 2 for i := 1 to n do 3 k := gcd(i, n); 4 P[k] := min(P[k], P[i]); 5 foreach i ∈Div(n) in decreasing order do 6 foreach d ∈Div(i) do 7 P[d] := min(P[d], P[i]); 8 foreach p ∈Div(n) do 9 if P[p] ≥p then p is a full op-period; Algorithm 2: Computing All Full Op-Periods of S. 1 T := op-PREF′; 2 for i := 1 to n do 3 k := gcd(i, n); 4 P[k] := min(P[k], P[i]); 5 foreach i ∈Div(n) in decreasing order do indices i ̸∈Div(n) to indices gcd(i, n) ∈Div(n). It is based on the fact that for d ∈Div(n), d | i if and only if d | gcd(i, n). Finally, we perform right-to-left processing as in Algorithm 1. However, this time we can afford to iterate over all divisors of elements from Div(n). Thus we arrive at the pseudocode of Algorithm 2. 5 Computing Smallest Non-Trivial Initial Op-Period 1 if S has a non-trivial strictly monotone op-period then 2 return smallest such op-period; ▷Theorem 17 3 p := the length of the longest monotone prefix of S plus 1; 4 while p ≤n do 5 k := op-LPPp(S); 6 if k = n then return p; 7 p := max(p + 1, k −p −1); 8 return min(pmon, n); 38:9 G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń Algorithm 3: Computing the Smallest Non-Trivial Initial Op-Period of S. 1 if S has a non-trivial strictly monotone op-period then 2 return smallest such op-period; ▷Theorem 17 3 p := the length of the longest monotone prefix of S plus 1; 4 while p ≤n do 5 k := op-LPPp(S); 6 if k = n then return p; 7 p := max(p + 1, k −p −1); 8 return min(pmon, n); G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń 8 return min(pmon, n); ▶Theorem 20. The smallest initial op-period p > 1 of a string S of length n can be computed in O(n) time. ▶Theorem 20. The smallest initial op-period p > 1 of a string S of length n can be computed in O(n) time. Proof. We follow the lines of Algorithm 3. If S is not strictly monotone itself, we can compute the smallest non-trivial strictly monotone initial op-period of S using Theorem 17. Otherwise, the smallest such op-period is 2. If S has a non-trivial strictly monotone initial op-period and the smallest such op-period is q > 1, then none of 2, . . . , q −1 is an initial op-period of S. Hence, we can safely return q. Let us now focus on the correctness of the while-loop. The invariant is that there is no initial op-period of S that is smaller than p. If the value of k = op-LPPp(S) equals n, then p is an initial op-period of S and we can safely return it. Otherwise, we can advance p by 1. There is also no smallest initial op-period p′ such that p < p′ < k −p −1. 5 Computing Smallest Non-Trivial Initial Op-Period Indeed, Lemma 19 would imply that p is strictly monotone if gcd(p, p′) = 1 (which is impossible due to the initial selection of p) and Theorem 3 would imply an initial op-period of S[1..p′] that is smaller than p′ and divides p′ if gcd(p, p′) > 1 (which is impossible due to Lemma 18). This justifies the way p is increased. Now let us consider the time complexity of the algorithm. The algorithm for strictly monotone op-periods of Theorem 17 works in O(n) time. By Lemma 9, k can be computed in O(k/p + 1) time. If k ≤3p, this is O(1). Otherwise, p at least doubles; let p′ be the new value of p. Then O(k/p + 1) = O((p + p′ −1)/p + 1) = O(p′ + 1). The case that p doubles can take place at most O(log n) times and the total sum of p′ over such cases is O(n). ◀ 5 Computing Smallest Non-Trivial Initial Op-Period If a string is not strictly monotone itself, it has O(n) such op-periods and they can all be computed in O(n) time. We use this as an auxiliary routine in the computation of the smallest initial op-period that is greater than 1. ▶Theorem 17. If a string of length n is not strictly monotone, all of its strictly monotone op-periods can be computed in O(n) time. ▶Theorem 17. If a string of length n is not strictly monotone, all of its strictly monotone op-periods can be computed in O(n) time. Let us start with the following simple property. Let us start with the following simple property. Let us start with the following simple property. ▶Lemma 18. The shape of the smallest non-trivial initial op-period of a string has no shorter non-trivial full op-period. ▶Lemma 18. The shape of the smallest non-trivial initial op-period of a string has no shorter non-trivial full op-period. Proof. A full op-period of the initial op-period of a string S is an initial op-period of S. ◀ Proof. A full op-period of the initial op-period of a string S is an initial op-period of S. ◀ Now we can state a property of initial op-periods, implied by Theorem 7, that is the basis of the algorithm. Now we can state a property of initial op-periods, implied by Theorem 7, that is the basis of the algorithm. ▶Lemma 19. If a string of length n has initial op-periods p > q > 1 such that p + q < n and gcd(p, q) = 1, then q is strictly monotone. ▶Lemma 19. If a string of length n has initial op-periods p > q > 1 such that p + q < n and gcd(p, q) = 1, then q is strictly monotone. Proof. Let us consider three cases. If n > pq, then by Theorem 7(a), both p and q are strictly monotone. If 2p < n ≤pq, then Theorem 7(e) implies that S[1..pq −1] is strictly monotone, hence p and q are strictly monotone as well. Finally, if p + q < n ≤2p, we have that q is strictly monotone by Theorem 7(f). ◀ G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń Algorithm 3: Computing the Smallest Non-Trivial Initial Op-Period of S. 6 Computing All Op-Periods An interval representation of a set X of integers is X = Ji1..j1K∪Ji2..j2K∪· · ·∪Jik..jkK where j1 + 1 < i2, . . . , jk−1 + 1 < ik; k is called the size of the representation. Our goal is to compute a compact representation of all the op-periods of a string that contains, for each op-period p, an interval representation of the set Shiftsp. p For an integer set X, by X mod p we denote the set {x mod p : x ∈X}. The following technical lemma provides efficient operations on interval representations of sets. (a) Assume that X and Y are two sets with interval representations of sizes x and y, respectively. Then the interval representation of the set X ∩Y can be computed in O(x + y) time. (b) Assume that X1, . . . , Xk ⊆J0..nK are sets with interval representations of sizes x1, . . . , xk and p1, . . . , pk be positive integers. Then the interval representations of all the sets X1 mod p1, . . . , Xk mod pk can be computed in O(x1 + · · · + xk + k + n) time. String Periods in the Order-Preserving Model String Periods in the Order-Preserving Model Algorithm 4: Computing a Compact Representation of All Op-Periods. 1 Compute op-Squaresp for all p = 1, . . . , n; ▷Lemma 22 2 for p := 1 to n do 3 Np := J1..n −2p + 1K \ op-Squaresp; 4 k := op-LCP(1, p + 1); ℓ:= op-LCS(n, n −p); 5 if k = n −p then Bp := Cp := J1..nK; 6 else Bp := J1..kK; Cp := Jn −ℓ+ 1..nK; 7 for p := 1 to n simultaneously do 8 Np := {(x −1) mod p : x ∈Np}; Bp := Bp mod p; Cp := Cp mod p; ▷Lemma 21(b) 9 Shifts1 := J0K; 10 for p := 2 to n do 11 Ap := J0..p −1K \ Np; 12 Shiftsp := Ap ∩Bp ∩Cp; ▷Lemma 21(a) 13 return Shiftsp for p = 1, . . . , n; Algorithm 4: Computing a Compact Representation of All Op-Periods. Algorithm 4: Computing a Compact Representation of All Op-Periods 1 Compute op-Squaresp for all p = 1, . . . , n; ▷Lemma 22 1 Compute op-Squaresp for all p = 1, . . . , n; ▷Lemma 22 2 for p := 1 to n do 3 Np := J1..n −2p + 1K \ op-Squaresp; 4 k := op-LCP(1, p + 1); ℓ:= op-LCS(n, n −p); 5 if k = n −p then Bp := Cp := J1..nK; 6 else Bp := J1..kK; Cp := Jn −ℓ+ 1..nK; 2 for p := 1 to n do 3 Np := J1..n −2p + 1K \ op-Squaresp; 4 k := op-LCP(1, p + 1); ℓ:= op-LCS(n, n −p); 5 if k = n −p then Bp := Cp := J1..nK; 6 else Bp := J1..kK; Cp := Jn −ℓ+ 1..nK; 7 for p := 1 to n simultaneously do 7 for p := 1 to n simultaneously do 8 Np := {(x −1) mod p : x ∈Np}; Bp := Bp mod p; Cp := Cp mod p; ▷Lemma 21(b) 9 Shifts1 := J0K; 10 for p := 2 to n do 11 Ap := J0..p −1K \ Np; 12 Shiftsp := Ap ∩Bp ∩Cp; ▷Lemma 21(a) 13 return Shiftsp for p = 1, . . . , n; ▷Lemma 21(a) 13 return Shiftsp for p = 1, . . . , n; ▶Lemma 22. String Periods in the Order-Preserving Model For a string of length n, interval representations of the sets op-Squaresp for all 1 ≤p ≤n/2 can be computed in O(n log n) time. Proof. Let us define the following two auxiliary sets. Lp = {i ∈J1..n −2p + 1K : S[i..i + 2p −1] is a left non-shiftable op-square} Rp = {i ∈J1..n −2p + 1K : S[i..i + 2p −1] is a right non-shiftable op-square}. Lp = {i ∈J1..n −2p + 1K : S[i..i + 2p −1] is a left non-shiftable op-square} Rp = {i ∈J1..n −2p + 1K : S[i..i + 2p −1] is a right non-shiftable op-square}. By Lemma 11, all the sets Lp and Rp can be computed in O(n log n) time. In particular, P |L | = O(n log n) By Lemma 11, all the sets Lp and Rp can be computed in O(n log n) time. In particular, P p |Lp| = O(n log n). Let us note that, for each p, |Lp| = |Rp|. Thus let Lp = {ℓ1, . . . , ℓk} and Rp = {r1, . . . , rk}. Let us note that, for each p, |Lp| = |Rp|. Thus let Lp = {ℓ1, . . . , ℓk} and Rp = {r1, . . . , rk}. The interval representation of the set op-Squaresp is Jℓ1..r1K ∪· · · ∪Jℓk..rkK. Clearly, it can be computed in O(|Lp|) time. ◀ We will use the following characterization of op-periods. ▶Lemma 21. (b) Assume that X1, . . . , Xk ⊆J0..nK are sets with interval representations of sizes x1, . . . , xk and p1, . . . , pk be positive integers. Then the interval representations of all the sets X1 mod p1, . . . , Xk mod pk can be computed in O(x1 + · · · + xk + k + n) time. STACS 2018 38:10 ▷Lemma 22 We will use the following characterization of op-periods. 0 1 2 3 4 5 6 6 6 6 6 6 9 8 7 12 11 10 S 1 2 3 3 5 4 A 1 2 3 5 3 4 B 1 2 3 3 5 4 C A B Figure 3 A string S = 0 12 6 1 11 6 2 10 6 3 9 6 4 8 6 5 7 6 is graphically illustrated above (the ith point has coordinates (i, S[i])). We have SH 6 = ABCABCABCA, where A = 1 5 3 2 4 3, B = 5 3 1 4 3 2, and C = 3 1 5 3 2 4. The shortest period of SH 6 is 3. Hence, 6 is a sliding op-period of S. Moreover, Lemma 27b implies that 3 is a period of SH 3, hence a sliding op-period of S. We will use the following characterization of op-periods. We will use the following characterization of op-periods. ▶Observation 23. p is an op-period of S with shift i if and only if all the following conditions hold: (A) S[i + 1 + kp..i + (k + 2)p] is an op-square for every 0 ≤k ≤(n −2p −i)/p, (B) op-LCP(1, p + 1) ≥min(i, n −p), (C) op-LCS(n, n −p) ≥min((n −i) mod p, n −p). (B) op-LCP(1, p + 1) ≥min(i, n −p), (C) op-LCS(n, n −p) ≥min((n −i) mod p, n −p). (C) op-LCS(n, n −p) ≥min((n −i) mod p, n −p). ▶Theorem 24. A representation of size O(n log n) of all the op-periods of a string of length n can be computed in O(n log n) time. ▶Theorem 24. A representation of size O(n log n) of all the op-periods of a string of length n can be computed in O(n log n) time. Proof. We use Algorithm 4. The sets Ap, Bp, and Cp describe the sets of shifts i that satisfy conditions (A), (B), and (C) from Observation 23, respectively. A crucial role is played by the set Np of all positions which are not the beginnings of op-squares of length 2p. It is computed as a complement of the set op-Squaresp. p Operations “mod” on sets are performed simultaneously using Lemma 21(b). All sets Ap, Bp, Cp have O(n log n)-sized representations. This guarantees O(n log n) time. ◀ 38:11 G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń 0 1 2 3 4 5 6 6 6 6 6 6 9 8 7 12 11 10 S 1 2 3 3 5 4 A 1 2 3 5 3 4 B 1 2 3 3 5 4 C Figure 3 A string S = 0 12 6 1 11 6 2 10 6 3 9 6 4 8 6 5 7 6 is graphically illustrated above (the ith point has coordinates (i, S[i])). We have SH 6 = ABCABCABCA, where A = 1 5 3 2 4 3, B = 5 3 1 4 3 2, and C = 3 1 5 3 2 4. The shortest period of SH 6 is 3. Hence, 6 is a sliding op-period of S. Moreover, Lemma 27b implies that 3 is a period of SH 3, hence a sliding op-period of S. 7 Computing Sliding Op-Periods For a string S of length n, we define a family of strings SH 1, . . . , SH n such that SH k[i] = shape(S[i..i + k −1]) for 1 ≤i ≤n −k + 1. Note that the characters of the strings are shapes. Moreover, the total length of strings SH k is quadratic in n, so we will not compute those strings explicitly. Instead, we use the following observation to test if two symbols are equal. ▶Observation 25. SH k[i] = SH k[i′] if and only if op-LCP(i, i′) ≥k. Sliding op-periods admit an elegant characterization based on SH k; see Figure 3. ▶Lemma 26. An integer p, 1 ≤p ≤n, is a sliding op-period of S if and only if p ≤1 2n and p is a period of SH p, or p > 1 2n and S[1..n −p] ≈S[p + 1..n]. For a string X, we denote the shortest period of X by per(X). ▶Lemma 27. Suppose that p = per(SH k[1..ℓ]) < ℓ. Then (a) p is also a period of SH k′[1..ℓ+ k −k′] for 1 ≤k′ ≤k, (b) q = per(SH k[1..ℓ+ 1]) satisfies p = q or p + q > ℓ. ▶Lemma 27. Suppose that p = per(SH k[1..ℓ]) < ℓ. Then (a) p is also a period of SH k′[1..ℓ+ k −k′] for 1 ≤k′ ≤k, (b) q = per(SH k[1..ℓ+ 1]) satisfies p = q or p + q > ℓ. We introduce a two-dimensional table PER, where: ▶Lemma 27. Suppose that p = per(SH k[1..ℓ]) < ℓ. Then (a) p is also a period of SH k′[1..ℓ+ k −k′] for 1 ≤k′ ≤k, (b) q = per(SH k[1..ℓ+ 1]) satisfies p = q or p + q > ℓ. We introduce a two-dimensional table PER, where: We introduce a two-dimensional table PER, where: PER[k, ℓ] = per(SH k[1..ℓ]) if per(SH k[1..ℓ]) ≤1 3ℓ, and PER[k, ℓ] = ⊥(undefined) otherwise. The size of PER is quadratic in n. However, Algorithm 5 computes PER column after column, keeping only the current column P = PER[·, ℓ]. The total number of differences between consecutive columns is linear. Hence, any requested O(n) values PER[k, ℓ] can be computed in O(n) time. We also use an analogous table PERR for the reverse string SR. ▶Lemma 28. Algorithm 5 is correct, that is, it satisfies the invariant. ▶Lemma 28. Algorithm 5 is correct, that is, it satisfies the invariant. 1 p := 1; 2 while p ≤1 2n do 3 if (q := PER[p, n −2p + 1]) = PERR[p, n −2p + 1] ̸= ⊥then 4 if p is a period of SH p[1..p + q] then report p; 5 p := min{p′ > p : p′ is a period of SH p[1..p + 2q]} 6 else if PER[p, ⌈3 4(n −2p + 1)⌉] = PERR[p, ⌈3 4(n −2p + 1)⌉] ̸= ⊥then p := p + 1; 7 else 8 if p is a period of SH p then report p; 9 p := min{p′ > p : p′ is a period of SH }; (q [p, p ]) [p, p ] if p is a period of SH p[1..p + q] then report p; p 5 p := min{p′ > p : p′ is a period of SH p[1..p + 2q]} 6 else if PER[p, ⌈3 4(n −2p + 1)⌉] = PERR[p, ⌈3 4(n −2p + 1)⌉] ̸= ⊥then p := p + 1; 7 else 5 p := min{p′ > p : p′ is a period of SH p[1..p + 2q]} 6 else if PER[p, ⌈3 4(n −2p + 1)⌉] = PERR[p, ⌈3 4(n −2p + 1)⌉] ̸= ⊥then p := p + 1; 7 else 8 if p is a period of SH p then report p; 9 p := min{p′ > p : p′ is a period of SH p}; 9 p := min{p′ > p : p′ is a period of SH p}; First, we consider the values PER[k, ℓ] for k < t. For this, we assume t > 1 and denote p = PER[t −1, ℓ−1]. Since p is a period of SH t−1[1..ℓ−1], Lemma 27a yields that p is also a period of SH k[1..ℓ] for k < t −1. We apply Lemma 27b for p′ = per(SH k[1..ℓ−1]). Since p′ + p ≤ℓ−1, we conclude that p′ = per(SH k[1..ℓ]), i.e., PER[k, ℓ−1] = p′ = PER[k, ℓ]. Now, we consider the value PER[t −1, ℓ]. Lemma 27b, applied for p = per(SH t−1[1..ℓ−1]) and q = per(SH t−1[1..ℓ]), yields p = q or p + q ≥ℓ. To verify the first case, we check whether SH t−1[ℓ] = SH t−1[ℓ−p]. In the second case, we conclude that q ≥2 3ℓ, so PER[t −1, ℓ] = ⊥ (and ℓ′ := 2ℓis also set correctly). ▶Lemma 28. Algorithm 5 is correct, that is, it satisfies the invariant. ▶Lemma 28. Algorithm 5 is correct, that is, it satisfies the invariant. Proof. First, observe that the invariant is satisfied after the first iteration. This is because per(SH k[1..1]) = 1 for each k and the initial values are not changed during this iteration. ( [ ]) Thus, our task is to prove that the invariant is preserved after each subsequent ℓth iteration. Let t = min{k : PER[k, ℓ−1] = ⊥} and t′ = min{k : PER[k, ℓ] = ⊥}. STACS 2018 String Periods in the Order-Preserving Model 38:12 Algorithm 5: Computation of PER[·, ℓ] from PER[·, ℓ−1]. Algorithm 5: Computation of PER[·, ℓ] from PER[·, ℓ−1]. 1 P[1..n] := [⊥, . . . , ⊥]; t := 1; ℓ′ := 3; 2 for ℓ:= 1 to n do 3 if t > 1 and SH t−1[ℓ] ̸= SH t−1[ℓ−P[t −1]] then 4 t := t −1; P[t] := ⊥; ℓ′ := 2ℓ; 5 if ℓ≥ℓ′ then 6 while per(SH t[1..ℓ]) = 1 3ℓdo 7 P[t] := 1 3ℓ; t := t + 1; ℓ′ := 2ℓ; ▷Invariant: P[k] = PER[k, ℓ] , t = min{k : P[k] = ⊥}, and per(SH t[1..ℓ]) ≥1 3ℓ′. [ ] 3 ; ; ; ▷Invariant: P[k] = PER[k, ℓ] , t = min{k : P[k] = ⊥}, and per(SH t[1..ℓ]) ≥1 3ℓ′. Algorithm 6: Computing the sliding op-periods p ≤1 2n. Algorithm 6: Computing the sliding op-periods p ≤1 2n. ▶Lemma 29. Algorithm 5 can be implemented in time O(n) plus the time to answer O(n) op-LCP queries in S. 7 Francine Blanchet-Sadri, Sean Simmons, Amelia Tebbe, and Amy Veprauskas. Abelian periods, partial words, and an extension of a theorem of fine and wilf. RAIRO - Theor. Inf. and Applic., 47(3):215–234, 2013. doi:10.1051/ita/2013034. G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń Next, we run Algorithm 6 to report sliding op-periods p ≤1 2n. Then, we iterate over p > 1 2n and report p if op-LCP(1, p + 1) = n −p. Correctness follows from Lemmas 30 and 26. The overall time is O(n) (Lemma 31) plus the preprocessing time of Lemma 10. ◀ G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń SH pi[pi +1..pi +ℓi], i.e., pi is a period of SH pi = SH pi[1..pi +ℓi] and pi is a sliding op-period due to Lemma 26. Moreover, any sliding op-period p′ > pi must be a period of SH pi (and, in particular, of SH pi[1..pi + 2q]) due to Lemma 27a. Consequently, p′ ≥pi+1, as claimed. In the second branch we only need to prove that SH pi[1..ℓi] ̸= SH pi[pi + 1..pi + ℓi]. For a proof by contradiction, suppose that we have an equality. The condition from Line 6 means that the length-⌈3 4ℓi⌉prefix and suffix of SH pi[1..ℓi] = SH pi[pi + 1..pi + ℓi] has the common shortest period q ≤1 3⌈3 4ℓi⌉≤⌈1 4ℓi⌉. The prefix and the suffix overlap by at least ⌈1 2ℓi⌉ characters, so we actually have q = per(SH pi[1..ℓi]) = per(SH pi[pi + 1..pi + ℓi]). Hence, in that case we would be in the first branch. Finally, in the third branch we directly use Lemma 26 to check if pi is a sliding op-period. Moreover, if p′ > pi is also a sliding op-period, then p′ is a period of SH pi, i.e., p′ ≥pi+1. ◀ Let us observe that PER[k, ℓ] and PERR[k, ℓ] is used in Algorithm 6 only for ℓ= n−2k+1 or ℓ=  3 4(n −2k + 1)  . These O(n) values can be computed in O(n) time using Algorithm 5. In [29] we show the following lemma. ▶Lemma 31. Algorithm 6 can be implemented in time O(n) plus the time to answer O(n) op-LCP and op-LCS queries in S. ▶Theorem 32. All sliding op-periods of a string of length n can be computed in O(n) space and O(n log log n) expected time or O(n log2 log n/ log log log n) worst-case time. Proof. First, we apply Lemma 10 so that op-LCP and op-LCS queries can be answered in O(1) time. Next, we run Algorithm 6 to report sliding op-periods p ≤1 2n. Then, we iterate over p > 1 2n and report p if op-LCP(1, p + 1) = n −p. Correctness follows from Lemmas 30 and 26. The overall time is O(n) (Lemma 31) plus the preprocessing time of Lemma 10. ◀ Proof. First, we apply Lemma 10 so that op-LCP and op-LCS queries can be answered in O(1) time. ▶Lemma 28. Algorithm 5 is correct, that is, it satisfies the invariant. Next, we consider the values PER[k, ℓ] for k ≥t. Since PER[k, ℓ−1] = ⊥, we have PER[k, ℓ] = ⊥or PER[k, ℓ] = 1 3ℓ. More precisely, PER[k, ℓ] = ⊥for k ≥t′ and PER[k, ℓ] = 1 3ℓfor t ≤k < t′. Thus, we check if per(SH k[1..ℓ]) = 1 3ℓfor subsequent values k ≥t. Since per(SH t[1..ℓ]) ≥1 3ℓ′, no verification is needed if ℓ< ℓ′. To complete the proof, we need to show that the update ℓ′ := 2ℓis valid if t′ > t. For a proof by contradiction suppose that r := per(SH t′[1..ℓ]) < 2 3ℓ. By Lemma 27a, r is a period of SH t[1..ℓ]. Since r + 1 3ℓ≤ℓ, Periodicity Lemma yields 1 3ℓ| r, and thus r = 1 3ℓ, which contradicts the definition of t′. ◀ ▶Lemma 29. Algorithm 5 can be implemented in time O(n) plus the time to answer O(n) op-LCP queries in S. ▶Lemma 30. Algorithm 6 is correct, that is, it reports all sliding op-periods p ≤1 2n of S. ▶Lemma 30. Algorithm 6 is correct, that is, it reports all sliding op-periods p ≤1 2n of S. Proof. Let pi be the value of p at the beginning of the ith iteration of the while-loop and let ℓi = n −2pi + 1. We shall prove that pi is reported if and only if it is a sliding op-period and that there is no sliding op-period strictly between pi and pi+1. + First, suppose that q = per(SH pi[1..ℓi]) = per(SH pi[pi + 1..pi + ℓi]) ≤1 3ℓi, i.e., we are in the first branch. If SH pi[1..q] = SH pi[pi + 1..pi + q], then we must have SH pi[1..ℓi] = 38:13 G. Gourdel, T. Kociumaka, J. Radoszewski, W. Rytter, A. Shur, and T. Waleń References 1 Tom M. Apostol. Introduction to Analytic Number Theory. Undergraduate Texts in Math- ematics, Springer, 1976. 2 Alberto Apostolico and Raffaele Giancarlo. Periodicity and repetitions in parameterized strings. Discrete Applied Mathematics, 156(9):1389–1398, 2008. doi:10.1016/j.dam.2006. 11.017. 3 Djamal Belazzougui, Adeline Pierrot, Mathieu Raffinot, and Stéphane Vialette. Single and multiple consecutive permutation motif search. 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https://openalex.org/W4249989623
https://wwwnc.cdc.gov/eid/article/18/7/pdfs/12-0055.pdf
English
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Electronic Event–based Surveillance for Monitoring Dengue, Latin America
Emerging infectious diseases
2,012
cc-by
2,449
The Study We focused on the geographic range of DENVs in Latin America and the Caribbean, where dengue is widespread and expanding in range. We attempted to identify areas contiguous with previously known dengue- endemic zones where new DENV transmission is occurring by using reports of recent outbreaks. The current dengue epidemic in Latin America represents a major threat to health. However, surveillance of affected regions lacks timeliness and precision. We investigated real-time electronic sources for monitoring spread of dengue into new regions. This approach could provide timely estimates of changes in distribution of dengue, a critical component of prevention and control efforts. Known dengue-endemic areas were defi ned as dengue risk areas identifi ed by the US Centers for Disease Control and Prevention (Atlanta, GA, USA) Health Information for International Travel (commonly referred to as the Yellow Book), 2010 (7) and 2012 (8) editions. Each edition of this book refl ects the known distribution of dengue risk in the prior 2 years. To characterize spread according to the Yellow Book, we identifi ed areas that were classifi ed as no known dengue risk in 2010 but were changed to risk areas in the 2012 edition (hereafter referred to as new dengue- endemic areas). D engue, a potentially fatal viral disease, has been recognized for >200 years (1). Once sporadic and limited geographically, dengue viruses (DENVs) and their mosquito vectors have spread globally, putting an estimated 2.5 billion persons at risk throughout the tropical and subtropical regions of the world (2). Factors contributing to the dramatic expansion of DENV activity include demographic changes such as population growth, urbanization, and globalization, and reductions in vector control and other public health measures (3). Because of the nature of passive surveillance, conventional systems have limited ability in identifying new epidemics quickly (1), thus suggesting a role for alternative information sources. D Outbreak data for December 1, 2009–March 18, 2011, were collected from HealthMap (http://www. healthmap.org/en/an), an open access online infectious disease outbreak monitoring system (9,10). HealthMap integrates outbreak-related data from >30,000 electronic sources, including the news media, ProMED-mail, and other electronic public health reporting sources, by using algorithms to classify the diseases and locations associated with each report. 1Current affi liation: The Geisel School of Medicine at Dartmouth College, Lebanon, New Hampshire, USA. Electronic Event–based Surveillance for Monitoring Dengue, Latin America dengue case counts (5,6). These event-based data sources can provide insight into new and ongoing public health challenges in areas of the world with limited public health reporting infrastructure. Few studies have investigated the value of unoffi cial sources for monitoring recent geographic expansion of infectious disease risk. Using dengue as a case study, we report on the utility of electronic outbreak surveillance for real-time monitoring of recent infectious disease spread. Anne G. Hoen,1 Mikaela Keller, Aman D. Verma, David L. Buckeridge, and John S. Brownstein Author affi liations: Children’s Hospital Boston, Boston, Massachusetts, USA (A.G. Hoen, J.S. Brownstein); Harvard Medical School, Boston (A.G. Hoen, J.S. Brownstein); Institut de Recherche en Informatique et Automatique, Lille, France (M. Keller); Université de Lille, Lille (M. Keller); McGill University, Montreal, Quebec, Canada (A.G. Hoen, A.D. Verma, D.L. Buckeridge, J.S. Brownstein); and Agence de la Santé et des Services Sociaux de Montreal, Montreal (D.L. Buckeridge) The Study Areas outlined with heavy black solid lines were classifi ed as high HealthMap alert density but were not identifi ed in either Yellow Book edition as dengue risk areas. Areas outlined with heavy black dashed lines were classifi ed as low HealthMap alert density but were identifi ed in the 2012 Yellow Book as areas at risk for dengue. Figure 1. A) Regions in Latin America analyzed for dengue. B) Central Mexico; C) central South America; D) eastern Amazonas, Brazil. Thin gray lines indicate boundaries of fi rst-level administrative units. Areas to which dengue was identifi ed in the 2010 Yellow Book are shaded in gray. New dengue-endemic areas identifi ed in the 2012 Yellow Book are outlined in blue. Dots indicate HealthMap dengue- related alerts. Modeled HealthMap alert probability density surface is shown in a gradient from yellow to red with yellow areas predicted as having lower alert densities and red areas predicted as having higher alert densities according to the model. Areas outlined with heavy black solid lines were classifi ed as high HealthMap alert density but were not identifi ed in either Yellow Book edition as dengue risk areas. Areas outlined with heavy black dashed lines were classifi ed as low HealthMap alert density but were identifi ed in the 2012 Yellow Book as areas at risk for dengue. distribution of new dengue-endemic areas identifi ed in the 2012 Yellow Book. Details of the datasets, models, and statistical methods are available in the online Technical Appendix. at elevated risk of endemicity, according to the dengue outbreak probability density estimated by our model. Of the 41 areas that remained unidentifi ed as dengue-endemic areas in the 2012 Yellow Book, our model classifi ed 35 (85%) as having reduced risk of endemicity. Figure 1 shows that high dengue outbreak activity occurred adjacent to previously recognized dengue- endemic zones in 6 states in central Mexico and in parts of northern Argentina, southern Brazil, Bolivia, and Paraguay. We used receiver-operating characteristic analysis with cross-validation (Figure 2) to set a threshold dengue report density that best identifi es new dengue- endemic areas (Figure 1; online Technical Appendix). Of the 19 new dengue-endemic areas reported in the 2012 Yellow Book, this threshold identifi ed 14 (74%) as being When compared with the Yellow Book, our model incorrectly classifi ed 6 areas as at elevated risk (Figure 1). The Study Because we wanted to identify spread into new dengue-endemic zones, we limited our analyses to areas that were identifi ed as having no known dengue risk in the 2010 Yellow Book but that were contiguous with >1 risk areas in the 2010 Yellow Book. We identifi ed 53 dengue outbreaks distributed in 60 of these areas. Free or low-cost sources of unstructured information, such as Internet news outlets, health expert mailing lists, social media sites, and queries to online search engines, when computationally fi ltered and mined, can provide detailed local and near real-time data on potential or confi rmed disease outbreaks (4). For dengue in particular, our group and others recently reported on a set of Google search terms that parallel temporal trends in offi cial We fi tted a bivariate Gaussian mixture model to the extracted HealthMap alerts to model a continuous surface of outbreak density (online Technical Appendix, wwwnc. cdc.gov/EID/pdfs/12-0055-Techapp.pdf). This modeled outbreak probability density surface represents a risk map of recent DENV spread into areas of previously unknown dengue endemicity according to the 2010 Yellow Book (Figure 1). We compared our map with the geographic Author affi liations: Children’s Hospital Boston, Boston, Massachusetts, USA (A.G. Hoen, J.S. Brownstein); Harvard Medical School, Boston (A.G. Hoen, J.S. Brownstein); Institut de Recherche en Informatique et Automatique, Lille, France (M. Keller); Université de Lille, Lille (M. Keller); McGill University, Montreal, Quebec, Canada (A.G. Hoen, A.D. Verma, D.L. Buckeridge, J.S. Brownstein); and Agence de la Santé et des Services Sociaux de Montreal, Montreal (D.L. Buckeridge) DOI: http://dx.doi.org/10.3201/eid1808.120055 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012 1147 DISPATCHES Figure 1. A) Regions in Latin America analyzed for dengue. B) Central Mexico; C) central South America; D) eastern Amazonas, Brazil. Thin gray lines indicate boundaries of fi rst-level administrative units. Areas to which dengue was identifi ed in the 2010 Yellow Book are shaded in gray. New dengue-endemic areas identifi ed in the 2012 Yellow Book are outlined in blue. Dots indicate HealthMap dengue- related alerts. Modeled HealthMap alert probability density surface is shown in a gradient from yellow to red with yellow areas predicted as having lower alert densities and red areas predicted as having higher alert densities according to the model. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012 The Study All alerts in these areas described outbreaks of cases acquired in a nearby known dengue-endemic region of the country. One alert also warned of the recent discovery of dengue vector mosquito larvae by the local surveillance program. The model also classifi ed 5 Yellow Book dengue- endemic areas as at reduced risk (Figure 1). Four of these areas were rural or isolated. Although other explanations Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012 1148 Electronic Event–based Surveillance for Dengue Figure 2. Receiver-operating characteristic plot of 5-fold cross- validated HealthMap alert density-based classifi cation with new dengue-endemic areas identifi ed by the 2012 Yellow Book as the standard. identify a reliable signal of dengue spread. Although this analysis was performed retrospectively, the timeliness of this signal far outperforms any traditional surveillance data stream. Passive case report-based surveillance systems typically operate at a delay of weeks to months, which limits their value for providing a picture of geographic spread, especially on an international scale where surveillance delays may be even more prolonged. We have demonstrated a novel approach to real-time monitoring of recent expansion of DENV activity in Latin America. Using outbreak reports captured by HealthMap, we identifi ed a signal of geographic expansion of dengue activity that would precede offi cial reports of the geographic distribution of dengue-endemic areas. Currently, no reliable surveillance system is in widespread use that reports the distribution of DENV activity on an ongoing basis and enables near real-time monitoring of trends in geographic expansion. Such a system should enhance the ability of regional and global public health authorities to dynamically allocate resources within a time frame that might effectively avert a full-blown epidemic. Like other large-scale surveillance data sources, our results must be interpreted cautiously. However, when used in conjunction with traditional surveillance methods, our approach has the potential to provide a timely estimate of changes in the geographic distribution of dengue, a critical component of targeted prevention and control efforts. Figure 2. Receiver-operating characteristic plot of 5-fold cross- validated HealthMap alert density-based classifi cation with new dengue-endemic areas identifi ed by the 2012 Yellow Book as the standard. likely exist, the low observed sensitivity in these areas illustrates certain limits of any system that relies on Internet- based information fl ow for monitoring disease spread. Acknowledgments Electronic event–based surveillance systems such as HealthMap and others are frequently used by public health authorities, travelers, physicians and patients, to gain a real-time understanding of global outbreak activity. The HealthMap dengue feed, DengueMap, is currently part of the online dengue information resource of the Centers for Disease Control and Prevention (http://www.cdc. gov/dengue/). Used in combination with traditional case reporting, HealthMap and other electronic surveillance systems have proven value for enhancing the timeliness of outbreak discovery and information dissemination (11). However, these information sources may also provide added value for monitoring ongoing spread. We thank Michael Johansson for critically revising the manuscript and providing Yellow Book data; and Katia Charland, Laura Pinheiro, Emily Chan, Susan Aman, Clark Freifeld, Katelynn O’Brien and Sumiko Mekaru for helpful discussions and database support. This study was supported by National Institutes of Health grants R01 LM010812 and G08 LM009776. Dr Hoen is an infectious disease epidemiologist in the Department of Community and Family Medicine at The Geisel School of Medicine at Dartmouth College. Her research interests include informatics-based approaches to understanding infectious disease risk, emergence and spread. Although the signal of DENV activity detected by HealthMap is relatively robust, it has certain limitations. First, the signal tends to be sparse in areas with limited reporting because of low population density or incomplete coverage by the news or social media. Second, the signal can be surrounded by background noise because separating reports caused by cases in travelers from true autochthonous transmission is diffi cult with automated methods. By limiting our analysis to areas contiguous with known dengue-endemic areas and smoothing outbreak alerts into an outbreak-density surface, we were able to 1. Gubler DJ. Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg Infect Dis. 1995;1:55–7. http://dx.doi. org/10.3201/eid0102.950204 2. World Health Organization. Fact sheet no. 117: dengue and dengue haemorrhagic fever. Geneva: The Organization; 2009. 1. Gubler DJ. Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg Infect Dis. 1995;1:55–7. http://dx.doi. org/10.3201/eid0102.950204 2. World Health Organization. Fact sheet no. 117: dengue and dengue haemorrhagic fever. Geneva: The Organization; 2009. 3. Gubler DJ. The global emergence/resurgence of arboviral diseases as public health problems. Arch Med Res. 2002;33:330–42. http:// dx.doi.org/10.1016/S0188-4409(02)00378-8 References 2. World Health Organization. Fact sheet no. 117: dengue and dengue haemorrhagic fever. Geneva: The Organization; 2009. 3. Gubler DJ. The global emergence/resurgence of arboviral diseases as public health problems. Arch Med Res. 2002;33:330–42. http:// dx.doi.org/10.1016/S0188-4409(02)00378-8 1149 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012 DISPATCHES 4. Morse SS. Global infectious disease surveillance and health intel- ligence. Health Aff (Millwood). 2007;26:1069–77. http://dx.doi. org/10.1377/hlthaff.26.4.1069 10. Brownstein JS, Freifeld CC, Reis BY, Mandl KD. Surveillance Sans Frontieres: Internet-based emerging infectious disease intelligence and the HealthMap project. PLoS Med. 2008;5:e151. http://dx.doi. org/10.1371/journal.pmed.0050151 5. Amarasinghe A, Kuritsky JN, Letson GW, Margolis HS. Dengue vi- rus infection in Africa. Emerg Infect Dis. 2011;17:1349–54. 11. Chan EH, Brewer TF, Madoff LC, Pollack MP, Sonricker AL, Keller M, et al. Global capacity for emerging infectious disease detec- tion. Proc Natl Acad Sci U S A. 2010;107:21701–6. http://dx.doi. org/10.1073/pnas.1006219107 6. Chan EH, Sahai V, Conrad C, Brownstein JS. Using web search query data to monitor dengue epidemics: a new model for neglected tropical disease surveillance. PLoS Negl Trop Dis. 2011;5:e1206. http://dx.doi.org/10.1371/journal.pntd.0001206 7. Centers for Disease Control and Prevention. CDC health informa- tion for international travel 2010. Atlanta: The Centers; 2010. 7. Centers for Disease Control and Prevention. CDC health informa- tion for international travel 2010. Atlanta: The Centers; 2010. Address for correspondence: Anne G. Hoen, Dartmouth-Hitchcock Medical Center, 1 Medical Center Dr, HB 7937, Lebanon, NH 03756, 8. Centers for Disease Control and Prevention. CDC health informa- tion for international travel 2012. Atlanta: The Centers; 2012. 8. Centers for Disease Control and Prevention. CDC health informa- tion for international travel 2012. Atlanta: The Centers; 2012. USA; email: anne.g.hoen@dartmouth.edu 9. Freifeld CC, Mandl KD, Reis BY, Brownstein JS. HealthMap: global infectious disease monitoring through automated classifi ca- tion and visualization of Internet media reports. J Am Med Inform Assoc. 2008;15:150–7. http://dx.doi.org/10.1197/jamia.M2544 Use of trade names is for identifi cation only and does not imply endorsement by the Public Health Service or by the US Department of Health and Human Services. 1150 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012 1150 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 7, July 2012
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An embryonic system to assess Wnt transcriptional targets
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An embryonic system to assess direct and indirect Wnt transcriptional targets Received: 10 May 2017 Accepted: 25 August 2017 Published: xx xx xxxx Received: 10 May 2017 Accepted: 25 August 2017 Published: xx xx xxxx Jahnavi Suresh1, Nathan Harmston2, Ka Keat Lim   2, Prameet Kaur1, Helen Jingshu Jin1, Jay B. Lusk1, Enrico Petretto2 & Nicholas S. Tolwinski   1,3 During animal development, complex signals determine and organize a vast number of tissues using a very small number of signal transduction pathways. These developmental signaling pathways determine cell fates through a coordinated transcriptional response that remains poorly understood. The Wnt pathway is involved in a variety of these cellular functions, and its signals are transmitted in part through a β-catenin/TCF transcriptional complex. Here we report an in vivo Drosophila assay that can be used to distinguish between activation, de-repression and repression of transcriptional responses, separating upstream and downstream pathway activation and canonical/non-canonical Wnt signals in embryos. We find specific sets of genes downstream of both β-catenin and TCF with an additional group of genes regulated by Wnt, while the non-canonical Wnt4 regulates a separate cohort of genes. We correlate transcriptional changes with phenotypic outcomes of cell differentiation and embryo size, showing our model can be used to characterize developmental signaling compartmentalization in vivo. Signaling pathways elicit cellular responses in part by regulating the transcription of specific cohorts of target genes. Signaling pathways that are crucial for development, homeostasis and tumorigenesis have both negative (repressive) and positive (activation and de-repression) effects on transcription1. Negatively regulated targets can have different biological activities from positively regulated targets2. The Wnt signaling pathway provides a striking example of this phenomenon where Wnt signals can elicit a variety of cellular responses including dif- ferentiation, growth, and polarity3–6. Increased Wnt signaling has apparently opposite roles on cell proliferation: excessive Wnt signaling leads to over proliferation of cancer cells, but it is also required to maintain undifferen- tiated, quiescent stem cells7, 8. As a result, therapeutic interventions that block Wnt in tumours are likely to have both beneficial and detrimental consequences; when cancer growth is inhibited, useful stem cells are likely to be lost as a side-effect9, 10. It is probable that these opposing effects occur through the transcriptional activation of different Wnt target genes, raising the intriguing possibility that therapies targeted downstream could avoid the detrimental effects of disrupting the whole pathway. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results D l Development of a naïve-embryo transcriptional assay system. We developed a transcriptional assay using the Drosophila embryo, where by using simple genetic manipulations we can create relatively naïve, homogeneous populations of cells, therefore minimizing the confounding effect of non-specific, secondary, and multiple signaling pathway effects that are often observed in gene expression studies35. In normal Drosophila development, eggs are provided with maternal patterning signals. These signals include anterior-posterior pat- terning molecules such as Bicoid and Nanos, terminal patterning determinants such as Torso and Torsolike (EGF pathway related), and dorsal-ventral signals such as Toll and Dpp (NFκB and TGFβ signaling pathways)36–38. These patterning signals determine the axes of the developing embryo and activate further signals that lead to specific cellular identities for each cell in the embryo. Removal of these basic patterning signals leads to eggs that develop a simple, un-patterned epithelium “naïve embryos”. For anterior-posterior patterning, we used a triple mutant (bicoid, nanos, torsolike) that eliminates anterior, posterior and terminal patterning respectively leading to highly compromised development (Supplementary Movies 1 and 2)39, 40. A further advantage of this system lies in the fact that these are maternal effect mutations, allowing the use of homozygous females that lay 100% mutant eggs, therefore removing the difficulty of identifying mutant embryos and avoiding the use of complicated germline clone techniques34, 41, 42. This experimental setup creates a condition where all the embryonic cells are identical with respect to Wnt signalling, and as these embryos do not gastrulate, additional complication of endo- dermal and mesodermal cells types is avoided. Basic phenotypic analysis of Wnt signaling. We introduced several genetic changes targeting specific components of the Wnt signalling pathway in otherwise wild-type embryos and assessed their consequences via a simple phenotypic assay (Fig. 1). This was accomplished by examining both the size of the embryos (nor- mal/small) and their differentiation status (naked or with denticles) (Fig. 1C). Wg overexpression (Wg++) was accomplished by using the GAL4/UAS system to establish the hyper-activated pathway condition43. A second approach to activate canonical Wnt signaling was to express arm∆N, an allele lacking the N-terminal region that contains phosphorylation sites for GSK3 and CK1 that target Arm for ubiquitination and proteasome mediated degradation44, 45. The opposite condition, or inactivation of Wnt signaling, was the expression of a dominant-negative allele (tcf∆N-short). An embryonic system to assess direct and indirect Wnt transcriptional targets f p g p y Wnt signalling and its dysregulation has been implicated in a variety of developmental disorders and dis- eases, including diabetes, Robinow Syndrome, cancer and aging11–13. Wnt regulation appears to be highly context-specific, affecting different genes in different cell types at different developmental stages14, and the fea- tures defining how a Wnt target gene is regulated are not fully understood15. Wnt signaling refers to a series of sig- naling pathways or networks divided into non-canonical and canonical. Non-canonical signaling is a collection of signal transduction pathways that do not use TCF/β-catenin for their transcriptional outputs16. These are associ- ated with planar or apical-basal polarity and calcium signaling. In vertebrates, these are driven by non-canonical Wnts-4, -5a, -5b, -6, 7a, -7b, and -11. In Drosophila, the main non-canonical or Planar Cell Polarity (PCP) path- way uses Wnt indirectly17, but there are two characterized Wnts that do not signal through the canonical path- way. Wnt5 binds to the receptor Ryk and mediates axonal pathfinding18, while Wnt4 functions through PTK7 in opposition to canonical Wnt119, 20 to regulate polarity, cell migration and invasion21, 22. 1Yale-NUS College, 12 College Ave West, #01- 201, Singapore, 138610, Republic of Singapore. 2Duke-NUS Medical School, 8 College Road, 169857, Singapore, Republic of Singapore. 3Department of Biological Sciences, National University of Singapore, Block MD6, Centre for Translational Medicine, Yong Loo Lin School of Medicine, 14 Medical Drive, Level 10 South, 10-02M, Singapore, 117599, Republic of Singapore. Jahnavi Suresh and Nathan Harmston contributed equally to this work. Correspondence and requests for materials should be addressed to N.S.T. (email: nicholas.tolwinski@yale-nus.edu.sg) Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 1 www.nature.com/scientificreports/ The canonical Wnt signaling pathway is largely mediated via β-catenin binding to the transcription factor TCF4, 5. TCF binds to DNA through a consensus sequence, CCTTTGATCTT, at genes it activates in conjunc- tion with β-catenin, and at the same site with Groucho/TLE at genes it represses. Genes at Gro/TCF sites can be de-repressed by Wnt signaling, where Gro and β-catenin competitively bind to TCF setting up a switch where β-catenin can remove Gro/TCF leading to derepression, or replace Gro/TCF with β-catenin/TCF leading to acti- vation23. Additionally, genes can be actively repressed upon signaling activation through TCF/β-catenin bind- ing to a novel consensus site, AGAWAW24, or through a second repressor Coop25. Specificity can be increased through helper sites bound by the C-clamp region15, 24, 26–31. An embryonic system to assess direct and indirect Wnt transcriptional targets In vertebrates, the four TCF gene family members function as both transcriptional repressors and activators32. Drosophila has only one gene that encodes TCF, which must perform both functions, making this system simpler to manipulate genetically. This feature that was recently used to study activation and derepression of Wnt targets in Drosophila tissue culture cells lacking TCF33. In studies of TCF in fly embryos, the difference between the two functions of TCF becomes apparent when loss of function mutants are compared to dominant negative TCF transgenes34. Loss of function embryos show a loss of patterning, but the embryos remain large34. In contrast, expression of dominant negative TCF leads to a small embryo that lacks patterning5. This finding led us to propose that the two roles of TCF might be separable at the transcriptional level, and led us to develop tools to analyse transcription in vivo. p p y p We focus on developing methods for assessing transcriptional programs downstream of Wnt signalling, and identifying the processes and mechanisms involved. To this aim, we developed a naïve embryo system in which we can activate or repress different forms of Wnt signaling at various levels in the different pathways. This system allowed us to dissect the effects of Wnt signalling at both the phenotypic level of the whole organism and at the molecular level. Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z Results D l This form of TCF lacks the Arm binding region, the Groucho binding sequence (GBS) and the C-clamp that are required for β-catenin binding, Gro repressor binding, and additional target specificity respectively (Supplemental Fig. 1)15, 27. This makes tcf∆N-short a strong dominant negative form of TCF as it is unable to release DNA, interact with Gro or recruit the transactivating domain of β-catenin effectively blocking both activation and de-repression and leading to small, un-patterned embryos (Fig. 1)3. We were unable to generate the perfect intermediate condition, or tcf maternally and zygotically mutant embryos as the genetics were too complicated, instead we overexpressed a longer form of tcf∆N (tcf∆N-long, S1). This TCF gene lacks only the N-terminal β-catenin binding domain and retains the GBS and C-clamp maintaining Gro dependent repression and de-repression, but lacks β-catenin dependent activation. This form of TCF does not respond to β-catenin and phenocopies loss of TCF in embryos (Figs 1 and S1)34.h p p p y g The classic wingless phenotype in Drosophila embryos shows a small denticle covered embryo46. Similar phe- notypes were observed for other strong loss of function alleles of Wnt signaling genes such as arm/β-catenin and dishevelled47, 48. tcf∆N-long expressing embryos, which lack transcriptional activation in response to Wnt activa- tion, are large34, whereas tcf∆N-short embryos, which lack activation in response to Wnts but retain repression of Wnt targets, are small5. Under both conditions, patterning and the cell-fate decisions are disrupted in the same way (all epidermal cells make denticles), which suggests that transcriptional activation is required for differenti- ation and cell-fate determination, while regulation of transcriptional repression is required for cell proliferation and embryo size (Fig. 1). y g Another way to establish the intermediate phenotype (large, un-patterned embryos) was the expressio a dominant negative version of Arm (DisArmed) where the transactivating region of the C-terminus is del 2 www.nature.com/scientificreports/ ntificreports/ Figure 1. Mutations affecting components of the Wnt signalling pathway lead to various developmental phenotypes. (A) Phenotypes of the Wnt signaling conditions analysed, with large denticle covered embryos (DisArmed, tcf∆N-long), large naked embryos (Wg++, arm∆N), and small denticle covered embryos (tcf∆N- short, wg, Wnt4++/Otk++) compared to wildtype (WT). (B) Schematic view of embryo size and denticle coverage as shown in black. (C) Embryos were classified based on their observed phenotype in terms of size (normal/small) and differentiation status (assessed by denticle coverage; covered/naked). Results D l Both are highly correlating indicating that they are located within the same branch of the Wnt signalling pathway (i.e. non-canonical signalling). Genes are highlighted depending on whether they are differentially expressed (absolute fold change > 1.5, FDR < 10%) in both conditions or only in one. Figure 2. Transcriptional profiling identifies patterns of gene expression reflecting differences in canonical and non-canonical Wnt signalling. (A) Hierarchical clustering of gene expression profiles (z-scores) revealed a well-defined clustering of samples by branch of the Wnt signalling pathway perturbed, and the identification of four distinct clusters of genes with differing patterns of response. (B) Comparison of fold changes observed comparing tcf∆N-short against WT versus tcf∆N-short-arm∆N against WT. Both are highly correlated indicating that they are located within the same branch of the Wnt signalling pathway (i.e. canonical signalling). Genes are highlighted depending on whether they are differentially expressed (absolute fold change > 1.5, FDR < 10%) in both conditions or only in one. (C) Comparison of fold changes observed comparing Wnt4++ against WT versus Otk++ against WT. Both are highly correlating indicating that they are located within the same branch of the Wnt signalling pathway (i.e. non-canonical signalling). Genes are highlighted depending on whether they are differentially expressed (absolute fold change > 1.5, FDR < 10%) in both conditions or only in one. g g p g yf y p ( g DR < 10%) in both conditions or only in one. (C) Comparison of fold changes observed comparing Wnt4++ gainst WT versus Otk++ against WT. Both are highly correlating indicating that they are located within the same ranch of the Wnt signalling pathway (i.e. non-canonical signalling). Genes are highlighted depending on whether hey are differentially expressed (absolute fold change > 1.5, FDR < 10%) in both conditions or only in one. Expression profiling reveals distinct transcriptional programs reflecting observed pheno- types. We examined changes in gene expression in all of the conditions using microarrays (see Methods). Our preliminary studies using wild-type embryos expressing Wnts showed huge numbers of gene expression changes (data not shown), so we turned to the “naïve” embryo system. In this developmentally restricted sys- tem we identified 1,360 genes whose expression was significantly altered upon perturbing either canonical or non-canonical Wnt signalling (False Discovery Rate (FDR) < 1%). Results D l These embryos are not bicoid, nanos and torsolike mutants. Figure 1. Mutations affecting components of the Wnt signalling pathway lead to various developmental phenotypes. (A) Phenotypes of the Wnt signaling conditions analysed, with large denticle covered embryos (DisArmed, tcf∆N-long), large naked embryos (Wg++, arm∆N), and small denticle covered embryos (tcf∆N- short, wg, Wnt4++/Otk++) compared to wildtype (WT). (B) Schematic view of embryo size and denticle coverage as shown in black. (C) Embryos were classified based on their observed phenotype in terms of size (normal/small) and differentiation status (assessed by denticle coverage; covered/naked). These embryos are not bicoid, nanos and torsolike mutants. along with a mutation in the Pygopus binding site24. Expression of DisArmed blocks signaling by binding to TCF but not forming any activating complexes as transcriptional machinery is not recruited. We observed that these embryos showed a patterning phenotype where all cells made denticles, but the embryos were still large similar to loss of TCF (Fig. 1). This suggests that DisArmed blocks transcriptional activation, but still allows de-repression similar to TCF mutants34.f In order to understand the effects of perturbing the non-canonical Wnt pathway, we used the non-canonical Wnt4 ligand that functions in opposition to the canonical Wg19, 20. We have recently shown that uniform expres- sion functions in conjunction with the co-receptor PTK7 (Protein Tyrosine Kinase 7, Drosophila Offtrack or Otk) to oppose canonical signals in Xenopus and Drosophila, resulting in small un-patterned embryos similar to tcf∆N-short and wg19. As Wnt4 opposes Wg, we hypothesized that this would provide a non-canonical, not functioning through arm/β-catenin and TCF, readout. Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 3 www.nature.com/scientificreports/ Figure 2. Transcriptional profiling identifies patterns of gene expression reflecting differences in canonical and non-canonical Wnt signalling. (A) Hierarchical clustering of gene expression profiles (z-scores) revealed a well-defined clustering of samples by branch of the Wnt signalling pathway perturbed, and the identification of four distinct clusters of genes with differing patterns of response. (B) Comparison of fold changes observed comparing tcf∆N-short against WT versus tcf∆N-short-arm∆N against WT. Both are highly correlated indicating that they are located within the same branch of the Wnt signalling pathway (i.e. canonical signalling). Genes are highlighted depending on whether they are differentially expressed (absolute fold change > 1.5, FDR < 10%) in both conditions or only in one. (C) Comparison of fold changes observed comparing Wnt4++ against WT versus Otk++ against WT. Results D l Hierarchical clustering of these genes revealed distinct expression patterns associated with perturbations affecting these separate branches of the Wnt signalling pathway (Figs 2A and S2A). Expression of DisArmed showed a milder phenotypic change compared to WT, and this was mirrored in the similarity of their expression profiles (Fig. 1A). Overall, the transcriptional changes segregated according to whether the genetic perturbation was affecting the canonical (arm∆N, tcf∆N-long, tcf∆N-short or Wg++), or non-canonical signalling (Wnt4++, Otk++). Full results of differential expression analyses and associated enrichments for biological processes and pathways are reported in Suppl. Tables 1 and 2 respectively. We compared our findings to a recent Drosophila Wnt-dependent transcription paper by looking at 42 genes (Fig. S3) that were reported as changing33. We find that known Wnt genes like fz3 and nkd are activated by Wg++ and arm∆N but blocked by tcf∆N-short. Interestingly, we find a strong activation of the patched gene in non-canonical Wnt4 activation. Within the canonical pathway, Arm primarily signals through TCF19, as such the expression profiles of tcf∆N-short-arm∆N and tcf∆N-short are highly similar (Figs 2A and S2A) and show highly correlated changes in gene expression compared to baseline expression in “naïve” embryos (referred to as WT for the purpose of expression analysis Fig. 2B), illustrating that these are epistatic. Wnt4 has been reported as primarily signalling via PTK7/Otk19. In keeping with this, we found that overexpression of Wnt4 and Otk had highly correlated tran- scriptional responses compared to WT (Fig. 2A,C), providing further evidence of their functional interaction, epistasis and location within the same branch of the Wnt signalling pathway.i p g g p y Across all conditions, we identified four major clusters of genes (I-IV, Figs 2A and S2B,C), each of which was associated with distinct enrichments for biological processes, pathways and transcription factor binding sites (TFBSs) (Fig. 3). Cluster I showed reduced expression upon stimulation of canonical Wnt signalling via Wg++ or arm∆N and increased expression upon overexpression of the non-canonical branch (Fig. S2D). This cluster was enriched for processes associated with development, differentiation, cell-cell communication and morpho- genesis (Fig. 3A), and contained several transcription factors (TFs). The enrichment for Trl and TCF motifs suggests that cluster I may contain direct targets of canonical Wnt-mediated repression (Fig. 3E). Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z Results D l Genes present in cluster II are downregulated by overexpression of Wnt4 or Otk (Figs 2A and S2D) and hence represent a set of genes that are putatively repressed by non-canonical Wnt signalling, but that also show upregulation upon the loss Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 4 www.nature.com/scientificreports/ Figure 3. Functional annotation of clusters of genes with different responses to perturbing Wnt signalling. (A) Gene ontology (GO) enrichment for cluster I (B) GO enrichment for cluster II (C) GO enrichment for cluster III (D) GO enrichment for cluster IV. Where applicable ten representative GO terms (FDR < 5%) are displayed to summarise the enrichment profile for each cluster (see Methods). (E) TFBS enrichment (FDR < 10%) of each cluster. Figure 3. Functional annotation of clusters of genes with different responses to perturbing Wnt signalling. (A) Gene ontology (GO) enrichment for cluster I (B) GO enrichment for cluster II (C) GO enrichment for cluster III (D) GO enrichment for cluster IV. Where applicable ten representative GO terms (FDR < 5%) are displayed to summarise the enrichment profile for each cluster (see Methods). (E) TFBS enrichment (FDR < 10%) of each cluster. of the ability of Wnt to derepress genes repressed by TCF. Intriguingly, this cluster was enriched for glutathione metabolism genes (Fig. 3B), as well as for binding sites of the known Wnt target Dll49 (Fig. 3E). Recent studies show a pathway activated downstream of canonical Wnts, but independently of β-catenin, through the Wnt/STOP pathway50. Cluster III corresponded to a set of 256 genes that were upregulated by Wg++ but not by arm∆N. This cluster was enriched for genes involved in processes and pathways relating to cell cycle and proliferation (Fig. 3C), potentially indicating the presence of the Wnt/STOP pathway in Drosophila51. Cluster IV reflected a set of genes that were strongly upregulated by Wg++ or arm∆N and was enriched for chitin metabolism (Fig. 3D). As expected this cluster was enriched for binding sites of known targets of canonical Wnt signalling, including ap and ind52 (Fig. 3E).f p ( g ) Several studies have found that canonical and non-canonical signalling exhibit antagonistic effects on each other17, 53, 54. Wg++ and Wnt4++ correspond to non-endogenous overexpression capable of driving these distinct antagonistic components of the Wnt pathway. Comparing expression of Wg++ against Wnt4++, we identified 1,798 genes as differentially expressed (Fig. Results D l 4A, absolute fold change > 1.5, FDR < 10%), with up- and down-regulated genes appearing to be associated with distinct biological processes and functions. Genes upreg- ulated in Wg++ compared to Wnt4++ were associated with processes associated with cellular growth or the cell cycle, while those showing decreased expression were linked to cell adhesion, polarity and morphogene- sis (Fig. 4B). Investigation of the promoter sequences of genes upregulated by Wnt4 compared to Wg revealed enrichment for binding sites of important developmental genes (i.e., Ubx and cad) (Fig. 4C). These results support the antagonism of canonical and non-canonical Wnt signalling and show Wg and Wnt4 as regulating vastly dif- ferent downstream transcriptional programs.fi p p g Despite the differences in the size of tcf∆N-short and tcf∆N-long embryos (Fig. 1), their expression profiles appeared to be highly similar (Fig. 2A). Surprisingly, only 222 genes were identified as differentially expressed between these conditions (Fig. 4D, absolute fold change > 1.5, FDR < 10%). Genes upregulated in tcf∆N-short were enriched for glutathione transferase activity genes (i.e., GstD4, GstD3, GstD9 and GstD6) and included genes known to be upregulated in response to severe stress (i.e., TotA, TotC and TotX)55, whereas the set of genes downregulated was enriched for proteolysis and chitin metabolism functions (Fig. 4E). These results suggest an upregulation of stress response and GSH depletion/redox state as a potential mechanism responsible for the dif- ferences in the sizes of tcf∆N-short and tcf∆N-long embryos. TCF occupancy confirmed at potential targets by HA-ChIP-qPCR. For those genes whose expression changed in response to perturbations in canonical Wnt-signalling, we investigated publicly available TCF ChIP-seq data from Drosophila embryos56. As canonical Wnt-signalling primarily signals via TCF/Pan, we expected to see an enrichment of genes bound by TCF in several sets of differentially expressed genes (i.e., Wg++, tcf∆N-short or tcf∆N-long). However, we observed that only those genes upregulated by Wg++ were Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 5 www.nature.com/scientificreports/ m/scientificreports/ Figure 4. (A) Volcano plot of differentially expressed genes identified between Wg++ and Wnt4++. Significantly differentially expressed genes (absolute fold change > 1.5, FDR < 10%) are highlighted in red or blue for upregulated and downregulated genes respectively. (B) GO enrichments and, (C) TFBS enrichments for genes identified as upregulated (red) and downregulated (blue) between Wg++ and Wnt4++ implicates Wg and Wnt4 as driving different downstream transcriptional programs. Results D l differences between whole embryos and our naïve system) could potentially explain this lack of enrichment for TCF binding. Th i l b i t h d l d ll HA t d i f f f t f i t t t b i t if between whole embryos and our naïve system) could potentially explain this lack of enrichment for TCF binding. The simple embryonic system we have developed allows HA-tagged isoforms of factors of interest to be intro- duced into the system, making it possible to perform ChIP experiments against chromatin binding factors that either lack or only have low quality antibodies. We randomly selected 23 genes whose expression profile mir- rored the size changes observed for tcf∆N-short, tcf∆N-long and Wg++ (Fig. 5A,B) (cluster IV). We performed ChIP-qPCR on their promoters from embryos expressing HA-tagged tcf∆N-long or HA-tagged tcf∆N-short con- structs (Fig. 5C), to investigate whether these genes are direct or indirect target of Wnt signalling in this system. In addition, we investigated whether H3K27me3, a histone mark associated with repressed and bivalent genes57, was present at this set of gene promoters (Fig. 5D). We identified two genes (CG13806 and CG7252) whose promot- ers were bound by tcf∆N-short-HA and showed high levels of H3K27me3 in tcf∆N-short embryos, suggesting that these genes are direct targets of Wnt/β-catenin repression in our system. Peritrophin-15A was found to lack tcf∆N-short-HA at its promoter but showed high H3K27me3 signal, suggesting that this gene is an indirect target of Wnt/β-catenin repression, whose expression is regulated, at least in part, via repressive histone modifications. Blocking Apoptosis restores cells to epidermis. The differences in size between the tcf∆N-long and tcf∆N-short can be explained at least in part by two processes, either the embryo is growing less or cells are undergoing more apoptosis. Our transcriptional analysis identified an upregulation of stress response in tcf∆N-short embryos (Fig. 4C,D), which could implicate either of the processes. To evaluate how expression of tcf∆N-long or tcf∆N-short influences cell division and apoptosis in our system, we performed immunostain- ing using anti-phospho-H3 antibody and anti-cleaved caspase 3, respectively. Immunostaining was performed on the embryos collected from flies overexpressing Wg and used as control in the experiment. As apoptosis begins at stage 11–12 during Drosophila embryogenesis58, we chose stage 14 embryos for immunostaining. Results D l (D) Volcano plot of differentially expressed genes identified between tcf∆N-short and tcf∆N-long. Significantly differentially expressed genes are (absolute fold change > 1.5, FDR < 10%) are highlighted in red or blue for upregulated and downregulated genes respectively. (D) GO enrichments for upregulated (red) and downregulated (blue) between tcf∆N-long and tcf∆N-short implicates an upregulation of genes involved in glutathione metabolism and stress in tcf∆N-short. Figure 4. (A) Volcano plot of differentially expressed genes identified between Wg++ and Wnt4++. Significantly differentially expressed genes (absolute fold change > 1.5, FDR < 10%) are highlighted in red or blue for upregulated and downregulated genes respectively. (B) GO enrichments and, (C) TFBS enrichments for genes identified as upregulated (red) and downregulated (blue) between Wg++ and Wnt4++ implicates Wg and Wnt4 as driving different downstream transcriptional programs. (D) Volcano plot of differentially expressed genes identified between tcf∆N-short and tcf∆N-long. Significantly differentially expressed genes are (absolute fold change > 1.5, FDR < 10%) are highlighted in red or blue for upregulated and downregulated genes respectively. (D) GO enrichments for upregulated (red) and downregulated (blue) between tcf∆N-long and tcf∆N-short implicates an upregulation of genes involved in glutathione metabolism and stress in tcf∆N-short. Figure 4. (A) Volcano plot of differentially expressed genes identified between Wg++ and Wnt4++. Significantly differentially expressed genes (absolute fold change > 1.5, FDR < 10%) are highlighted in red or blue for upregulated and downregulated genes respectively. (B) GO enrichments and, (C) TFBS enrichments for genes identified as upregulated (red) and downregulated (blue) between Wg++ and Wnt4++ implicates Wg and Wnt4 as driving different downstream transcriptional programs. (D) Volcano plot of differentially expressed genes identified between tcf∆N-short and tcf∆N-long. Significantly differentially expressed genes are (absolute fold change > 1.5, FDR < 10%) are highlighted in red or blue for upregulated and downregulated genes respectively. (D) GO enrichments for upregulated (red) and downregulated (blue) between tcf∆N-long and tcf∆N-short implicates an upregulation of genes involved in glutathione metabolism and stress in tcf∆N-short. 6 Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z www.nature.com/scientificreports/ Figure 5. HA-ChIP followed by qPCR in embryonic system identifies putative targets of Wnt/TCF mediated repression. (A) Cluster IV is contains a series genes that showed strong upregulation in Wg++ and arm∆N, mild downregulation in tcf∆N-long, and strong downregulation in tcf∆N-short indicating that these genes are under regulation by canonical Wnt signalling. Results D l (B) Expression profiles of 23 randomly selected genes from cluster IV (C) Promoter occupancy by HA ChIP of binding of tcf∆N-short and tcf∆N-long at selected promoters, and (D) H3K27me3 enrichment at selected promoters in tcf∆N-short and tcf∆N-long identifies CG13806 and CG7252 as genes which are potentially directly repressed by Wnt/TCF signaling. Figure 5. HA-ChIP followed by qPCR in embryonic system identifies putative targets of Wnt/TCF mediated repression. (A) Cluster IV is contains a series genes that showed strong upregulation in Wg++ and arm∆N, mild downregulation in tcf∆N-long, and strong downregulation in tcf∆N-short indicating that these genes are under regulation by canonical Wnt signalling. (B) Expression profiles of 23 randomly selected genes from cluster IV (C) Promoter occupancy by HA ChIP of binding of tcf∆N-short and tcf∆N-long at selected promoters, and (D) H3K27me3 enrichment at selected promoters in tcf∆N-short and tcf∆N-long identifies CG13806 and CG7252 as genes which are potentially directly repressed by Wnt/TCF signaling. enriched for TCF binding. Both technical (e.g., antibody specificity) and biological factors (e.g. differences between whole embryos and our naïve system) could potentially explain this lack of enrichment for TCF binding. The simple embryonic system we have developed allows HA-tagged isoforms of factors of interest to be intro- duced into the system, making it possible to perform ChIP experiments against chromatin binding factors that either lack or only have low quality antibodies. We randomly selected 23 genes whose expression profile mir- rored the size changes observed for tcf∆N-short, tcf∆N-long and Wg++ (Fig. 5A,B) (cluster IV). We performed ChIP-qPCR on their promoters from embryos expressing HA-tagged tcf∆N-long or HA-tagged tcf∆N-short con- structs (Fig. 5C), to investigate whether these genes are direct or indirect target of Wnt signalling in this system. In addition, we investigated whether H3K27me3, a histone mark associated with repressed and bivalent genes57, was present at this set of gene promoters (Fig. 5D). We identified two genes (CG13806 and CG7252) whose promot- ers were bound by tcf∆N-short-HA and showed high levels of H3K27me3 in tcf∆N-short embryos, suggesting that these genes are direct targets of Wnt/β-catenin repression in our system. Peritrophin-15A was found to lack tcf∆N-short-HA at its promoter but showed high H3K27me3 signal, suggesting that this gene is an indirect target of Wnt/β-catenin repression, whose expression is regulated, at least in part, via repressive histone modifications. enriched for TCF binding. Both technical (e.g., antibody specificity) and biological factors (e.g. Results D l We could not detect much apoptosis at these stages, but we did observe a large number of cell divisions occurring in wild type and Wg++ embryos (Fig. 6A). There was a small increase in apoptosis in tcf∆N-short as quanti- fied in Fig. 6B. These differences indicate increased apoptosis as a potential mechanism to explain the smaller embryos in tcf∆N-short. An increase in apoptosis downstream of singnaling loss does lead to smaller embryos59. Additionally, Wg affects cell growth through Myc regulation bringing together growth and apoptosis as a possible explanation for why we see effects on embryo size60, 61. Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 7 7 www.nature.com/scientificreports/ Figure 6. Comparison of cell division and apoptosis markers in developing embryos. Embryos of the three canonical Wnt signaling conditions along with wildtype stained for the cell division marker phosphohistone H3 and apoptosis marker cleaved caspase3 as compared shown as whole embryos and with confocal close-ups of dividing nuclei and apoptosing cells. Quantification: done by counting dividing and dying nuclei where all comparisons show some level of significant difference with p values: PhosphoHistoneH3 tcf∆N-short vs wt (0.001751243), tcf∆N-long vs wt (0.008122443), Wg++ vs wt (0.01965572). Caspase - tcf∆N-short vs wt (5.328096e-06), tcf∆N-long vs wt (0.001070419), Wg++ vs WT (0.0001124644). Figure 6. Comparison of cell division and apoptosis markers in developing embryos. Embryos of the three canonical Wnt signaling conditions along with wildtype stained for the cell division marker phosphohistone H3 and apoptosis marker cleaved caspase3 as compared shown as whole embryos and with confocal close-ups of dividing nuclei and apoptosing cells. Quantification: done by counting dividing and dying nuclei where all comparisons show some level of significant difference with p values: PhosphoHistoneH3 tcf∆N-short vs wt (0.001751243), tcf∆N-long vs wt (0.008122443), Wg++ vs wt (0.01965572). Caspase - tcf∆N-short vs wt (5.328096e-06), tcf∆N-long vs wt (0.001070419), Wg++ vs WT (0.0001124644). Discussion We show that our naïve embryo system is amenable to quantitative analysis of transcriptional responses to pertur- bations targeting specific components and branches of the Wnt signalling pathway. The Wnt signalling pathway is of particular interest for this type of analysis as it consists of a canonical pathway with a well-defined mechanism of signal transduction, and a series of cell polarity pathways regulating a variety of cellular behaviours13, 62. These pathways can be thought of as a signalling network63, 64, where upon signal activation a poorly defined mechanism selects the pathway and outcome. Our system allowed the observation of specific transcriptional profiles that were clustered depending on which branch and which component was perturbed, illustrating clear differences in the sets of regulated genes and the involved biological processes. g g g p For the canonical pathway, much but not all of the cellular response is mediated through β-catenin and TCF transcriptional activation and repression51. For a strong activation of canonical Wnt signaling, we used overex- pression of Wg, which resulted in full embryo growth (aside from a head involution defect58). Phenotypically Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 8 www.nature.com/scientificreports/ embryos generated by overexpression of Wg appear the same as those with a gain of function Arm allele. However, we found a marked difference between the two conditions with a large number of genes activated by Wg++ but not by arm∆N (cluster III, Fig. 2A). These genes were associated with cell proliferation and the cell cycle. The results from our transcriptional analysis therefore suggest that Arm independent transcriptional activation occurs downstream of Wg, resulting in a gene cohort similar to the Wnt/Stop pathway, but occurring through a transcriptional rather than a protein stability mechanism50, 51, 53. g p p y For the strong loss of signaling condition, we performed two experiments using tcf∆N-short alone and tcf∆N-short along with arm∆N. We observed that the transcriptional profile was very similar illustrating that all Arm dependent transcription requires a form of TCF that can interact with Arm (Fig. 2B). This re-establishes the Arm/TCF interaction as the main source of transcriptional activity due to Arm transactivation5. For the intermediate loss of signaling condition, we expressed a tcf∆N-long construct, a condition where we observe a loss of patterning, with most epidermal cells producing denticles but without a strong effect on embryo size. The identical phenotype was produced by the dominant negative DisArmed allele24. Discussion As this is a highly-expressed form of the Arm protein that is immune to standard ubiquitin-mediated degradation and fails to act in transcriptional activation, the most likely explanation is that DisArmed binds to TCF, either sequestering it or preventing TCF from taking part in transcription. Either way, it perfectly phenocopies the absence of TCF (Fig. 1), and shows a very similar transcriptional profile to tcf∆N-long especially in clusters III and IV (Fig. 2A).hf y p pi g p y g The fourth condition was the use of a non-canonical Wnt4 molecule that signals through a different receptor (PTK7/Otk) opposing Wg. We found that in all three conditions Otk expression, Wnt4 expression, and Wnt4/Otk expression a similar cohort of genes was regulated (Fig. 2A), and that the highly-correlated expression profiles of Otk and Wnt4 compared to WT support that they reside within the same section of the Wnt signalling pathway (Fig. 2C). The set of genes upregulated by perturbing Wnt4/Otk did not correspond to those upregulated by the canonical Wg pathway, and instead represent a new gene set involved in morphogenesis, cell:cell communication and adhesion (Figs 3 and 4), a finding that is in keeping with and correlates with the polarity pathways that deter- mine cell shape and organization during epidermal development65–75. p g g p p Recently, Wang and colleagues found that redox state in germ line stem cells was regulated by Wnt signaling76. Different cellular states (i.e. proliferation, apoptosis, differentiation) have been associated with different redox states77, 78. Our transcriptional analysis indicated that glutathione metabolism, a metabolic pathway associated with regulation of redox potential in the cell, was regulated by Wnt signaling in our system. tcf∆N-short embryos showed an increase in the expression of genes associated with both glutathione metabolism and response to stress, whereas such a similarly strong change in expression was not observed in tcf∆N-long and Wg++. This finding suggests that embryonic Wnt signaling is required to modulate redox metabolism and its dysregulation in tcf∆N-short might result in increased stress and apoptosis79. g p p Our gene expression and ChIP data do not support a simple explanation for which genes are activated and which are repressed. Previous studies attempting transcriptional profiling of genes downstream of Wnt have found a wide range of results and thousands of genes14, 35, 80. Discussion For example, an early study looking at developmen- tally important transcription factors in Drosophila embryonic development found more than 1,000 sites where TCF was bound by ChIP-Chip35. Since these genes are not expressed in the same way in different cells, it is likely that a complex combinatorial system with multiple transcription factors or epigenetic regulation is in place. p y p p p g g p Overall, we present a useful in vivo Drosophila system that allowed us to characterize and bring together sev- eral aspects of Wnt signaling. We have looked at transcriptional repression and activation, moderate and strong canonical signaling conditions, and at the effects of opposing Wnt ligands. Showing the utility of our experimen- tal system, our transcriptional analysis led us to identify a novel, Drosophila β-catenin independent set of genes activated by overexpression of Wg and completely different gene cohorts downstream of Wnt4 and Wg. We envi- sion that detailed cellular and molecular studies in this naïve embryo system will allow to identify and test specific transcription factors and binding sites, and to delineate different signaling outcomes from different perturbations of Wnt and other signaling pathways. Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z ate a s a d et ods Fly strains and transgenics.  The bicoid, nanos, torsolike strain (bcdE1, nosL7, tsl146)40 was recombined with DaGal4 flies to make a triple mutant with Gal4 driver. UAS-Otk-3XHA19, UAS-Wnt481, UAS-DisArmed24, UAS- arm-∆N42, 44, 82, 83 were described previously. g TCF Long REV—TGAAACGCTAATAACGCCGTTATCGGAAGAh Enrichment for TFBS motifs was performed using AME (from the MEME suite89) against the JASPAR 2016 database90. Promoter regions were defined as 1 kb upstream/downstream of a gene’s Ensembl-annotated TSS (dm6, Ensembl version 86). p-values were corrected using fdr, with a TFBS classified as significant at an FDR of 10%. ChIP-seq analysis. ChIP-seq data for TCF at embryonic 0 h–8 h and 16 h–24 h was downloaded from modENCODE and lifted over from dm5 to dm656. A gene was defined as been bound by TCF if there was at least one identifiable peak within 2 kb of the gene’s Ensembl-annotated TSS (dm6, Ensembl version 86). A hyper- geometric test was used to calculate if TCF binding was overrepresented in defined sets of genes. Peaks were confirmed by ChIP-qPCR for 23 selected genes by comparing enrichment of precipitated chromatin to input chromatin. Embryo Collection and immunostainings. Embryos were collected 4–16 hrs after egg deposition and dechorionated with bleach and fixed with 4% formaldehyde in presence of heptane and sodium phosphate buffer and vortexed at maximum speed. Embryos were devitellinized in methanol/heptane and stored at −20 c until needed. Immunostainings were performed by standard methods with respective antibodies and Alexa Fluor dyes71–73. Whole embryo images were taken under 20X magnification, and confocal images were obtained at 63X. Quantification of fluorescence was done using ImageJ software tools91. FFT band pass filter was applied to the images for correction of any uneven illumination and horizontal scan lines acquired by phase contrast micro- scope followed by conversion to 40 pixels. For fluorescence quantification in the cells, small structure default pixels were optimized to 3 pixels and tolerance threshold was set at 5% using binary process function. Intensity density was obtained by using the particle analyser tool. Standard error was calculated using data from n = 3 for each condition and error bars were plotted. Antibodies. The following antibodies were used in the study: polyclonal Anti-phosphorylated histone H3 (Millipore, #06-570) and Cleaved caspase 3 (Cell signaling Technology, #9661) for embryo staining as cell divi- sion marker and apoptosis marker respectively. Hoechst stain was used to image nuclei (Invitrogen). Rabbit poly- clonal to HA tag (Abcam, #ab9110) antibody, mouse monoclonal (mAbcam6002) to Histone H3 (tri methyl K27) and Anti RNA polymerase II (Millipore #05-623B) were used in chromatin immunoprecipitation experiments. Real-Time PCR. g TCF Long REV—TGAAACGCTAATAACGCCGTTATCGGAAGAh g The PCR products were cloned into pENTR vectors (Invitrogen) and recombined using Gateway technology (Invitrogen) into pUASg.AttB.3XHA vectors for fly injection84. The DNA was injected into strain P[CaryP]attP2 68A4 by BestGene Inc California85. Microarray. We collected approximately 50 embryos per microarray experiment, with the control (WT) embryos being non-expressing naïve embryos. Embryos were staged to approximately 4–16 hour stages to allow for early and mid-stage expression. Extracted mRNA from the various genetic conditions was then analysed on Affymetrix Drosophila 2 microarrays by standard procedures. For each condition, we performed two biologi- cal replicates. Microarrays were normalised using GC-RMA. Prior to differential expression analysis, probesets were filtered by 1) removing probesets not mapping to a gene 2) removing probesets which mapped to multiple genes 3) if a gene had multiple probesets assigned to it the probeset with the largest IQR was used 4) probesets not showing expression greater than 2.5 in at least two samples and those mapping to non-canonical chromo- somes were removed. Following these preprocessing steps, differential expression analysis was performed using LIMMA51. Clustering of gene expression profiles was performed by converting gene expression to z-scores and clustering them using (1 − cor)/2 as a dissimilarity measure. To assess the stability of sample level clustering we used pvclust with 10000 iterations to calculate approximately unbiased (AU) p-values (Fig. S2A). All of the major expected clusterings remained stable. The set of samples relating to perturbation of the non-canonical pathway (Wnt4++, Otk++, Wnt4Otk++) did not form a stable cluster but were highly unstable between each other. The optimum number of clusters to cut the gene-associated dendrogram was determined by calculating the mean silhouette width over a number of different cluster sizes (Fig. S2B,C). f g Enrichment for Gene ontology was performed using a hypergeometric test from the GOStats package86. Results from GO enrichments were simplified for presentation purposes by filtering terms with a high seman- tic similarity87, all significant results (adjusted p-value < 0.05) from the enrichment analyses are available in Supplemental Table S2. Enrichment for pathways was performed using ReactomePA88, all significant results (adjusted p-value < 0.05) are available in Supplemental Table S2. For pairwise comparisons, enrichments per- formed using the set of genes used in the differential expression analysis as the background, whereas for the enrichments based on the clustering (Fig. 2A) the background was the set of genes identified as differentially expressed over all conditions. Materials and Methods Fly strains and transgenics Fly strains and transgenics. The bicoid, nanos, torsolike strain (bcdE1, nosL7, tsl146)40 was recombined with DaGal4 flies to make a triple mutant with Gal4 driver. UAS-Otk-3XHA19, UAS-Wnt481, UAS-DisArmed24, UAS- arm-∆N42, 44, 82, 83 were described previously. 1. wgIG22; Df (3L) H99 2. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-tcf∆N-long-3XHA 3. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-tcf∆N-short-3XHA 4. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Wg 5. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-DisArmed 6. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Wnt4 7. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Otk 8. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Wnt4, UAS-Otk 9. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-tcf∆N-short-3XHA, UAS-∆N-Arm 10. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-arm-∆N 11. bcdE1, nosL7, tsl146, da-Gal4 females x Ubi-NLS-GFP; UAS-myr-Tomato The TCF transgenes were made by PCR amplification of DNA from an ovarian library using primers: TCF Short FOR—CACCATGGTTTCTGGAATTTTCGGGCTAAGTCAA TCF Short REV—CGTTGTCGATCTGTCTTTTTTTCGCTTTTT 1. wgIG22; Df (3L) H99 2. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-tcf∆N-long-3XHA 3. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-tcf∆N-short-3XHA 4. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Wg 5. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-DisArmed 6. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Wnt4 7. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Otk 8. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-Wnt4, UAS-Otk 9. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-tcf∆N-short-3XHA, UAS-∆N-Arm 10. bcdE1, nosL7, tsl146, da-Gal4 females x UAS-arm-∆N 11. bcdE1, nosL7, tsl146, da-Gal4 females x Ubi-NLS-GFP; UAS-myr-Tomato TCF Short REV—CGTTGTCGATCTGTCTTTTTTTCGCTTTTT Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 9 www.nature.com/scientificreports/ g TCF Long REV—TGAAACGCTAATAACGCCGTTATCGGAAGAh RT qPCR primers set which can amplify 150–200 base pair fragments were designed (NCBI primer design tool) for the 23 short listed genes for evaluating ChIP assays from the indicated genomic regions. Realtime PCR was carried in a PikoReal96 Real Time PCR system (Thermo Scientific) following the manufactur- er’s instructions. Gene-specific transcription levels were determined in a 10 µl reaction volume in triplicate using QuantiFast SYBR Green and qPCR was conducted at 95 °C for 7 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 1 min. Two biological replicates were used to perform the experiment and results have been replicated. The specificity of the reaction was verified by melt curve analysis. Primer sequences are available in Supplemental Data 3. qPCR changes were calculated using the ∆∆Ct method92. Scientific REPOrTS | 7: 11092 | DOI:10.1038/s41598-017-11519-z 10 www.nature.com/scientificreports/ Chromatin Immunoprecipitation (ChIP). Embryos staged around 14–16 hrs were collected and cross linked with 1.8% formaldehyde in presence of heptane. Cell and nuclear lysis was done by respective lysis buff- ers (Easy Magna Chip kit (Millipore)) and Wheaton Dounce homogenizer was used to achieve uniform lysis. Chromatin was sheared for 18 cycles (30 sec ON and 30 Sec OFF) by sonication (Diagenode Bioruptor®) to a size range of 200 bp −1 kb chromatin fragments and the size was checked on a 2% agarose gel. Anti-HA tag (Abcam, #ab9110) and H3K27me3 (Abcam, #6002) antibodies have been used to immunoprecipitate the DNA. Chromatin was diluted 10 fold in Chip dilution buffer, control sample was saved and immune complexes were prepared and incubated at 4 °C overnight with respective antibody and protein A/G magnetic beads (Millipore). Subsequent washing of immune complexes was performed with low salt, high salt, LiCl immune complex wash and TE buffer, and then eluted in Elution Buffer. After reverse cross-linking and Proteinase K treatment, ChIP and control DNA samples were prepared and purified with columns (Millipore). IgG and IgM were used as negative controls in the ChIP assay. Samples were quantified by Quantitative PCR.: We designed RT qPCR primer sets that amplify 150– 200 base pair fragments (designed with the NCBI primer design tool) for the 23 short listed genes for evaluating ChIP. All reactions were carried out in technical triplicates and biological duplicates. Specificity of the reaction was verified by melt curve analysis. Primer sequences are available in Supplemental Data 3. g TCF Long REV—TGAAACGCTAATAACGCCGTTATCGGAAGAh Input DNA samples (cross-linked but not immunoprecipitated) were used as positive controls, whereas samples incubated with IgG antibody were used as negative controls. Percent input method was used to analyze the ChIP-qPCR data (https:// www.thermofisher.com/br/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/ chromatin-immunoprecipitation-chip/chip-analysis.html). Data availability. 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Tolwinski. g We thank Xiaoping Liu who made the original TCF constructs. We thank Eric Wieschaus and Ken Cadigan for fly strains, and J. Bischof and K. Basler for constructs. This work was supported by an Academic Research Fund (AcRF) grant (MOE2014-T2-2-039) from the Ministry of Education, Singapore to N. Tolwinski. g We thank Xiaoping Liu who made the original TCF constructs. We thank Eric Wieschaus and Ken Cadigan for fly strains, and J. Bischof and K. Basler for constructs. This work was supported by an Academic Research Fund (AcRF) grant (MOE2014-T2-2-039) from the Ministry of Education, Singapore to N. Tolwinski. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-11519-z Supplementary information accompanies this paper at doi:10.1038/s41598-017-11519-z Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Analyses of body composition charts among younger and older Chinese children and adolescents aged 5 to 18 years
BMC public health
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6,358
RESEARCH ARTICLE Open Access * Correspondence: fgxni@graduate.hku.hk 3Department of Orthopaedics and Traumatology, Nanfang hospital, Southern Medical University, Guangzhou 510515, China Full list of author information is available at the end of the article © 2012 Xiong et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Childhood obesity has become a major public health problem in China. The objective of this study was to examine the effect of age and sex on the relationship between fat-free mass (FFM) and fat mass (FM), fat-free mass index (FFMI) and fat mass index (FMI) in Chinese children using body composition chart analysis, and to compare the changing pattern with Caucasian and Japanese counterparts. Methods: A total of 1458 children (790 boys and 668 girls) between 5 and 18 years of age were studied to determine a body composition by bioelectrical impedance analysis. The relationship of FFM and FM, FFMI and FMI were delineated by body composition charts. Results: Different changing patterns in body composition were observed during 5-11y (younger age group), and 12-18y (older age group), with non-significant sex difference with FM and FMI for the younger age group and significant sex and age differences for the older age group. For the younger age group, simultaneous increase of FFM and FM was found in both genders. However, for the older age group, the increase in weight and BMI with age is largely due to the increment of FFM and FFMI in boys, and of FM and FMI in girls. In addition, different changing patterns in body composition exist between Chinese children and their Caucasian and Japanese counterparts, largely due to the higher fat mass component in Chinese subjects. Conclusions: Our results indicate that age- and gender-related changing patterns of body composition in Chinese children may differ at different growth stage, and differ with those in Caucasian and Japanese children at the same age period. Such changing patterns should be considered when designing the intervention proposal for childhood obesity in China. rds: Body composition chart, Body mass index, Fat-free mass index, Fat mass index, Chinese childre Analyses of body composition charts among younger and older Chinese children and adolescents aged 5 to 18 years Kai-Yu Xiong1, Hui He1, Yi-Ming Zhang2 and Guo-Xin Ni3* Kai-Yu Xiong1, Hui He1, Yi-Ming Zhang2 and Guo-Xin Ni3* Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Measurements All th All the measurements were taken in a local fitness club by a single well-trained examiner (H.H.) between October and November, 2011. Subjects were dressed in light sports attire. Height was measured to the nearest 0.01 cm with barefoot, using a stadiometer fixed to a wall in the laboratory. Body weight was measured to the nearest 0.01 kg on an electrical digital scale. Currently, there are a number of methods developed to assess the body composition [13]. It is rarely possible to make measurements on large samples with sophisti- cated techniques, like dual energy X-ray absorptiometry or deuterium dilution. Bioelectrical impedance analysis (BIA) is relatively inexpensive and easy to use, and therefore suitable for epidemiological studies. It can dis- tinguish between lean and fat tissue on the basis of their different conductance and impedance characteristics [14], and has been shown to be reliable and valid, even in children [15-17]. The body composition was assessed with bioelectrical impedance analysis (BIA) using a multi frequency bioelec- trical impedance analyzer (InBody J10, Biospace, Seoul, Korea). The measurements were performed according to general recommendations from the manufacturer, at least 2 h after ingestion of a light breakfast and after voiding the urinary bladder before the measurement. The body measurements and BIA were conducted in the morning on the same day. The body composition chart (BCC) was developed to delineate the relationships between FM (FMI) and FFM (FFMI) [18,19]. This simple chart provides four kinds of information at the same time: FM (FMI), FFM (FFMI), body mass index (BMI), and fat percentage, by which those who have a small BMI (slim) without excess fat can be distinguished from the slim who maintain a large FM. Wells [20] used BCC to examine the relationship of BMI and body fatness in reference Caucasian infants and children aged 1–10 years. More recently, Hattori et al. [19] reported the age-related patterns of change in FM/FMI and FFM/FFMI in Japanese subjects aged 11–59 years. Since it is known that human body composition is ethnicity dependent, these body composition charts may not be applicable to children of other ethnicity. This study was therefore carried out to examine the effect of age and sex on the relationship between FM and FFM, and FFMI and FMI in Chinese children in age ranging from 5 to 18 years using body composition chart analysis. Subjects and methods Subjects Based on the age-related changing patterns in both body composition charts, analysis of variance was used to in- vestigate the effect of sex and age on FFM and FM, as well as FFMI and FMI for two growth stages (5-11y, and 12-18y). All statistics were calculated using the SPSS statistical package (version 16.0). A total of 1458 individuals, 790 males and 668 females, between 5 and 18 years of age, living within the urban area of the municipality of Chongqing city, China partici- pated in this study. All participants were recruited from six local primary and secondary schools. Those who are willing to attend this project were selected. They were apparently healthy, and those engaged in any intense sport training programs were excluded. Full informed consent was obtained from the parents/guardians of each child before the start of the study. Ethical permission was Background the importance of growth patterns of body compos- ition. However, little is known regarding the age- and sex-related pattern of changes in body composition in Chinese children [5]. Obesity has become a major public health problem glo- bally, especially in children [1]. In China, with the recent economic development and lifestyle changes, the preva- lence of childhood obesity has increased rapidly [2,3]. National epidemiological survey showed that the preva- lence of childhood obesity in 2006 was 8.9% for boys and 5.3% for girls, which was 3.6 and 4.7 times higher than that of 1996 for boys and girls respectively [4]. The ongoing epidemic of childhood obesity has highlighted Growth involves the deposition of both fat mass (FM) and fat-free mass (FFM) components. Quantitative infor- mation on FM and FFM has been extensively reported on different population [6-9]. However, there is relatively little discussion of the way by which such data is expressed. The body mass index (BMI, weight/height2) is commonly used as a simple way of assessing excess weight. Nevertheless, there are well-known limitations regarding the use of BMI as it cannot distinguish FFM and FM, and not sensitive to the change in adiposity Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Page 2 of 9 granted by the Human Subject Review Committee, Beijing Sport University. granted by the Human Subject Review Committee, Beijing Sport University. during childhood [10-12]. BMI is not able to distinguish whether it is an increase in FM or a decrease in FFM that causes the increase in the body mass of a child, but it can be partitioned into the fat and lean components of FMI (FM index=FM/height2) and FFMI (FFM index=FFM/ height2). It is therefore desirable to investigate the rela- tionship of both compartments (FM and FFM, FMI and FFMI) when analyzing the body composition data. Measurements All th A secondary aim was to compare the results of Chinese children with those of Caucasian and Japanese counterparts at the same age period. Two types of body composition charts were used in this study. The details have been described elsewhere [19]. Briefly, the means of FFM and FM for male and female subjects of every age group were plotted on the body composition chart 1 in common units (kg). The x-axis represents FFM and the y-axis FM, with add- itional diagonal lines indicating weight and fat percent- age (% fat). Therefore, four kinds of information were provided at the same time in this chart: FM, FFM, weight, and % fat. The values of body compartment of FM (kg) and FFM (kg) measured with BIA were used to calculate two indices of height-normalized body composition: FFMI, calculated as FFM/height2, and FMI, calculated as FM/height2. The means of FFMI and FMI for male and female subjects are plotted on body composition chart 2 in common units (kg/m2). The x-axis represents FFMI and the y-axis FMI, with add- itional diagonal lines indicating BMI and % fat. As such, four kinds of information were provided at the same time in this chart: FMI, FFMI, BMI, and % fat. Results According to the general changing pattern, two age groups (younger age group: 5-11y, and older age group: 12-18y) were analyzed separately, and the results of ana- lysis of variance showed different changing patterns in body composition with age and sex. Although the FFM showed significant sex and age differences for both groups, the FM for sex difference was non-significant, but was significant for age difference for the younger age group, whereas significant sex and age differences for the older age group (Table 3). Between 5 y and 10 y, the age-related patterns in the boys and girls are similar with simultaneous increase of FFM and FM. During this period, boys exhibit higher FM and FMM than girls at each age point, except 8 y old with higher FM in girls. Age 11 appears to be the only age point where the girls have higher FM and FFM than boys. However, totally different changing patterns were found afterwards, char- acterized by a sharp rise of FM with a relatively slight in- crease in FFM for the girls, and a sharp rise of FFM with a relatively slight increase in FM for the boys. Results The means and standard deviations of height, weight, FM, FFM, BMI, FFMI, FMI and fat percentage were pre- sented in Tables 1 and 2. Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Page 3 of 9 Page 3 of 9 Table 1 Body size and body composition of Chinese boys by age group Age group N Height Weight BMI FM FMI FFM FFMI FAT Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 5 44 112.82 5.23 22.38 3.79 17.48 1.90 5.66 2.33 4.38 1.59 16.72 1.91 13.10 0.67 24.48 6.41 6 70 118.97 5.20 25.11 4.84 17.67 2.69 6.32 3.36 4.43 2.23 18.79 2.28 13.24 0.92 24.08 8.82 7 105 124.53 5.45 27.73 5.27 17.78 2.50 6.62 3.46 4.21 2.04 21.11 2.60 13.57 0.95 22.73 7.72 8 48 130.61 5.56 30.14 5.16 17.62 2.45 7.08 3.80 4.13 2.07 23.07 2.40 13.49 0.78 22.50 8.07 9 60 133.17 6.51 31.98 6.25 17.94 2.62 7.72 4.05 4.32 2.13 24.26 3.36 13.62 0.92 23.09 7.84 10 49 136.90 6.04 35.37 8.56 18.70 3.44 9.14 5.63 4.77 2.75 26.23 3.76 13.93 1.07 23.98 9.43 11 52 141.75 7.59 37.40 −8.71 18.45 3.04 8.78 5.11 4.32 2.29 28.62 4.99 14.13 1.19 22.31 8.15 12 63 151.85 9.45 47.42 13.61 20.28 4.13 11.35 7.10 4.85 2.83 36.07 8.51 15.42 1.99 22.61 8.69 13 60 158.95 8.27 52.45 10.98 20.64 3.36 10.69 6.44 4.23 2.50 41.76 7.05 16.41 1.57 19.42 8.29 14 50 165.06 5.54 59.52 11.53 21.79 3.85 12.62 7.05 4.62 2.57 46.90 6.50 17.17 1.92 20.10 8.06 15 55 169.92 5.81 63.83 10.54 22.07 3.25 13.63 6.28 4.73 2.20 50.20 6.16 17.34 1.53 20.63 6.78 16 50 169.15 4.56 64.42 8.37 22.48 2.50 12.59 4.92 4.38 1.66 51.83 5.11 18.10 1.47 19.37 5.76 17 39 170.79 9.28 65.13 11.84 22.16 2.92 12.73 6.52 4.31 2.08 52.41 7.55 17.84 1.50 18.83 6.29 18 45 169.07 5.05 67.79 9.60 23.72 3.27 14.49 7.14 5.10 2.60 53.30 4.89 18.62 1.15 20.85 7.69 Table 1 Body size and body composition of Chinese boys by age group Figure 1 shows the age-related changes in FM and FFM in male and female subjects from 5 to 18 y old. Results In terms Table 2 Body size and body composition of Chinese girls by age group Age group N Height Weight BMI FM FMI FFM FFMI FAT Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 5 24 110.76 4.22 20.99 2.62 17.08 1.66 5.28 1.88 4.29 1.45 15.70 1.26 12.82 0.59 24.63 6.17 6 35 116.52 4.94 22.60 4.27 16.56 2.42 5.41 3.07 3.93 2.15 17.19 1.79 12.63 0.63 22.59 8.74 7 44 121.76 6.77 24.86 4.33 16.64 1.76 5.72 2.70 3.81 1.68 19.14 2.45 12.87 0.76 21.96 7.23 8 43 128.19 5.66 28.50 5.87 17.24 2.69 7.25 3.77 4.36 2.12 21.25 2.77 12.88 0.84 24.17 7.78 9 48 133.56 5.75 29.38 4.12 16.43 1.63 6.40 2.39 3.60 1.37 22.99 3.09 12.83 0.91 21.56 6.40 10 35 139.27 7.57 35.07 9.20 17.85 3.23 8.95 4.97 4.51 2.25 26.11 5.06 13.34 1.34 24.08 7.78 11 44 147.48 8.43 41.31 10.37 18.75 3.05 10.71 5.57 4.80 2.16 30.60 5.57 13.95 1.19 24.70 7.21 12 66 152.80 7.81 43.76 8.40 18.61 2.37 10.67 3.92 4.53 1.51 33.09 5.54 14.08 1.28 23.77 5.52 13 52 155.34 10.13 49.75 8.42 20.50 2.23 13.93 4.53 5.76 1.70 35.82 5.45 14.74 1.11 27.64 5.58 14 50 156.72 6.51 50.53 6.87 20.57 2.59 14.84 4.01 6.07 1.76 35.69 4.36 14.50 1.21 29.07 4.86 15 66 158.17 6.07 54.55 7.80 21.78 2.68 17.51 4.79 7.00 1.88 37.04 4.02 14.78 1.05 31.70 4.91 16 61 159.20 5.20 55.19 6.78 21.74 2.11 17.52 4.24 6.91 1.60 37.66 4.02 14.83 1.05 31.58 5.26 17 57 159.14 5.57 54.70 6.35 21.58 2.09 17.60 3.93 6.95 1.51 37.10 3.59 14.63 1.01 31.92 4.44 18 43 157.55 5.04 52.25 4.97 21.06 1.97 15.92 3.58 6.44 1.54 36.33 3.18 14.62 0.90 30.40 5.14 Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Xiong et al. BMC Public Health 2012, 12:835 Page 4 of 9 Figure 1 Age-related change of the relation between FM and FFM in Chinese boys and girls aged 5-18y. M: male, F: female. d change of the relation between FM and FFM in Chinese boys and girls aged 5-18y. M: male, F: female. of fat percentage, it remains relatively unchanged for both sexes before 12 y, however, it decreases slightly for boys while it increases rapidly for girls afterwards. indicating a consistent increase of FFMI with relatively little change in FMI in the period. Results In contrast, the plots from 12 y onwards move toward the upper side of the chart for girls, which were characterized by a sharp rise of FMI with relatively little change in FFMI. At each age point, the boys have higher BMI than girls. The increase in male BMI between 5 and 18 y is due to increasing FFMI, while FMI remains relatively unchanged during this period. On the other hand, however, the girls show an increase in both components. The height-normalized changes in FFM and FM in both male and female subjects are shown in Figure 2. Table 4 showed the results of analysis of variance, with age and sex as the factors, of FFMI and FMI of two age groups. Although the FFMI showed signifi- cant sex and age differences for both groups, the FMI for sex difference was non-significant, but was signifi- cant for age difference for the younger age group, whereas significant sex and age differences for the older age group. Although boy has higher FFMI at each age point between 5 y and 11 y, plots are dense in a narrow range for both sexes, indicating that the body composition change is proportionate to the change in height squared. In male subjects, the plots from 11 y onwards move directly toward the right-hand side, Correlations of BMI with FM, FFM, FMI, and FFMI are shown in Table 5. BMI was positively and signifi- cantly correlated with FM and FMI weight for each sex at all ages. Correlations were moderate to high between BMI and FFM and between BMI and FFMI for each sex at each age, with significant difference from zero with the exception to FFM for girls at age 5. Results Table 3 Results of analysis of variance, with age and sex as the factors, of FFMI and FMI of 5–11 y group and 12–18 y group df FFMI FMI SS ms F P SS ms F P 5-11y Sex 1 43.67 43.67 45.28 <0.01 4.84 4.84 1.06 0.3 Age group 6 87.19 14.53 15.07 <0.01 40.87 6.81 1.49 0.18 Sex X age group 6 6.28 1.05 1.09 0.37 25.28 4.21 0.92 0.48 Residual 687 662.55 0.96 3140.92 4.57 12-18y Sex 1 1331.26 1331.26 659.07 <0.01 480.42 480.42 106.96 <0.01 Age group 6 285.48 47.58 23.56 <0.01 151.71 25.29 5.63 <0.01 Sex X age group 6 142.81 23.8 11.78 <0.01 184.46 30.74 6.84 <0.01 Residual 743 1500.78 2.02 3337.25 4.49 lysis of variance, with age and sex as the factors, of FFMI and FMI of 5–11 y group and 12–18 y Table 3 Results of analysis of variance, with age and sex as the factors, of FFMI and FMI of 5–11 y Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Xiong et al. BMC Public Health 2012, 12:835 Page 5 of 9 Figure 2 Age-related change of the relation between FMI and FFMI in Chinese boys and girls aged 5-18y. M: male, F: female. Figure 2 Age-related change of the relation between FMI and FFMI in Chinese boys and girls aged 5-18 e-related change of the relation between FMI and FFMI in Chinese boys and girls aged 5-18y. M: male, F: female. Figure 3 shows the changing pattern in body compos- ition in Caucasian [20,21] and Chinese subjects between 5 and 10 y. Although both Chinese and Caucasian sub- jects exhibited simultaneous increase of FFM and FM with age, differences exist in a number of aspects. Cau- casian girls had higher weight and BMI than boys, which is opposite to Chinese children. In addition, Chinese boys and girls have considerably higher weight and BMI than their Caucasian counterparts. The fat percentage ranges 20-30% for Chinese children, whereas 10-20% for Caucasian children. Moreover, although Caucasian chil- dren had constantly lower FM and FMI at each age point, by 10 y, they had similar FFM and higher FFMI than their Chinese counterparts. FFM and FFMI were in Japanese girls, however, even higher FM and FMI in Chinese girls led to the higher weight and BMI than their Japanese counterparts. Results Dur- ing 11–14 y, Japanese boys had constant FM, and an ob- vious decline in FMI. However, during the same period, FM increases and FMI remains almost unchanged in Chinese boys. Discussion In the current study, the relationship between FM and FFM, and FFMI and FMI in Chinese children was, for the first time, examined using body composition chart analysis. These charts graphically present the age and sex-related patterns of changes in body composition of Chinese children aged 5–18 years in this cross-sectional study. Although changes with age cannot be accurately determined from cross-sectional data due to potential cohort or generational differences, the current study Although the general trend in the changing pattern of body composition was similar, great disparity was also observed between Chinese children and their Japanese counterparts [19] between 11-17y (Figure 4). Higher Table 4 Results of analysis of variance, with age and sex as the factors, of FFM and FM of 5–11 y group and 12–18 y group df FFM FM SS ms F P SS ms F P 5-11y Sex 1 104.34 104.34 9.20 <0.01 7.58 7.58 0.463 0.50 Age group 6 11212.71 1868.79 164.74 <0.01 1429.40 238.23 14.53 <0.01 Sex X age group 6 275.07 45.85 4.04 <0.01 170.93 28.49 1.74 0.11 Residual 687 7793.28 11.34 11261.76 16.39 12-18y Sex 1 1331.26 1331.26 659.07 <0.01 480.42 480.42 106.96 <0.01 Age group 6 285.48 47.58 23.56 <0.01 151.71 25.29 5.63 <0.01 Sex X age group 6 142.81 23.8 11.78 <0.01 184.46 30.74 6.84 <0.01 Residual 743 1500.78 2.02 3337.25 4.49 Table 4 Results of analysis of variance, with age and sex as the factors, of FFM and FM of 5–11 y group and 12–18 y group analysis of variance, with age and sex as the factors, of FFM and FM of 5–11 y group and 12–18 y Table 4 Results of analysis of variance, with age and sex as the factors, of FFM and FM of 5–11 y Xiong et al. Discussion BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Page 6 of 9 Table 5 Correlations of BMI with Body Composition Variables Age group Boys Girls FM FMI FFM FFMI FM FMI FFM FFMI 5 0.93* 0.94* 0.59* 0.60* 0.93* 0.95* 0.39 0.62* 6 0.91* 0.93* 0.47* 0.56* 0.97* 0.97* 0.55* 0.60* 7 0.93* 0.93* 0.59* 0.64* 0.92* 0.93* 0.39* 0.51* 8 0.95* 0.96* 0.47* 0.68* 0.96* 0.97* 0.59* 0.76* 9 0.94* 0.95* 0.46* 0.66* 0.90* 0.87* 0.36* 0.55* 10 0.97* 0.96* 0.70* 0.73* 0.95* 0.94* 0.74* 0.83* 11 0.96* 0.95* 0.64* 0.79* 0.97* 0.96* 0.76* 0.86* 12 0.94* 0.90* 0.69* 0.79* 0.90* 0.86* 0.66* 0.81* 13 0.92* 0.90* 0.52* 0.71* 0.87* 0.87* 0.45* 0.66* 14 0.89* 0.90* 0.70* 0.81* 0.91* 0.91* 0.45* 0.81* 15 0.91* 0.92* 0.60* 0.81* 0.94* 0.96* 0.55* 0.86* 16 0.82* 0.79* 0.58* 0.75* 0.87* 0.86* 0.50* 0.63* 17 0.93* 0.92* 0.62* 0.82* 0.84* 0.89* 0.47* 0.74* 18 0.97* 0.96* 0.50* 0.79* 0.90* 0.92* 0.32* 0.71* * Significantly different from zero P<0.05. percentage decreases in boys, but increases in girls. Such gender differences appear to be largely due to the greater production of estrogen by the females and testosterone by the males [26]. The age-related changing patterns are generally characterized by a sharp rise of FM with a rela- tively slight increase in FFM for girls, and a sharp rise of FFM with a relatively slight increase in FM for boys. Un- like the previous stage, the increase in weight and BMI with age for this stage is largely due to the increment of FFM and FFMI in boys, and of FM and FMI in girls. Therefore, boys’ physiques become more lean and mus- cular in this growing period. Our findings were in ac- cordance with the report by Chumlea et al. [27], who investigated body composition for children aged 10–18 y, and found that boys have a constant annual increase in lean body mass without a constant annual increase in total body fat; whereas, girls have a small annual change in lean body mass with a constant annual increase in total body fat. Ethnic differences have been extensively reported on human body composition [28,29]. In the current study, different changing pattern in body composition was also found between Chinese children and their Caucasian and Japanese counterparts using BCC analysis. Discussion Between 5 and 10 y, Chinese boys and girls have considerably higher weight and BMI than their Caucasian counter- parts, which is almost entirely due to the higher fat mass components in Chinese children. Similarly, the higher weight and BMI in Chinese boys and girls in comparison to their Japanese counterparts between 11-17y is largely due to the higher FM and FMI components. Taken to- gether, our results revealed that the different changing pattern in body composition between Chinese children and their Caucasian and Japanese counterparts largely lies in the higher fat mass component found in Chinese subjects. Such ethnicity-dependent differences may be attributed to a number of factors, including the molecu- lar genetic factor and many other obesogenic environ- mental factors [29]. indicates several trends in body composition changes during growth period in Chinese children, which are somewhat different from Caucasian and Japanese sam- ples at the same age period. From 5 to 18 y, the weight and BMI tend to increase with age for Chinese subjects, and Chinese boys tend to have higher weight and BMI than girls. Similar trend has also been reported in a number of studies for Chinese children [22-24]. In Chinese traditional opinion, boys tend to receive extra attention in early life, and are expected to be strong, while girls should be slim. Hence, boys prefer to have a larger body size than girls [25]. What is more, using body composition chart analysis, this current study further illustrates how growth may affect lean and fat development differentially at different times. Between 5 and 11 y, concurrent increase of FFM and FM is generally observed, and the fat percentage was constant for both genders. Therefore, the weight gain is due to both FM and FFM components. The results of analysis of variance, with sex and age group as the fac- tors, revealed that the FM and FMI showed insignificant sex difference, whereas the FFM and FFMI showed sig- nificant sex and age differences. As higher FFM is found in boys both on an absolute and height-normalized basis, it is suggested that boys gain relatively more lean mass components than girls during this growing period. With the rapid economical development, the Chinese population, especially those living in urban areas, is undergoing a remarkable but undesirable nutrition, physical and behavioral transition, closely associated with obesity and many other disorders [30-32]. Discussion Being aware of this increasing health problem, a number of interventions have been taken for the prevention or treatment of overweight among children and adolescents since the 1990s [33-36]. Nevertheless, the efficacy of these interventions has been mainly inconclusive [37]. As recommended by numerous other studies, our find- ings also highlight the importance of intervention tar- geted at the overweight of Chinese children. More importantly, several implications from our findings may be useful for the interventional strategy. Firstly, at early Of interest, totally different changing pattern was found between 12 and 18 y. The results of analysis of variance for this period indicated that FM/FMI and FFM/FFMI showed significant sex and age differences with significant interactions. From 11 to 13 y, fat Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Page 7 of 9 Figure 3 Age-related change of the relation between FM and FFM (UP), FMI and FFMI (BELOW) in Chinese (solid lines) and Caucasian subjects (dashed lines, data from Ref. 21) between 5–10 ys. M: male, F: female. Figure 3 Age-related change of the relation between FM and FFM (UP), FMI and FFMI (BELOW) in Chinese (solid lines) and Caucasian subjects (dashed lines, data from Ref. 21) between 5–10 ys. M: male, F: female. Figure 3 Age-related change of the relation between FM and FFM (UP), FMI and FFMI (BELOW) in Chinese (solid lines) and Caucasian subjects (dashed lines, data from Ref. 21) between 5–10 ys. M: male, F: female. childhood period, Chinese boys tend to have larger body size than girls, which is opposite to that in Caucasian children. Such disparity is very likely due to an indistinct pre-school fatness loss phase [38] for Chinese boys, im- plying that our intervention should be taken on children, especially boy, as early as possible. Secondly, during early to mid-childhood, the gain in FFMI in Chinese children is much less than Caucasian children. Also, the lean mass components are constantly lower in Chinese girls than Japanese girls from 11 to 17 years. What is more, a typical preadolescent fat loss phase [36], which was observed in Japanese boys between 11 to 14 years, is less evident in Chinese boys. As such, special attention should be paid on the particular component(s), time point(s) and gender(s) when designing the intervention proposal. There are some limitations to our study. Abbreviations Abbreviations FFM: Fat-free mass; FM: Fat mass; FFMI: Fat-free mass index; FMI: Fat mass index; BMI: Body mass index; BIA: Bioelectrical impedance analysis; BCC: Body composition chart. Competing interest The authors declare no conflict of interest. Conclusion In conclusion, our results indicate that age- and gender- related changing patterns of body composition in Chin- ese children may differ at different growth stage. In addition, the changing pattern of body composition in Chinese children is different from that in Caucasian and Japanese counterparts at the same age period, largely due to the higher fat mass component found in Chinese subjects. Our findings have some important clinical Discussion Firstly, our study was carried out in 2011, about 10 years later than Harriot’s [19], and 30 years later than Fomon’s [21]. In addition, BIA was used to assess body composition in our study, whereas, deuterium dilution and densitometry in Fomon’s [21] and Harriot’s [19], respectively. Such inconsistence probably underestimated the differences between Chinese children and their Caucasian and Japanese counterparts. Another concern is that this study was conducted only in Chongqing city. There are regional variations and urban–rural difference in the growth of Chinese children [22,23,31]. Whether or not our findings Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 Page 8 of 9 Figure 4 Age-related change of the relation between FM and FFM (LEFT), FMI and FFMI (RIGHT) in Chinese (solid lines) and Japanese subjects (solid lines, data from Ref. 19) between 11–17 ys. M: male, F: female. Figure 4 Age-related change of the relation between FM and FFM (LEFT), FMI and FFMI (RIGHT) in Chinese (solid lines) and Japanese subjects (solid lines, data from Ref. 19) between 11–17 ys. M: male, F: female. can be generalizable to children in China needs further investigations on children from different regions of China. implications when designing the intervention proposal for childhood obesity in China. 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Horlick M: Body mass index in childhood–measuring a moving target. J Clin Endocrinol Metab 2001, 86:4059–4060. 36. Tian B, Lu S, Qian L, Zhang W, Zhang J: Impact evaluation on obesity control among primary school students in 4 cities in China. Chin J School Health 2006, 27:869–871. 12. Franklin MF: Comparison of weight and height relations in boys from 4 countries. Am J Clin Nutr 1999, 70:157S–162S. 37. Authors’ contributions bl f KYX was responsible for the study design. YMZ coordinated the study conduct. HH coordinated the study personnel, the recruitment phase and was responsible for data processing. GXN performed the statistical analysis Page 9 of 9 Page 9 of 9 Xiong et al. BMC Public Health 2012, 12:835 http://www.biomedcentral.com/1471-2458/12/835 and prepared the manuscript. All authors read and approved the final manuscript. 22. Chen TJ, Modin B, Ji CY, Hjern A: Regional, socioeconomic and urban–rural disparities in child and adolescent obesity in China: a multilevel analysis. Acta Paediatrica 2011, 100:1583–1589. 23. Pan H, Jiang Y, Jing X, Fu S, Jiang Y, Lin Z, Sheng Z, Cole TJ: Child body mass index in four cities of East China compared to Western references Ann Hum Biol 2009, 36(1):98–109. Acknowledgements This work was supported by National Science & Technology support program during the Eleventh Five-Year Period (2009BAK62B03). 24. Navder KP, He Q, Zhang X, He S, Gong L, Sun Y, Deckelbaum RJ, et al: Relationship between body mass index and adiposity in prepubertal children: ethnic and geographic comparisons between New York City and Jinan City (China). J Appl Physiol 2009, 107(2):488–493. Author details 1 h 1Teaching experiment center, Beijing Sport University, Beijing, China. 2Department of Anatomy, College of Sport Science, Beijing Sport Univers Beijing, China. 3Department of Orthopaedics and Traumatology, Nanfang hospital Southern Medical University Guangzhou 510515 China 1Teaching experiment center, Beijing Sport University, Beijing, China. 2Department of Anatomy, College of Sport Science, Beijing Sport University, 25. Li Y, Hu X, Ma W, Wu J, Ma G: Body image perceptions among Chinese children and adolescents. Body Image 2005, 2:91–103. Beijing, China. 3Department of Orthopaedics and Traumatology, Nanfang hospital, Southern Medical University, Guangzhou 510515, China. Beijing, China. 3Department of Orthopaedics and Traumatology, Nanfang hospital, Southern Medical University, Guangzhou 510515, China. 26. Forbes GB (Ed): Human body composition: growth, aging, nutrition, and activity. Springer-Verlag: New York; 1987. Received: 14 April 2012 Accepted: 26 September 2012 Published: 1 October 2012 27. Chumlea WC, Siervogel RM, Roche AF, Webb P, Rogers E: Increments across age in body composition for children 10 to 18 years of age. Hum Biol 1984, 55:845–852. Published: 1 October 2012 References 28. Morrison JA, Barton BA, Obarzanek E, Crawford PB, Guo SS, Schreiber GB: Racial differences in the sums of skinfolds and percentage of body fat estimated from impedance in black and white girls, 9 to 19 years of age: The National Heart, Lung, and Blood Institute Growth and Health Study. Obes Res 2001, 9:297–305. 1. Dehghan M, Akhtar-Danesh N, Merchant AT: Childhood obesity 1. Dehghan M, Akhtar-Danesh N, Merchant AT: Childhood obesity, l d i N J 2005 4 24 1. Dehghan M, Akhtar-Danesh N, Merchant AT: Childhood obesity, prevalence and prevention. Nutr J 2005, 4:24. prevalence and prevention. Nutr J 2005, 4:24. prevalence and prevention. Nutr J 2005, 4:24. 2. Ji CY: The prevalence of childhood overweight/obesity and the epidemic changes in 1985–2000 for Chinese school-age children and adolescents. Obes Rev 2008, 9(Suppl1):78–81. 2. Ji CY: The prevalence of childhood overweight/obesity and the epidemic changes in 1985–2000 for Chinese school-age children and adolescents. Obes Rev 2008, 9(Suppl1):78–81. 29. Liu A, Byrne NM, Kagawa M, Ma G, Kijboonchoo K, Nasreddine L, Koon Poh B, Ismail MN, Hills AP: Ethnic differences in body fat distribution among Asian pre-pubertal children: a cross-sectional multicenter study. BMC Publ Health 2011, 11:500. 3. Ji CY, Cheng TO: Epidemic increase in overweight and obesity in Chinese children from 1985 to 2005. Int J Cardiol 2009, 132:1–10. 3. Ji CY, Cheng TO: Epidemic increase in overweight and obesity in Chinese children from 1985 to 2005. Int J Cardiol 2009, 132:1–10. 4. Ding ZY: National epidemiological survey on childhood obesity, 2006. Zhonghua Er Ke Za Zhi 2008, 46:179–184. 4. Ding ZY: National epidemiological survey on childhood obesity, 2006. Zhonghua Er Ke Za Zhi 2008, 46:179–184. 30. Cui Z, Hardy LL, Dibley MJ, Bauman A: Temporal trends and recent correlates in sedentary behaviours in Chinese children. Int J Behav Nutr Phys Act 2011, 8:93. g 5. Li S, Zhang M, Yang S, Okada T, Iwata F, Harada K: Age- and sex-specific body composition of Chinese children. Acta Paediatrica 2005, 94:1139–1142. g 5. Li S, Zhang M, Yang S, Okada T, Iwata F, Harada K: Age- and sex-specific body composition of Chinese children. Acta Paediatrica 2005, 94:1139–1142. 31. Zhang J, Seo DC, Kolbe L, Middlestadt S, Zhao W: Associated trends in sedentary behavior and bmi among chinese school children and adolescents in seven diverse Chinese provinces. Int J Behav Med 2012, 19(3):342–350. 6. References Li M, Li S, Baur LA, Huxley RR: A systematic review of school-based intervention studies for the prevention or reduction of excess weight among Chinese children and adolescents. Obes Rev 2008, 9:548–559. 13. Wells JCK, Fewtrell MS: Measuring body composition. Arch Dis Child 2006, 91:612–617. 14. Chumlea WC, Guo S: Bioelectrical impedance and body composition: present status and future directions. Nutr Rev 1994, 52:123–131. 38. Garn SM, Clark DC: Trends in fatness and the origins of obesity Ad Hoc Committee to Review the Ten-State Nutrition Survey. Pediatrics 1976, 57:443–456. 15. Tyrrell VJ, Richards G, Hofman P, Gillies GF, Robinson E, Cutfield WS: Foot-to-foot bioelectrical impedance analysis. A valuable tool for the measurement of body composition in children. Int J Obes 2001, 25:273–278. doi:10.1186/1471-2458-12-835 Cite this article as: Xiong et al.: Analyses of body composition charts among younger and older Chinese children and adolescents aged 5 to 18 years. BMC Public Health 2012 12:835. 16. Li S, Zhang M, Yang S, Okada T, Iwata F, Harada K: Age and sex-specific body composition of Chinese children. Acta Paediatr 2005, 94:1139–1142. 17. Prins M, Hawkesworth S, Wright A, Fulford AJC, Jarjou LMA, Prentice AM, Moore SE: Use of bioelectrical impedance analysis to assess body composition in rural Gambian children. Eur J Clin Nutr 2008, 62:1065–74. 18. Hattori K, Tatsumi N, Tanaka S: Assessment of body composition by using a new chart method. Am J Hum Biol 1997, 9:573–578. 19. Hattori K, Tahara Y, Moji K, Aoyagi K, Furusawa T: Chart analysis of body composition change among pre- and postadolescent Japanese subjects assessed by underwater weighing method. Int J Obes 2004, 28:520–524. 20. Wells JC: A Hattori chart analysis of body mass index in infants and children. Int J Obes Relat Metab Disord 2000, 24:325–329. 21. Fomon SJ, Haschke F, Ziegler EE, Nelson SE: Body composition of reference children from birth to age 10 years. Am J Clin Nutr 1982, 35(5 Suppl):1169–1175.
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Budkavlen 2005: Special issue
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A Special Issue of A Special Issue of A Review for Swedish Ethnology and Folkloristics in Finland Founded by the Brage Society in 1922 A Review for Swedish Ethnology and Folkloristics in Finland Founded by the Brage Society in 1922 Published by the Institute For Folklife Research in Abo Published by the Institute For Folklife Research in Abo A SPECIAL ISSUE OF BUDKA VLEN Participants in this issue: Phil. Dr. Beate Binder, Berlin, Germany Hum. kand Nicklas Hagen, Abo Ph.D Sven-Erik Klinkmann, Vasa FD Lena Marander-Eklund, Abo FM Ann-Charlotte Palmgren, Abo Prof emeritus Nils Stora, Abo FM Ann-Helen Sund, Mariehamn Prof Ulrika Wolf-Knuts, Abo Prof Anna-Maria Astrom, Abo The Council of editorial staff John Hackman Lena Marander-Eklund Ulrika Wolf-Knuts Maria Schulman Anna-Maria Astrom Published with contributions from the Society of Swedish Literature in Finland and the Publishing Board at Abo Akademi University. ISSN 0302-2447 Abo 2005 Oy Formato Print Ab, Borga ISSN 0302-2447 Abo 2005 Oy Formato Print Ab, Borga Abo 2005 Oy Formato Print Ab, Borga special issue Binder's article is followed by one from Anna-Maria Astrom. In her article Astrom analyses the structures and identity of the Finnish capital Helsingfors, writing about the capital as symbol and meaning, and as a constructed and lived space. The article maintains the notion that the city of Helsingfors does not possess a negative identity, does not belong to either east or west, but rather is a point of integration where certain bridge-building functions and national elements combine to create the basis for a positive identity. One basis for a positive identity can be found in the residents' own accounts of their city, in which the city's different elements, buildings and suburbs, as well as symbols for social situations, nature, climate and daily life are used for the appropriation of space. The identity of the urban residents is based upon their own experiences of the city. This special issue of Budkavlen in English consists of articles that have been published in Swedish earlier in this journal. The articles fall into three themes, very different but of current interest in the fields of ethnology and folkloristics at Abo Akademi University. The themes are urbanity, narration and masculinity. The issue begins with eight interesting articles on these subjects, and finishes with two thorough presentations offolkloristics and ethnology at Abo Akademi. The theme of urbanity starts with an article written by Beate Binder. Binder writes about the re-structuring of the city's landscape using the German capital Berlin as an example. In her discussion about the re-structuring of the city's landscape, Binder takes two public events as starting points in order to explore the symbolic re- structuring of the city over recent years. The events cited are the day of German unification and the opening of the Mex- Artes Festival. In order to understand the cultural logic underlying this symbolic re- structuring of the city and its landscape, Binder proposes to concentrate not only on discourses concerned with old and new architectures and the (re-) naming of streets and places, monuments and commemoration sites, but to take into account the special quality of cultural performances and their "contribution" to the symbolic structure ofa city's landscape as well. The third article connected to urbanity is Nils Stora's article about fish fairs in Finland. This article discusses the history of fish fairs in Finland, and especially in the city of Abo as an urban environment. CONTENT CONTENT Ann-Charlotte Palmgren Budkavlen 2005, special issue Beate Binder National Tasks and Cosmopolitan Dreams. Constructing the "New, Old" Capital of Germany . Anna-Maria Astrom The Capital as symbol and Meaning. Helsinki as a con- structed and lived space Nils Stora Commerce and Carnival: The Fish Fair as a MeetingPlace . Lena Marander-Eklund The Significance ofNarratives and Narrating . Ann-Charlotte Palmgren In Search of the New Man. Masculinity in the New Men's Magazines. 5 8 . 21 37 . 48 . 54 Ann-Helen Sund The Formula 1 Race as a Pheno- menon. Aspects of Masculinity and Modernity . . 70 Sven-Erik Klinkmann Elvis, "Coolness" and Masculinity in the 50s USA . 86 Nicklas Hagen Narrative Analysis of Football Reports in a Finland-Swedish Daily Newspaper . . 102 ******************************* Finland Swedish Folklore Studies Ulrika Wolf-Knuts 114 What is a Swedish Etnology in Finland? Anna-Maria Astrom 121 Ann-Charlotte Palmgren Budkavlen 2005, special issue Beate Binder National Tasks and Cosmopolitan Dreams. Constructing the "New, Old" Capital of Germany . Anna-Maria Astrom The Capital as symbol and Meaning. Helsinki as a con- structed and lived space Nils Stora Commerce and Carnival: The Fish Fair as a MeetingPlace . Lena Marander-Eklund The Significance ofNarratives and Narrating . Ann-Charlotte Palmgren In Search of the New Man. Masculinity in the New Men's Magazines. 5 8 . 21 37 . 48 . 54 Ann-Helen Sund The Formula 1 Race as a Pheno- menon. Aspects of Masculinity and Modernity . . 70 Sven-Erik Klinkmann Elvis, "Coolness" and Masculinity in the 50s USA . 86 Nicklas Hagen Narrative Analysis of Football Reports in a Finland-Swedish Daily Newspaper . . 102 ******************************* Finland Swedish Folklore Studies Ulrika Wolf-Knuts 114 What is a Swedish Etnology in Finland? Anna-Maria Astrom 121 special issue In his article Stora asks the following questions: What attracts people to the fairs? Who are the visitors? What is the significance of the fish fairs today? In order to illuminate these questions a review of the concept of the fair is needed, and Stora gives a presentation of the fish fair from the 19th century to 1997 - a year in which a 5 5 total of 4 7 fish fairs were organised in Finland. Stora points out that during the long history of fairs, their nature has varied. Fish fairs are held both in towns and in the countryside, mostly on the coast, but also inland. The fairs mostly take place on market squares and in harbours, or along the banks of a river, where goods can be sold directly from boats. Stora stresses that the fair concept can be explored in terms of its financial, social and commercial, as well as carnival elements. at how it is reported, and at the same time, even if more indirectly, by looking at how the reporting role of the mass media is dealt with. In reporting on soccer, the articles assume a certain position of expertise and, paradoxically enough, both "objectivity" and solidarity with the team. Soccer is spoken of in terms of its own time, space and actors: expectations, solidarity, conflicts, ideas about fairness and feelings of joy or disappointment are expressed. A third theme, that of masculinity, is represented in three articles. Ann-Charlotte Palmgren's study is primarily focused on the male ideals in the new men's magazines Stitz and MG that began to be published in Finland in the middle of the 1990s. Palmgren looks at what male ideals the readers were being encouraged to aspire to and she wonders if these men's magazines are entering into a search for the new man. She therefore analyzed the men's magazines and interviewed readers and non-readers. Even if the intended group of readers differ between the two magazines, the ideal man is relatively stereotypical. He is fixated on gadgets and the body, sporty and at the same time distinguished and domestic when it comes to delicious recipes. Palmgren comes to the conclusion that it is the culture as a whole that has changed, and not only ideas about masculinity. The theme of narration is the subject of two of the articles included in this volume. special issue In her article, Lena Marander-Eklund discusses why it is important to be able to tell stories, and what significance narratives have for each individual. Marander-Eklund attempts to answer these questions using the three principal functions which Albrecht Lehmann (1978) distinguishes in personal experience narratives. She has taken her examples from her research on stories of childbirth (Marander-Eklund, 2000). Marander-Eklund further asks: Why are narratives an important form of communication - so important that they are used in drama-inspired pedagogy in some of today's schools? This is done with the help of the fable beast Baltus, which is a visual characterization of the basic structure of a story. It is used to teach the components which comprise a narrative - how one recounts a message in the form of a story. Ann-Helen Sund attempts, in her article, to describe Formula 1 as a modern/post- modern phenomenon: in part as a phenomenon in and of itself, and in part in terms of how the average TV audience experiences and describes it. The concept of manliness is also studied: what sort of manliness is manifest in this sport, and what is meant when Formula 1 is referred to as a "manly" sport? This article is based on interviews with students who are interested in Formula 1 and watch Formula 1 races on television. A small portion of the informants was not interested in Formula The other article on narration is written by Nicklas Hagen. This article analyzes the report of soccer articles about the team FF Jaro in the Finno-Swedish regional newspaper, Jakobstads Tidning. The basis used for the analysis is William Labov's theory about a complete narrative comprising six elements (abstract, orientation, complicating action, evaluation, result or resolution and coda), but focussing on the perspective of the reports. The objective is to obtain an understanding of what makes soccer newsworthy by looking 6 1. The study shows that the concept of prestige is central in Formula 1, and attempts to determine what this prestige encompasses. The significance of nationality in Formula 1 is also discussed, and how different nationalities are organized in the spectator's consciousness to build a whole entity. The article also briefly addresses the Formula 1 computer game and the experience of witnessing a Formula race in person. scene of the 1930's. BEATE BINDER On 3rd October 2002, the youngest German national holiday was celebrated in Berlin: the day of German unity. This newly established national holiday (having begun in 1991) is meant to commemorate the re- unification of Germany which took place on 3rd October 1990. A state-ceremony took place in the opera house and a street was held at the Pariser Platz and around the Brandenburg Gate. The most important symbolic center of the re-unified city turned into the grounds for a huge open-air festival. Like every year, German counties were encouraged to present their regional or local specialties, in the first place food and drink, such as fish from Hamburg, Hessian apple wine, Bavarian white sausages and so on, and to show folk dances, music or other "traditions" from their "home" countries. Dancing companies, cabaret, music or folklore groups presented their programs during the day on a huge stage which was put up for this occasion. The Ministries and the government had information desks and gave informative materials and gadgets to punters. A parade took place in the afternoon, which was also meant to present "Germany's feast" as a celebration of the re-unification and to show the richness and diversity of Germany's regions: folklore groups, groups wearing traditional costumes, and some brass bands walked along the alley Unter den Linden and showed the richness of German regional "heritage." This year, the celebration was even bigger, because Berlin hosted the central festivity which is the honor of that country - the chairman of the Bundesrat, the upper house of the German parliament which houses all the representatives of the German counties. On this occasion, the Berlin government thought of a special event to celebrate re-unification day. Thus the focal point of the celebration was the uncovering of the Brandenburg Gate, which had been under reconstruction during recent years. The famous German designer Willy Bogner lifted the covering out of the air and the rebuilt monument came to the fore - new and bright. The symbolic structure of this event is not very sophisticated: the day is meant to celebrate the national self-image of Germany as a federal state, and to commemorate the re-unification as an important turning point of national history. It aims at showing the progress of the unification process and stages the joy at the fall of the wall and the overcoming of the division of Germany. special issue Accordingly, one also has good reason to argue that coolness, as a quality in youth, is related to an entirely unique range of cultural formations which, at the beginning of the 1800' s in Europe and America, had a very specific and strong effect on the formation of popular culture. In this article, Sven-Erik Klinkmann discusses the history of the concept of "cool" as a complicated process of conversions, manifest in discourse, from signifying a complex black masculinity, a black bearing, with many meanings, to symbolizing Elvis Presley's rather playful and outer coolness and masculinity, expressed in his public 50's persona, the carnival-like King of Rock 'n' Roll. The third article on this theme is written by Sven-Erik Klinkmann. In his study of coolness as the central significant sign for youth in the American 50' s, the semiotician Marcel Danesi demonstrates that the concept of "cool" has its roots in the jazz 7 BEATE BINDER In the terms of Don Handelman, it is a public event "that presents the lived-in world". 2 Thus the celebrations and the feast offer Berliners as well as city visitors a pleasant atmosphere of consumption which enables to experience the "German-ness" of Germany and its new capital. Another short glimpse at another public event: the opening of the Mex-Artes festival which took place at the House of World 8 Cultures in the summer of 2002. The House of World Cultures describes itself as one of the leading centers for contemporary art of non-European origin. It provides a platform for projects and themes transcending borders and frontiers. This is also what the opening ceremony aims at. It announces the beginning of a rich cultural program - that is an art exhibition, concerts and film programs, round table discussions and symposiums all concerned with the contemporary culture of Mexico and German-Mexican relationships in terms of cultural exchange over the centuries. Cultures in the summer of 2002. The House of World Cultures describes itself as one of the leading centers for contemporary art of non-European origin. It provides a platform for projects and themes transcending borders and frontiers. This is also what the opening ceremony aims at. It announces the beginning of a rich cultural program - that is an art exhibition, concerts and film programs, round table discussions and symposiums all concerned with the contemporary culture of Mexico and German-Mexican relationships in terms of cultural exchange over the centuries. stages an atmosphere of multiculturalism and creativity, of importance and a "Good to be here" -feeling in terms of networking and showing oneself to the public. These two events are taken out of a huge amount of similar happenings which have taken place in Berlin during the last few months more or less randomly. While talking about the restructuring of the city's landscape, I shall take them as starting points in order to discuss the symbolic re- structuring of the city during the last years. Street party on the occasion of the arrival of the government - September 1999. Photo: Beate Binder BEATE BINDER In order to understand the cultural logic underlying this symbolic re-structuring of the city and its landscape, I propose to concentrate not only on discourses concerned with old and new architectures, the (re-)naming of streets and places, monuments and commemoration sites (in short, with the whole "architexture"3 of the city), but to take into account the special quality of cultural performances and their "contribution" to the symbolic structure of a city's landscape as well. Cultural Performances commemorations, About 1000 people came to the House of World Cultures that very evening. Advertised all over the city, the event obviously was able to pull in a crowd, and it offered a stage for all those who are involved in culture (politics), who are simply interested in culture and/or Mexico, and, last but not least, for all Mexicans and Latin Americans living in Berlin. Framed by the speeches of national representatives of both Germany and Mexico, the festival 9 Invitation card for the opening of the Mexartes Festival. events are also important "agents" in the structuring and defining of urban space. They offer stages for a special way of experiencing a city and, in addition, they take part in the production of a "discerning eye", which is what the anthropologist Yi Fu-Tuan named the eye which is able to read and interpret the meaning of urban space, and which perceives the historical heritage incorporated in the urban structure. 5 Of course - public events have always been of great interest for ethnographic research. For example, Emile Durkheim, Milton Singer, Victor Turner, or Clifford Geertz proposed that they are keys for the investigation of societies, because they enable ethnographers to grasp the symbolic structure, collective understandings or principles of social structures underlying a societal order. Or, as Don Handelman put it: "They constitute dense concentrations of symbols and their associations, which are of relevance to a particular people". 6 demonstrations, or in short all kinds of modem public "rituals" - inscribe meaning into the urban landscape on the one hand, and offer a space which allow people to experience the city in a particular way on the other. Per-Markku Ristilammi4 proposed that we compare the space of public events with those spaces that Foucault called heterotopias - spaces which reflect and comment on the hopes and fears of society. Invitation card for the opening of the Mexartes Festival. BEATE BINDER "Events", writes Ristilammi, "are inextricably linked to the surrounding world because they are designed to reflect and make an impression on the world outside. The visualizing of an [sic] utopian past and future is often a technique used in the managing of events." At the same time, public events bear mechanisms of opening and closure within them. They show who belongs and who will not. And last but not least the special atmosphere of public events effects people and opens them up for agency. Thus the space-time structure of public events allows people to join together, to confess to celebrated goals and symbols and to conceive of decisions for their engagement in something because of the emotional surplus which taking part of the event produces. p p p Today these assumptions are being criticized. Whereas Clifford Geertz 7 interpreted the Balinesian cock fight as a key symbol of the whole society, and Milton Singer8 saw cultural performances as elementary for the great tradition of Hindu Indian culture (to mention only those two classic accounts on cultural performances), these holistic interpretations are questioned today. It does not at seem to be clear if it is possible to "read a culture from the symbols of a cultural performance," warns for example Bailey. 9 And Don Handelman calls for careful consideration that a cultural performance or public event "among a particular people communicates only a version of their social order." Different versions or interpretations, according to Handelman, "overlap and conflict with one another, in the knowledge and experience, and affect they convey. If events contain keys to codes, then these unlock many doors, as much to labyrinths Therefore, as I would suggest, public 10 as to great halls and cosy kitchens." 10 is very much "in the making." Thus the transformation Berlin is undergoing on a material level is accompanied by transformation processes concerning the symbolic content of the urban landscape. For the former front-line city, a socialist capital and a capital waiting to become a capital and metropolis in its own right, Berlin must modify or even change the symbolic structure of its image and its representations in public spaces. as to great halls and cosy kitchens. Nevertheless and with this warning in mind, I suggest taking public events more seriously not only in ethnography as such but especially in urban anthropology. BEATE BINDER But before talking more about the theoretical framework which investigates the study of public events with sense, I would like to return to the city of Berlin for a moment where those above-mentioned events were set onto the scene. The special situation of Berlin during the last decade gives reason for the assumption that studying public events allows one to explore the question how the new-old capital of Germany is re- defined and - in a certain sense - produced. This means that the process of defining spaces and structuring the urban landscape is not only at work "as usual", but is also very dynamic and accompanied by a whole range of conflicts and discussions. Indeed, since the fall of the wall in 1989, the re- unification of Germany and Berlin, and last but not least the decision to move the parliament seat from Bonn to Berlin in 1991, turned Berlin's inner-city into an immense collection of cranes, construction fences and ditches and more. As soon as the first plans were rendered, the construction work was accompanied by a discourse on questions of national representation, the appropriateness of the planning and the emerging new tasks and functions of the city. Politicians as well as newspapers, panel discussions, exhibitions and "normal citizens" engaged in these questions of who, where and what of the transformation, the re-definition of the city and its urban landscape. Building a "new, old capital" - the symbolic transformation of Berlin The two events, the celebrations of 3rd October and the opening ceremony of the Mexartes festival, belong together insofar as both offer spaces in which the "New Berlin" is staged. They are meant to show different aspects of this newness in that they talk about a twofold problem which Berlin is confronted with today while trying to find a place on the mental map of European Capitals as well as in the international network of metropolises. And they are meant to produce places which are appropriate for staging these events. Since the fall of the wall in 1989, German unification in 1990 and the decision to move the seat of parliament from Bonn to Berlin, the city has been undergoing a process of symbolic transformation which is set on the scene in different places. Because of this, Berlin offers a special frame for investigation. The city is a "zone in transition" 11 , and this, the city's liminality (Turner), is visible last but not least in the very fact that the public spaces are not finally defined and classified in terms of symbolic structure and in terms of their appropriate uses. In this sense Berlin On the one hand, the task of national representation is questioned. Since the early 19th century, European capitals have been distinguished sites of national politics and culture. They have represented themselves as national cities, and they are still treated as products of different national histories. London was - and in a certain sense still is - an English, Paris a French, Helsinki a Swedish-Finnish, and Berlin a German city in the first place. Even though modern European cities have al ways been international, heterogeneous and 11 Map of the City Center: 1 House of World Cultures, 2 Pariser Platz In this context, the discourse produced the metaphor of the "Berliner Republic", which opened up a symbolic space for a bundle of different interpretations and meanings of the national. The metaphor structures time by separating the new period from its predecessors, namely the "Bonner Republik". Despite the somehow felt beginning of a new era, the discourse emphasizes the continuities between the old Federal Republic of Germany and the unified Germany in terms of democratic consent, the reliability of German politics, and the state's orientation towards Europe and the West. Building a "new, old capital" - the symbolic transformation of Berlin At the same time, the metaphor "Berliner Republic" structures space and emphasize the importance of the capital as a central site of national representation. Thus it refers to the city as symbol and as the output of the new political situation in general. Due to the high degree of "social present time reality" ascribed to the city, the discourse stresses cosmopolitan sites, European capitals have been first of all localized manifestations or peculiar urban versions of different national cultures and histories. It is not only the architecture and the cultural grammar of the built landscape which represents changing images of the national self. The capital serves at the same time as a stage for national representations, feasts and ceremonies which stage the central values and convictions the "imagined community" of the nation which it will follow. Indeed, since 1991 the question of what consequences the move of parliament will have for national and international politics has been under discussion. How will other countries perceive the new German capital and its buildings? Which kind of national representation is necessary and appropriate for a nation in the middle of Europe at the turn of the millennium in general and appropriate especially for Germany with its national past? 12 the question of whether the past is still alive in Berlin. Thus, while the discourse on the "Berliner Republic" opened a space for negotiations about national self-images and the possibility and necessity of national pride, even for Germans, it has to cope with the problem of representation. For in many respects Berlin represents an undesired national continuity, which is of history, or even better, the continuing effect of history on contemporary politics. By moving from Bonn to Berlin, the German government as well as the "Germans" in general are perceived as being confronted with the "burden" of German history more than ever (mit der Last der Vergangenheit). Advertisement for the new capital in a shop window in Tiibingen, October 1999. Photo: Beate Binder Building a "new, old capital" - the symbolic transformation of Berlin Whereas Bonn was a "neutral" place in terms of history and therefore able to represent the West-German will to begin anew in 1949, many consider Berlin as a place where history "really took place", and that means German fascism and the killing of millions of people, the beginning and the end of World War II, the Cold War and the division of Germany, of Europe, and, in the end, of the whole world, the fall of the wall and German and European unification - just to mention the most important turning points of20th century history. And all these events have left their traces in the spatial and symbolic structure of the cityscape. Therefore, for the former "front-line-city", the socialist capital, and the old centre of power to become a true capital city in its own right, Berlin must structure its historical narrative in a new way and find new modes of national representation in and through the city. And that is what the metaphor of the "Berliner Republic" tells us about. But apart from this national narrative t h d ith th t h "B li most important scenes of current social and political transformations taking place in late capitalism and post-socialism. A whole range of sociological as well as anthropological accounts narrates the story of the late modern city and renders pervasive pictures of the dramatic changes the political and cultural economy ofurban space is undergoing. On the one hand, they tell us about the emergence of a new urban underclass, living in damaged or ruined urban quarters, about slums and ghettos, about poverty and social exclusion. On the other hand, there are the new urban shopping and entertainment centres as well as the business quarters with their malls, sky-scrapers, and bank towers representing richness and economic growth. But apart from this national narrative touched on with the metaphor "Berliner Republic", a second metaphor, namely the "New Berlin", tells us about another story and about another problem the city has to cope with. Because cities, and especially big cities and metropolises, are now the The changes which urban landscapes are undergoing are reflections of the ongoing processes of social, political, and economic transformation which normally underline terms of globalization and/or second modernization. Many authors in this 13 metaphor of the "New Berlin" tells us about the "cosmopolitan dream" of becoming an economic and cultural metropolis. Building a "new, old capital" - the symbolic transformation of Berlin This dream is a strong force, and it structures the rhetoric of change and legitimizes a lot of different activities that are concerned with the rebuilding of the city. By envisioning the future role of Berlin as a "turntable between East and West" and as an important economic as well as political knot in the network of European cities, Berlin tries to render the attractiveness of its location. context have pointed out the emergence of a new symbolic economy and new city marketing strategies, which are mainly based on culture and cultural politics. In as far as the imaginability of cities "becomes the new selling point" - as Christine Boyer put it12 - the aesthetic and design of the urban landscape, its incorporated heritage and its new zones of attraction are strong and powerful resources for marketing a city. p g y Efforts to create a symbolic, selling economy produce social, political and cultural conflicts between local economies and the logic of globality. Cities have to accumulate ever more political, economic, and cultural functions, making them international, or ( even better) cosmopolitan. Thus the representation of cosmopolitanism within a city offers pervasive symbolic capital in the world-wide struggle for hegemony; it enables cities to enter and to take part in the global competition for economic and symbolic capital successfully. Insofar as Berlin encounters both the task to become more national and more global at the same time, the symbolic transformation of the city appears to be extremely dramatic. Behind the question of how to cope with the contradictory tasks of becoming national and cosmopolitan at the same time hides a field of political and cultural struggle and conflicts which accompany the restructuring not only of the city and its landscape but of the entire society. And this is what the term of the "New Berlin" tells us too. It tries to bundle all those efforts undertaken during the last decade in order to attract investors, developers and, last but not least, tourists to leave their money in the city. The economic situation of Berlin changed dramatically after German unification. Building a "new, old capital" - the symbolic transformation of Berlin While West Berlin used to function largely on the basis of an extensive politics of subsidies and East Berlin, as the capital of the GDR, had access to larger resources than any other East German city, Berlin now must enter the general economic and cultural competition of cities in order to gain ground on a national as well as on an international scale. Whereas the old production industry broke away on a large scale, the city aims at building up an economy mainly based on service industries. Tourism, media, and information and communication technologies are the main areas of engagement. Thus a lot of different efforts of the last decade aim at the modernization of the entire city. The This is even more so because of the former division of the city into two halves. Today two societies with different experiences and starting points come together within in the city of Berlin. And that is what the metaphor of the "New Berlin" tells us about as well: the vision of the re-unification of the two city halves and of the will of rejoining the divided metropolis into one smoothly functioning urban structure. With the two metaphors of the "Berliner Republic" and the "New Berlin" I have rendered the central problem that faces Berlin today. In short, the "New Berlin" aims at constructing a new image which is supposed to fit the tasks of representing both the national and the global. To reinvent the "new/old capital" means to produce new images which can tum the German and the Prussian Berlin into a world city by keeping its local traditions at the same time. To follow this field of struggles between 14 The Dome of the Reichstag building is open for public. Photo: Beate Binder. industrial production and the increasing importance of deal-making and selling investment have fundamentally changed the working and meaning of the symbolic economy of cities. While it is most important for cities to construe a legible and identifiable image nowadays, Zukin emphasizes that the material landscape itself became the most important visual representation of cities and thus plays a fundamental role in the construction of cities. The new symbolic economy is based on the appropriation and use of culture for the development and material reproduction of cities. Building a "new, old capital" - the symbolic transformation of Berlin This means that the very social and cultural conflicts in contemporary societies take place as struggles about modes of representation, aesthetics and their connected meanings. The new symbolic economy structures cities by making "decisions about what - and who - should be visible and what should be not, on concepts of order and disorder, and on uses of aesthetic power", to quote Sharon Zukin again. 14 Thus understanding cities as texts means taking the representational different symbolic systems and their respective logics, I propose starting with the texture of the city, or even better, the changing pattern(s) of this texture. Understanding cities as texts means to interpret the buildings, places, streets, parks, monuments - in short the whole urban street design and built environment - as a culturally encoded text of urbanity. The "architexture" of a city represents historical imaginations, political visions and myths, social memories and cultural nostalgia living in the society. Cities with their spatial order, their architecture, buildings, and commemoration sites, represent historical imaginations and political visions, social hierarchies and cultural constructions of the self. And as far as cities are places and locations of cultural myths, memories and nostalgia as well as of contemporary power and hegemony, the constantly changing physical environment tells of the social, political, and cultural changes a society is undergoing. As Sharon Zukin pointed out in her book "The Cultures of Cities", 13 the decline of 15 World Cultures is located close to the newly built Kanzleramt, the seat of the Chancellor. The Pariser Platz, which is maybe a 15 minute walk away from this place, is situated close to the Reichstags-building. practices of different social groups and the aesthetics or visual means of inclusion and exclusion as starting points for further investigation. But, as I have proposed at the beginning, not only the architecture and design of a city structures its texture, but so do social practices themselves and public events which take place in urban space. In the remainder of this article I would like to concentrate on this last point. Because, in the context of the symbolic transformation processes of Berlin, new public events are invented and/or incorporated into the urban landscape. These public events are meant to make the content and possibilities of "New Berlin" visible, and they open these contents and possibilities up for experience. Building a "new, old capital" - the symbolic transformation of Berlin As cultural representations these events become real in order to define and structure urban space, and in order to articulate political positions and power. Despite their closeness, the two places belong to different symbol systems in terms of the national and the cosmopolitan. Gerhard Schroder, the German chancellor, characterizes the House of World Cultures as follows: "When we say that Germany has to become more international, then the House of World Cultures is doing real pioneering work." 15 While the House of World Cultures represents the world's cultural diversity, it simultaneously shows the openness of Berlin towards the diversity of its own citizens. It stages new ways of cultural hybridization, products of cultural contact and its influence - especially on the high arts. First and foremost, it opens a space for the intellectual exchange on questions of cultural globalization. Or, as Homi Bhahba put it last year: "In this momentous of transition, the House of World Cultures - like Berlin itself - is becoming a meeting place for dialogues between cultures, a bridge between the past and the present, East and West, North and South." 16 To be able to follow up these mechanisms of public events, I will have a somewhat closer look at the events mentioned in the beginning. I can't describe them in complete detail, so they are not meant to prove the thesis of the evidence of public events in any systematic sense. But they allow me to discuss some preconditions and problems concerning the investigation of public events in urban settings. My considerations focus on the question of the effect which public events have on the production of locality as outlined above. In contrast to the House of World Cultures, the Pariser Platz represents the local and national heritage of Berlin. The reconstruction of the buildings around the place followed the guidelines of historical reconstruction issued in the beginning of the 1990s, even though interpretations of these guidelines vary from building to building. Thus the two more postmodem houses designed by Behnisch and Gehry were discussed heavily and had to change at least their front fa9ade to better fit the rules of historical reconstruction. Nevertheless, the Pariser Platz is perceived as the "parlor" of the city for now. The Berlin Newspaper Tagesspiegel named the square a magical site and the "first address Public Events re-visited: The 3rd of October and the opening of the Mexartes Festival Both events, Germany's Feast on 3rd October and the opening ceremony of the Mexartes Festival, serve different purposes and speak different languages. And they are set on the scene in different parts of the inner city, even though both locations are not far away from each other. The old congress hall which hosts the House of 16 of Germany", last but not least because of the four Bank houses, the French and the planned American embassies, the Hotel Adlon. And, the Tagesspiegel announces, "Besides the nearby Potsdamer Platz, the citadel of the globalized urban planning, the Pariser Platz appears as a national forum made of stone. " 17 In short, the Pariser Platz is a German place - not only because of its architecture but because of the events taking place there. It is not so much a place of daily routines of the citizens but a place for special events and, of course, for tourists looking at the Brandenburg Gate or watching the official guests of Berlin residing in the Hotel Adlon on their way from the car to the entrance. city. All these events belong to the process of the festivalization of city politics, which Hartmut HauBermann and Walter Siebel have identified as an emerging new marketing strategy of cities. 18 Even though they all have different promoters and organizers, these events are part of the city's marketing because of their attractiveness not least of all for tourists who will come to the city and leave their money in the city. All of these events are interesting by themselves, as they stage different messages and produce different modes of agency. But in light of the restructuring of urban space they belong together. In the line of these events, the process of restructuring becomes visible. In this sense they are interconnected as they together produce the text of multicultural diversity, of openness towards the world and of the rich mixture and creativity Berlin is able to offer. Combined, they draw a mental map of the city in which all of those places where these events take place are integrated. This map tells about the joyful atmosphere of festivities which are able to satisfy curiosity and the lust for adventurous tours through the diversity of the world. The title of this map is "through the world within the city". Public Events re-visited: The 3rd of October and the opening of the Mexartes Festival Thus a perfect alliance exists between the two events and their respective locations. On the one hand, both belong to different symbol systems; on the other, the two locations are able to investigate the events with legitimization and authority, whereas the events themselves structure and reify the symbolic content of these locations. The opening of the Mexartes Festival does not stand alone. In fact, the efforts to stage Berlin as multicultural, as open to the world and as enjoying its diversity structure a whole range of different public events which are put on during the year. Parades - like the Love Parade of the techno kids, the Carnival of Cultures - a parade of different migrant organizations -, the Christopher Street Day, which is the parade of the gay and lesbian community, or- to some extent - the Berlin Marathon. These all are the biggest and most important of these public events which operate on arguments of cultural diversity, multiculturalism and internationalism. Besides these, every year events are performed. There are a lot of single occasions, like the opening ceremony of the newly built Debis and Sony Center at the Potsdamer Platz, which try to produce an atmosphere of cultural diversity and to show difference as a central resource of the The yct of October belongs to another map of the city and to another atmosphere of discovery. It is the map of national heritage and of those events which enable one to experience the "imagined community" (Anderson) of the nation. And this map also creates its own centres and places. The Pariser Platz, the Brandenburg Gate and the famous Alley Under den Linden, the Reichstags-Building and in a certain sense the Museumsinsel are built at the very centre of this map. The events taking place in these inner city spaces do not only strengthen the national meaning and perception of these places, but they do make it a lively space of encounter and negotiation framed by features of national self-image. 17 "The Wrapping is Peeled Off", Tagesspiegel 4.10.2002 "The Wrapping is Peeled Off", Tagesspiegel 4.10.2002 "The Wrapping is Peeled Off", Tagesspiegel 4.10.2002 And while the public event itself represents only one single version, namely the one which has gained the most authority, the discussions taking place (whether in the media or in face-to-face debates) offer a wide range of different interpretations, while showing strategies to legitimize positions and interpret the staged performance in terms of content, sense and meaning. In short, they evaluate and reify the very meaning of the public event and thus help to uncover the problems and conflicts underlying the performance itself. In consequence, these debates restructure the event and reflect on their "improvement." The discourses on events become themselves part of the following stagings. The first is concerned with the question of whether it should be allowed to pass the Brandenburg Gate by car in future. This discourse touches questions of everyday practices taking place in public urban space in general, and in this square in particular. Both the pro-car party as well as those who want to have the Gate car-free try to strengthen their argument by pointing out the national importance and symbolic content of the Brandenburg Gate. The second discourse is concerned more directly with the symbolic content of the Gate and the square as national locale. It asks who shall be allowed to use the Pariser Platz as site of demonstrations and as a stage for festivities. In 2000, this discussion became extremely dynamic because of the right wing party NPD, which marched through the Brandenburg Gate. 19 During the days following the event, the media, especially the Berlin newspapers, and some politicians asked for stronger restrictions on the right' s ability to demonstrate in the middle of this newly built government quarter. And somehow it seemed that the walk of the NPD through the Gate was even worse than the very fact that their right wing positions gained ground in Germany at all. In the following weeks, counter- demonstrations and meetings were arranged in which thousands of people joined. Of course, this took place at the Pariser Platz. It was a symbolic occupation of the somehow "contaminated" place, a public demonstration of the "other", that is democratic Germany, and, at the same time, a self-assurance that those democratic forces are stronger than the NPD and its followers. Especially the Pariser Platz became the locale for staging the national during the last years, thereby using the symbolic content the Brandenburg Gate as national monument bears and strengthening it at the same time. Already in 1991 the official opening of the reconstructed Gate was meant to set the national symbol anew. The farewell ceremonies for the allied troops took place at the Pariser Platz in front of the Brandenburg Gate in 1994, and the official celebration of the 9th ofNovember, the day of the opening of the wall, is normally staged at the Pariser Platz, to mention but a few festivities which the place hosts. More and more, the Gate and the Pariser Platz became the central stage for celebrating national holidays and commemoration ceremonies in public. Located next to the Reichstags-building, the Pariser Platz and especially the Brandenburg Gate seem to offer the "right" setting for the staging of these national holidays. At the same time, these public events help to strengthen the role of Berlin as the new national capital, which, in turn, found in a way its public stage in the Pariser Platz. Historical narratives which talk about the varied history this part of the city experienced established finally and strengthen constantly the image of the Pariser Platz as the "parlor" of the nation. Especially the celebrations on 3rd October 2002 made this mechanism obvious. Even though the renovation work was finished some weeks ago, the uncovering took place during the celebration of German unity and, thus, was embedded in the national narrative of unification. Probed in advance and more than once announced in the newspapers, people were put in the mood of expectation. Finally, the Brandenburg Gate was given back like a gift to the Berliners and their visitors, given to them as a site of identification and belonging. But the process of defining this national urban space was structured not only by the public events themselves but by those 18 with legitimacy and gave them their strength. discussions accompanying and commenting on them as well. At least two discourses structure the staging of yct October at the Pariser Platz in 2002. To sum up: Discussions between events are most often concerned with the question of whether the setting, the framing and carrying through of the event fulfilled its purposes. 20 Here as well, the localized urban history and the newly constructed national content of the Pariser Platz worked together and invested the demonstrations And the discourses offer a key to follow up the process of the symbolic transformation of an urban landscape. Coming back to my starting point (namely that public events may help to investigate in the symbolic landscape of cities), I suggest the following: Ethnological investigation of cities finds in public events a useful starting point for investigation, but has to keep in mind that public events can no longer be interpreted as a single key to a society. That is why not only the events themselves and their immanent symbolic language are important sites of investigation, but also the discourse following up these single events investigates them with sense and meaning. Lead by the question of how it is possible to stage an event at all, the reflexive interconnectedness between event and discourse seems to be a key feature. It contributes to producing the symbolic structure of the event and is the most important frame for understanding the input 19 zum balinesischen Hahnenkampf, in: ders.: Dichte Beschreibung. Frankfurt/M. 1987, 202- 260. and outcome of a single event. Last but not least, the series of events during the year as well as over the years offer a key to investigate the process of (re-)structuring an urban landscape. viii Milton Singer: When a Great Tradition Modernizes: An Anthropological Approach to Indian Civilization, London 1972, 67-80. REFERENCES ix F. G. Bailey: Cultural Performance, Authenticity, and Second Nature, in: D. Parkin u.a. (Eds.): The Politics of Cultural Performance, Oxford 1996, 1-17. i My lecture emerged from a research project at the Institute of European Ethnology at the Humboldt-University, Berlin. The project, entitled "Stagings of Power: The making of the 'governmental area' a representational space" is concerned with the restructuring of Berlin following the move of parliament from Bonn to Berlin. For further information see http:// www2.huberlin.de/ethno/seiten/forschen/ projekte/dfg/buehnen/binder.htm u.a. (Eds.): The Politics of Cultural Performance, Oxford 1996, 1-17. x Handelman 1990, 9. ANNA-MARIA ASTROM The capital as symbol and meaning. Helsinki as a constructed and lived space. An analysis of the structures in Helsinki which convey meaning to both outsiders and to those who live there must be based on the history of the town, and on the capi- tal as an incarnation of the country itself. Historically, Finland was part of Sweden until 1809, and between that and 1917 it was part of Russia. During that period, in 1812, Helsinki was proclaimed the capital of the Grand Duchy of Finland. In the latter part of the 19th century, many national institutions were established in the capital. The independent national era began in 1917. We can thus point to the historical roots in Helsinki in different ways, and we can also look at the formation of its identity and the inhabitants' identities on a symbolic plane. I will start with a brief introduction to more general standpoints and concentrate then on how the ordinary townsman formulates his or her local identity, and what its foundations could be. The debate on the feeling of identity of the Finns has frequently been focussed on their agrarian background and, more recent- ly, even pagan features such as shamanism have come to the fore. The search has been for something that transcends the language and the country. At the same time, it has been maintained that the Finns have always had to define themselves in a negative manner, as not Swedish and not Russian, belonging neither to the West nor to the East; the folklore specialist Pertti Anttonen argues for this thought. He points out that such a definition, based on negation, has not allowed any positive history or positive narration to evolve. There is no heroic history, but throughout the history of the country, external threats have provided the uniting factor (Anttonen 1996). The Kalevala epic, and a small farmers' menta- lity have of course been stressed, and equally, the lack of a "European" finesse and an unability to play at different abstract levels (Apo 1995). But a negative identity must perforce be uncertain. In what ways can an urban dimension alter this picture? Meanwhile, the daily life of the people and their interpretations of the town form another cornerstone of the history of the city. I would thus want to stress the picture the local residents of Helsinki have of their home town and the meanings they give to the symbols that are thought to constitute Helsinki. x Handelman 1990, 9. xi Rolf Lindner: Berlin - Zone in Transition, in: Anthropological Journal on European Cultures, Bd.2, Nr.2, 1993, 99-112. xii Christine Boyer: Merchandising History at South Street Seaport. In: Michael Sorkin (Ed.): xii Christine Boyer: Merchandising History at South Street Seaport. In: Michael Sorkin (Ed.): Variations on a Theme Park. The New American City and the End of Public Space. New York 1992, 181-204. Variations on a Theme Park. The New American City and the End of Public Space. New York 1992, 181-204. ii Don Handelman: Models and mirrors: toward an anthropology of public events. Cambridge, New York, Melbourne 1990. xiii Sharon Zukin: The Cultures of Cities. Cambridge/Mass., Oxford 1995. iii Maoz Azaryahu: Die Umbenennung der Vergangenheit. Oder: Die Politik der symbolischen Architextur der Stadt Ost-Berlin 1990-1991, in: Zeitschriftfar Volkskunde 88, 1/ 1992, 16-29. xiv Zukin 1995, 7-8. xiv Zukin 1995, 7-8. xv www.hkw./external/en/Profil/c index.html (October 2002). (October 2002). xvi www.hkw./external/en/Profil/c index.html (October 2002). (October 2002). iv Per-Markku Ristiliammi: Cultural Bridges, Events, and the New Region, in: Per Olof Berg, Anders Linde-Laursen, Orvar Lofgren (Eds.): Invoking a Transnational Metropolis. The Making of the 0resund Region. Lund 2000, 95- 108, 96. xvii Tagesspiegel 31.12.2000. xviii Hartmut Hauflermann, Walter Siebel: Die Politik der Festivalisierung und die Festivalisierung der Politik, in: dies. (Hg.): Festivalisierung der Stadtpolitik. Leviathan Sonderheft 13, 1993, 7-31. v Yi-Fu Tuan: Space and Place. The Perspective of Experience. Minneapolis 1977, 192. xix Cf. e.g. tageszeitung 20.10.2000. vi Handelman 1990, 9. xx Cf. e.g. tageszeitung 8.3.2000, vii Clifford Geertz: "Deep Play": Bemerkungen 20 ANNA-MARIA ASTROM The capital as symbol and meaning. Helsinki as a constructed and lived space. Through these memories Helsinki can be seen as the scene for everyday life. The urban elements (the architecture and city life), the impact ofnature (the sea, the four seasons), the monuments, the multicultu- ralism of the 1950th (Finnish, Swedish, Russian, Jewish) are the cornerstones providing a base for a positive story on which the identity can be based. Much of what was written down as answers to my questions was based on the memory of the inhabitants, of how they remembered their childhood town after the Second World War and what they thought about it today. 21 The Cathedral on the Senate Square constitute the premier landmark. Helsinki City Mu- seum its position as the capital of Finland is the result of a conscious goal-oriented process during almost two hundred years. During this period, the town has been endowed with its national buildings reflecting symbolic values: the university, the Cathedral, the government palace, all in the early 19th century, the Bank of Finland, the House of the Estates, in the late 19th century, and the Railway Station and the Parliament building in the first and second decade of independence, and many others. All these are incarnations and symbols of the nation's road towards the independence and its stabilization. The buildings in themselves are active efforts to represent the nation as something positive and permanent. As far as the identity of the capital and the Helsinki townspeople are concerned, there are nevertheless features pointing in the opposite direction, at a tenacious - possibly even spasmodic - striving for self- esteem, and this reflects both national and international goals. Helsinkis identity could be regarded as having become crystallized in opposing the negative features, in various synthetizing and thus more favourable straits, and also in various efforts of secu- ring a national security and identity. The townspeople themselves also participate in this identity-building by means of their local identifications. On the other hand, Helsinki has also remains of both the Swedish and Russian era. In the course of its history Helsinki has not been especially keen on removing signs of the foreign rulers. The fortress Sveaborg is still a reminder of the Swedish era although it was renamed Suomenlinna (The Fortress of Finland) in the 1920s. The Russian era is symbolized by the ortodox church, and a statue of Tzar Alexander the first and a monument of tzarevna Maria. ANNA-MARIA ASTROM The capital as symbol and meaning. Helsinki as a constructed and lived space. The names of many streets still bear the name of relatives of the tzar's families (Alexander-, Dagmar- ' Elisabeth-, Anne-, Catharinastreet etc.) The names Wladimir- and Andrestreets have nevertheless been changed to Kaleva- and Lonnrotstreet, national names per se. But altogether one could say that the forementioned cultural threats have been wowen into the history of the city. The names of the streets are still in both Finnish and Swedish -the Russian names where however excluded after 1917. Officially Helsinki has still two names: Helsinki and Helsingfors. I base my discussion on Herman Bausinger, who says that one has an identity insofar as one is sure of oneself and is capable of tying one's past in with one's future, and one is fully accepted by the others. In a transposed sense, a person may then be said to have a home region, that is, a place or locality in which he or she feels a deep confidence, and this place is, in the last instance, not only the basis of the personal identity but in fact its very essence (Bausinger 1985, 89). The individual identities are a result of interaction with others but also of interaction with a place or in some way of "producing the place". During the 20th century Helsinki has been changed to a more homogenus ethnic town at the same time as the industrial featuers has given way to high tech. The influx of people and the growth of the popu- lation figure has, by and by, made Helsinki As far as the town Helsinki is concerned, 22 TABLE 1. POPULATION OF HELSINKI IN INNER AND OUTER REGIONS 1946-1998 1946 1950 1960 1970 1995 Inner city* 243.116 247.140 212.387 171.046 115.534 Old suburbian areas** 43.380 48.357 56.325 49.932 42.446 Suburbs*** 51.054 62.631 164.813 286.069 346.651 337.550 358.128 433.525 507.047 489.212 6vrig befolkning 4.013 12.834 14.790 16.630 11.134 S:a 341.563 370.962 448.315 523.677 515.765 The inner city and the old suburbian areas are called Inner city *city districts 1-15 **city districts 16-27, 52-53 ***city districts 28-51,54 TABLE 1. POPULATION OF HELSINKI IN INNER AND OUTER REGIONS 1946-1998 "history", a narration of a kind. At the same time, the Finns have striven to launch Helsinki as an attractive city for all kinds of international conferences. ANNA-MARIA ASTROM The capital as symbol and meaning. Helsinki as a constructed and lived space. Thus, the intentions of the national leaders, townplanners and the tourist leaflets as a mediating forum, produce different ways of pushing and presenting the town, of creating an image which brings out the positive features. There is nothing remarkable in this. Similar efforts are made by almost all European and other cities trying to distinguish themselves. The result is pictures for the outsiders. What is remarkable is the effort - or sloweness to react - that leads to incorporation even of foreign or even in some senses negative traits. Today we still have a Leninpark and a somewhat grotesque statue of peace and freedom presented as a gift from the Soviet Union in the 1980s. An historical awareness represents on the other hand the failure of an anachronic effort to install a Lenin monument in 1999. In this way Helsinki has not forgotten its history and compared to many other cities this history can still be seen in its monuments. change, from a small town to the only real city in Finland, with half a million inhabitants. This fast development may also be a source of naive pride - especially when acceptance is looked for elsewhere. Hel- sinki has also changed remarkably with regard to its linguistic distribution: still in the 1870s, the majority of the population spoke Swedish, whereas in the 1990s, the Swedish-speaking Finns were only about 7%, due to the vast immigration of Finns. Some of the old minorities, Jews, Russians, Tatars and Gypsies, have received ad- ditional influxes, mainly in the 1990s of completely new ethnic groups. The new immigrants now consitute 3,2 % of the population. Although the town is now mostly Finnish-speaking, neither the municipal authorities nor the old inhabitants in general will exclude Swedish as the second language. The expression "the Helsinki spirit" was coined during the first SALT meetings in 1969 between American and Russian delegates, to denote a relaxed atmosphere, in which they met. In the course of the numerous meetings - the U.S. and the Russian Presidents have met twice in Helsinki -, a joint diplomatic chronology has eventually been created. This could also be seen as one expression of a wish to bring about a positive, neutral diplomatic The voices of the townspeople One of my points is thus that the city perhaps naively and perhaps unconsiously has worked to develop and synthetizise a 23 pictures which at most stress history, there is a vantage-point in the personal memories and in the process of recalling the town. All informants refer to their own experiences and memories of the town of bygone days. Only individuals can have memories and base their ideas of the town on them whereas institutions prouduce only museums and archives ( cf. Eriksen 1997, 130). In the memories of the Helsinki residents, the place as such has great importance (Astrom 1997). The place becomes equal to roots. This is explained with rare lucidity by one of the informants: number of symbols and that the politicians have worked to give a positive image of the city internationally - but my main point will be that the citizens themselves have also taken the symbolic dimensions to their hearts in a deeply personal way and they have in their narratives also produced "places of their own". During the mid- l 990s, I have collected a material consisting of about 500 narratives, where Helsinki townspeople describe their surroundings, their everyday life and their opinions of the town. These narratives are replies to four different letters of enquiry and they can be looked upon as outcomes of what Henri Lefebvre calls lived space. Lefebvre is also of the opinion that this kind of space, e.g. the lived one, can be expressed only in art - but I would argue that we have not been listening and questioning the ordinary citizen, he and she can also express him- or herself. Helsinki, to me, means "roots". In life, a person may feel at home anywhere, but those pictures which were built into our brains in childhood follow us and remain alive throughout our lives. These are sights, atmospheres, human relations, various experiences communicated by our senses. They come into our minds as short glimp- ses or as entire series of events. Time changes them. For each one of us, they are unique. To me, as I am born in Helsinki, the town is the sum of the images of my childhood. IV: 16, 1 p Looking at the different aspects dealt with in the texts, we may apply David Harvey's "grid of spatial practices" to the material. The voices of the townspeople This narrows down to the townspeople's descriptions of personal space and mental maps of occupied space, or descriptions of familiarity, open places and places of popular spectacle, that is, streets, squares and markets. On the other hand, the narratives of the townspeople also include official places, for instance monumental spaces and constructed places of ritual, i.e. just those places that tourist brochures have a predilection for (Harvey 1989, 220-221). Helge Gemdt mentions four kinds of symbols or markers in urban settings: urban space and buildings, symbols of the social situation, symbols of the geographic position and the climate, and symbols of everyday life. In the narratives they mingle together, but I would like to threat them in two blocks - the more material first and the more social later -just for the sake of clarity (Gerndt 1985). Urban space, buildings and the geographic position I will now look for general, symbolically important elements and study how attitudes are developed as personal standpoints and as part of the personal histories of the townspeople. Another very lucid passage of this same informant is the following: As I grew older, Helsinki was the scene for my growing independence and for widening the sphere of the world. Adventurous tours around the town, to the Suomenlinna fortress and to newly incorporated areas at the end stops of the blue buses, were part of both my own development and of the expanding picture of my home town and its many different Moreover, in contrast to the official 24 faces. Even then I could already guess that being a Helsinki inhabitant could be approached from many different angles, from Kaivopuisto (the diplomats' area), from Kallio (a workers' area), from Maunula (a suburb), and all these pictures would be different, but true. There were workers, gentlefolks, rural immigrants, and all from Helsinki. I was not sure about my own place: at times I wanted to come from one kind of place, and at other times from another. Master of Arts, IV: 16, 3 one see the tower of the Cathedral. It comes out, takes form, you can see it from far away, it shines. This is always heart-stirring to me, I am coming home, I am coming to Helsinki. Author, born in 1954, IV:32 The city's social heterogeneity, the mix of old and new and Finnish and Russian; Enso's head quarter designed by Alva, Aalto and theUspenski Cathe- dral in the back- ground. Helsinki City Museum. Helsinki as a vast area, containing of different districts with different social stamp is obvious as well as the dynamic of the city in the formative years of the 1950s and 1960s. The entity of the Helsinki stories is on the wholle created by the mixture of descriptive parts containing important urban elements, and other parts where the feelings of freedom, security - and insecurity - and beauty are verbalized. Sometimes criticism of the developement of the town is expressed. Also the geographical position comes out as references to the weather, the differenses between winter and summer and the fact that Helsinki lies on a peninsula with many islands and inlets. The sea, and the town as seen from the sea with the silhouette against the sky, are perhaps the strongest element in the positive pictures. The city's social heterogeneity, the mix of old and new and Finnish and Russian; Enso's head quarter designed by Alva, Aalto and theUspenski Cathe- dral in the back- ground. Helsinki City Museum. Urban space, buildings and the geographic position Unemployed, born in 1941, IV:14, 7 The most important impressive houses are, to my mind, the Senate Square with the surrounding buildings, the Uspenski cathedral, the Railway Station, the National Theatre, the art museum Ateneum, the Par- liament Building and the National Museum. The Temppeliaukio church is impressive in its own way. In Eira, there are many beautiful houses, and also in Kruunuhaka. The Suomenlinna fortress is an impressive sight. Also, the wooden house areas in Valli/a and Kapyla illustrate the times when they were built. The Glass Palace, the bus station building and the Tennis Hall are part of the picture. Maybe this is where you can see the Helsinki spirit. But this spirit also includes the main market on the south harbour and the Kappeli outdoor restaurant, the Halmniementori square and Tyovaentalo (the workers' house), and the townspeople, the tramcars and the buses. Labour safety worker born in 1941, IV:41, 4 The environment as a totality made up of urban elements is stongly. We are still witnessing a city where traffic is a main element everywhere (Benjamin 1990, 93). But also a city that is captured by bicycle, an occupation that was the fate of many young in the 1950s and 1960s. A young person of today, just moved in and quickly rooted, will start in the opposite way: Is there anything good in Helsinki? Well yes, definitely I A beautiful and active lazy summer can be spent in many ways in the Kaivopuisto park. As a matter of fact I "move out" there every summer. There is park gymnastics, Roller blading, picnics and chess. The stairs to the Cathedral and its surroundings are a real "living-room" for the Helsinki townspeople ... Like in tourist brochures the informant also mentions the towndwellers themselves but in a more initiated manner. He knows the places and he has made up his opinions of them. In the lists of buildings, we thus frequently have stress on the same buildings as in the tourist brochures, but the places and buildings have been worn into the minds in the course of long lives. How the central areas in town were appropriated may also have to do with the first workplace of the person: But she continues with the same references to symbolically important buildings, but mentions also important meating places for the inhabitants: As a town for representation, Helsinki is unnecessarily shy. Urban space, buildings and the geographic position I quote two descriptions of the same phenomenon: one see the tower of the Cathedral. It comes out, takes form, you can see it from far away, it shines. This is always heart-stirring to me, I am coming home, I am coming to Helsinki. Author, born in 1954, IV:32 The town is thus by these literary open persons seen as beautiful and inviting, and even as the symbol of home-coming. The built environment of the market square is meating the natural elements as the boat moves into the inlet harbour. The sea as a natural element also comes up as references to chlidrens playing at the shore or moving dangerously on the ice floes. Another frequently occurring mention is the shores as experienced by walking along them. They have become familiar to the informants during walks at various stages in life. Some also talk about walks on the sunlit ice and about skiing to islands off the coast. Summertime is on the other hand depicted with a lazy tone, as hot asphalt and again trips to the outshore islands. The most important element for me is the proximity to the sea. The sea shore -the Kaivopuisto shores. The image of the town itself: the seaway into the harbour. The silhouette of the town with the Cathedral and other towers and familiar buildings. There is no sight to equal this in beauty. It is like being embraced, wrapped into strong arms. MA, author, born in 1935, IV:24 The architecture in itself is seen as beautiful, especially around the Senate Square, with the Cathedral. The center of the city is described as exciting. The fact that buildings listed in contemporary tourist brochures have a real meaning for the townspeople is revealed by the long lists of As I arrive in Helsinki by boat from the sea, the morning mist will at first only let 25 buildings which are mentioned as incarnating the town: Aleksi (slang for the Aleksanterinkatu stre- et), the Kaivohuone building and the centre of Helsinki at night ... The picture of the town consists of the buildings, the traffic, the streets and the people. Unemployed, born in 1941, IV:14, 7 Aleksi (slang for the Aleksanterinkatu stre- et), the Kaivohuone building and the centre of Helsinki at night ... The picture of the town consists of the buildings, the traffic, the streets and the people. Urban space, buildings and the geographic position I would like some more life and movement. The symbols of the town are the Railway Station, the Cathedral and the Uspenski cathedral. Also, the (new modern art museum) Kiasma and the house of Parliament, and the Stadion with its tower. For the local people, the symbols are the clock outside the Stockmann department store and the compass at the main Railway Station. The town derives its character from the sea, the embassies and representations, the tipping stations in Hakaniemi-Kallio and the cafes where you can have beer. The spirit of Helsinki is best condensed in the I moved around the centre quite a lot with my work: first I was a bicycle messenger, and then I worked in a restaurant, travelling to and from work. What comes to my mind is the Railway Station, the Central Post Office, the Hankkija building, the Bus Station, the "Sausage house" (christened because of an architectural feature), the 26 Helsinki with Suomenlinna in the background. Here is Helsinki's most popular walking thoroughfare. Helsinki City Museum. towndweller (stadilainen) admires the flowerbeds at the Esplanad. Informatitian b. 1931, III:29, 3 towndweller (stadilainen) admires the flowerbeds at the Esplanad. Informatitian b. 1931, III:29, 3 towndweller (stadilainen) admires the flowerbeds at the Esplanad. Informatitian b. 1931, III:29, 3 Kaivopuisto park on a sunny summer's day, with many people, events, things to do, cliques. A kiosque vendor born in 1971, IV:43, 2,6 But negative phenomena are also mentioned: the noise and the pollution, the graffiti, the drugs, the roadworks and the destruction. Some regard the change, with the extinction of local shops, as particularly unhappy, whereas others rejoice that the city area has become so attractive, with new cafes and a generally more open atmopshere. Some speak up against the drunkenness in the steets, the ugly language used and the misplaced architecture. In many narratives the parks, both those opening to the sea and those lying in the centre, and the three different markets for greens and vegetables are said to contribute to a sense of homeliness and belonging. One narrative takes us to all parks with the same pride as in all former narratives: The parks, the lungs of the city, where of utter importance for our wellbeing.In my opionion also large green areas belong to the parks; Kaivopuisto, Alppipuisto, Seurasaari and the other islands as well as the graveyards. Symbols of social situations and everyday life We are now already moving with descriptions that illuminate also the social side of the the everyday life.What makes these lists and descriptions more than a kind of vote for beautiful and less beautiful values is the perspective that the informants have. They pronounce their opinions on a town which they mostly know since childhood, the buildings and places they mention are things that they say mean much to them personally. The question "is there a special Helsinki spirit" is very rarely associated with the diplomatic sense of the expression. Instead the search for the Helsinki spirit often leads the thoughts to something more timeless and permanent: the new possibilities offered by the modem arts museum Kiasma next to the Main Post Office, whereas others abhor this building. All phenomena are part of everyday experiences of the citizens, former experiences mingled with experiences of the city of today. Also, the "stonetown" itself, consisting of blocks and of stone, is mentioned. These mentionings take the stonetown as granted, but fill them with the life they used to witness, in this earlier stage, only one street is here depcited: I think that the best-known symbol of the town is the Havis Amanda statue. There are so many who want to be photographed next to it. The Helsinki spirit is concentrated in my mind as I enter the Esplanaadi park, there I have reason to remember ... Helsinki has many great things, but there is also peace. Foreman born in 1925, IV:18 I think that the best-known symbol of the town is the Havis Amanda statue. There are so many who want to be photographed next to it. The Helsinki spirit is concentrated in my mind as I enter the Esplanaadi park, there I have reason to remember ... Helsinki has many great things, but there is also peace. Foreman born in 1925, IV:18 On the Iso Roobertinkatu street there was this milkbar Valio, with its "pirtelo ", nowadays called milkshake. There was also a shop for rubber wares ... and at least three cinema theatres ... and in the vicinity there were still many more, Gloria, Edison, Merana and Alice on Fredrikinkatu. Jso Roobertinkatu has stayed in my memory as a colouful distinctive street ... At that street there was also a second hand bookshop, where we used to spend hours after school. Economist, born 135. Urban space, buildings and the geographic position Wtih the children we used to go to the Sinebrychoff park and aslo the graveyard of Hietaniem was a superb place. There you could sit and feed the wild ducks and the squirrels and enyoy the landscape and the vegetation. Of course every Especially the innovations of the 1960s, with a department store and an inbuilt parking house facing the respectworthy Railway Station, and a main office block designed by Alvar Aalto in the centre of the South Harbour area, irritate some townspeople. Again, some looked forward with trust and curiosity to 27 enrich the town. Are they the missing heroes? Symbols of social situations and everyday life Was she unable to see the rich stimulating environment that I lived in? I:55, 13 My Helsinki is from the time when I was a child, when the courtyards were alive with street singers, trash bin divers, potato sellers, long lines of washing and angry caretakers. We, the children, lived all this and saw all kinds of things. We became tolerant but perhaps a bit hard. The Swedish language was also definitely part of my Helsinki, as were the grocery shops, the meat shops, the milk shops and the paper shops. I loved the library in the Ludviginkatu street, and the swimming hall in the Yrjonkatu street. I learnt the basic rules for living close to one another. I loved the shop windows, because at that time they were beautiful and offered a child visual pleasures. The Helsinki of my youth offered. .. art exhibitions on Sunday mornings, movies, the marvellous homes of friends in personal blocks of flats, the tram trips, the hurry to catch the last bus, the spring evenings in the Kaivopuisto park, and the sea, the sea. Lecturer, born in 1945, IV:9, 1 An everyday greyness, densely populated small dwellings, crowds of children, adults at work, life being more of everyday routines than celebration. No lustre, but as seen from the present day, alive with the gold of memories. That was Kallio of my childhood and youth, where I took root as a slow-growing rowan tree in a tiny nook of the Vesilintu hill. In those days, like today, Helsinki is to me the town. I:70, 10 So I lived in the old Rooperi area. Our block lay between the streets Sepankatu, Albertinkatu, Pursiniemenkatu and Laivurinkatu. Actually, "home" was not just the dwelling, but to me, "home" was the whole building with the yard and Sepankatu street as an outer yard. We spent much time there. But the borders of my area went further. They included Punavuori, Eira, Kaivopuisto, Tahtitorninmaki, Ullanlinna. Since we spent much time out of doors, in those days, those were the areas where we walked. The streets, the people were familiar.. There also lay my school, my hobbies .. We knew a few of the "kings of the street" .. To me, the sea also belonged to us. Symbols of social situations and everyday life III:45,4 The informants describe both the changes in the outlook of the town and the permanent features. The styles used, which are sometimes literary, give the narratives a tone of recognition and of identification with the town. The informants refer to types of behaviour that have remained the same for several years. A Swedish- speaking elderly woman from the workers' district relates of Helsinki as a place for visitors and events: The fact that it has a somewhat old- fashioned and traditional imprint in the central part is appriciated. One informant says that no skyscrapers are needed. The human format, the public transports, the social services and the limited city area all contribute to the feeling of security. Even the townspeople themselves are said to The main market was something that absolutely had to be visited during the fish market days, since every year, one simply had to taste the black bread from the Aland islands ... Every New Year's night, the whole 28 courtyard. I could not understand that one of my aunts used to expound on my "ill fate to have to grow up in a stone desert like this one". Was she unable to see the rich stimulating environment that I lived in? I:55, 13 family would walk to the Senate square to listen to the Mayor's speech. There was no tram, but we walked in the cold and the wind from Hermanni along all of the Hameentie street and up the Unioninkatu street. Book-keeper, born in 1915, IV:6,1 The street as the towndwel/ers living room with nationally important buildings in the background: here the Parliament, and on the previous page the Govern- ment Palace on the Senate Square. Helsinki City Museum. Many of the informants revert to the days of their childhood in the 1950s and describe their impression of the town oftheese days, not paying attention to the official or modem image: courtyard. I could not understand that one of my aunts used to expound on my "ill fate to have to grow up in a stone desert like this one". Was she unable to see the rich stimulating environment that I lived in? I:55, 13 courtyard. I could not understand that one of my aunts used to expound on my "ill fate to have to grow up in a stone desert like this one". The street as the towndwel/ers living room with nationally important buildings in the background: here the Parliament, and on the previous page the Govern- ment Palace on the Senate Square. Helsinki City Museum. Symbols of social situations and everyday life We often made trips to the shores by The home district as parts of the town is characterized in warious ways: I loved Katajanokka and its smell of asphalt on a hot day, the echoes of children 's clear voices in the pitlike inner courtyards, strictly separated from one another by high walls; the mysterious lofts and basement cellars; the din from the harbour; the architectural details and the familiar people moving around our own 29 Kaivari, and to the little island Uunisaari. This marine imprint distinguished our area from so many others. I:41, 4, 6 A characteristic place in the town: The Pentagon (in Swedish Femkanten) on the border of Punavuori and Ullanlinna, the district cinemas also constitute dear "place for memory", in the picture cinema Merano. Helsinki City Museum. A characteristic place in the town: The Pentagon (in Swedish Femkanten) on the border of Punavuori and Ullanlinna, the district cinemas also constitute dear "place for memory", in the picture cinema Merano. Helsinki City Museum. a service station. The caretaker with his family lived by the other staircase. His sister Iita kept order in the whole building and especially among the children. I:73a, 2 Our building had quite many people with academic degrees: a meteorologist, a forester, a professor of history, a master of languages, a high-school engineer, a mas- ter of business sciences, and so on. But there were also shift workers, a night watchman, an entrepreneur, one-parent families, pensioners. Actress Ruth Snellman made a special impression on me, and so did the European boxing champion Eelis Ask; both lived by our staircase. Actually, the only uniting factor was the children in the buil- ding who were diligently cruisingfrom one home to another, thereby also making their parents acquainted with one another. I:42, 2 The people living in the building were quite colourful: downstairs the Jews, whose boys we were teasing, and the Jews on our floor who had beautiful dark-haired daugh- ters, the upstairs Jewish ballet dancer, Mot- her "rallakka" (a Salvation Army officer with an adopted son), Vartsila engineers (the building may have been owned by the Vartsila company), the genteel family of the manager of the building, families with children my own age, whose workplaces I was ignorant of The father of my friend was a painter, their home was one room, a kitchen and a toilet, and four children. There were few spacious dwellings. A characteristic place in the town: The Pentagon (in Swedish Femkanten) on the border of Punavuori and Ullanlinna, the district cinemas also constitute dear "place for memory", in the picture cinema Merano. Helsinki City Museum. Symbols of social situations and everyday life I:22, 1 Kaivari, and to the little island Uunisaari. This marine imprint distinguished our area from so many others. I:41, 4, 6 Stone in itself - or nature - are phenomenons to relate to. Only tree glimses of the lists of the citizens of ones own house will show how the social situation is imprinted in the memory: The building was mainly inhabited by working people. Family fathers and most mothers were working outside the home. I cannot remember anyone unemployed living there at the time. They were decent and quiet workers: drivers, workers especially from the Kone lift factory, tram drivers and ticket collectors, tramway track cleaners, night watchmen and people working at a printer's. The women worked either in factories or in nearby shops. There were also some manual workers such as a dental technician, who had a car as early as the '50s, seamstresses and a tailor. The other car was owned by a family who had The social, linguistic and ethnic cultures of the town, and the dichotomies, recur as a background element, but sometimes also find articulation, first a bourgeois view, then a worker: In the same way it must be said that the Helsinki of my youth was the bourgeois town, which was only spiced by the 30 Sia in sl lein s s laa laas. Relsmgin hienom railiovamau lmlkee tau. sl eissilli I • an e1n. Se pysihlyy Raulalientorilla lu,.tunneln. a illes ineen. Kiy dsiin. lpuuofl Tie taaa pitltliD•. Baatadeutorllla ,...._1a 11 .15 J• 17-21 Job uldJiiiYi J• 1a-1m1.a 11-17; Ooppenduolla Tlldli• JU I• l:nppalorllla piaoi.1ta. s,wa1io11 oa IDattll'fiaa yWIJIIIIJooa. hlla 040-548 0175 I.Ill La._ aqpll....ao 1114 07420, BOO.! 1:altyilajoa •i .-falllllm, laaaalaala a&a aJalla 31,1,,31,7, Advertisment for Sinebrychoffs tram, which funtion as a pub. Thetextsay:The city's most stylish tram runs again. It stops at the (railway)station every hour. Drop in! workers' quarters in Rooperi (slang for Punavuori). The other workers' areas, Kallio, Valli/a, Hermanni and Toukola, were in fact unknown to me, foreign districts which, in my early youth, were even slightly frightening. .. The landscape of my youth was the centre of Helsinki, the cafes and the streets. Development manager born in 1952, IV:20, 5, 6 quarters, is very strong in my memories. Here, you learnt to understand that everyone did not have the same starting poi- nt, and that some had more than others. Advertisment for Sinebrychoffs tram, which funtion as a pub. Thetextsay:The city's most stylish tram runs again. It stops at the (railway)station every hour. Drop in! Symbols of social situations and everyday life This was easy to accept, since the whole of the area where I lived was similar, so this is part of being a townsperson. Unemployed restaurant worker born in 1941, IV: 14, 2 However, what the informants say really belongs to them is neither the public face of the city nor the atmosphere of the cent- re, and not even the quarters of the childhood scene but more specific spots, such places as "daddy's house in the Punavuori area" (IV:24), "my uncle's and aunt's record shop on the Viiskulma squa- re" (IV:9), the cemeteries (IV: 10, IV: 14, IV:23), a crevice at the south end of the Seurasaari outdoor museum (IV:31), and various places with a view (the Kallio church, IV: 13; the Observatory hill, IV:23). These are places where the informants felt Perhaps the occurrence of the Swedish language and Swedish people in Helsinki has lowered the threshold to meeting foreigners, regardless of their skin colour. I do not think there were ever any problems in this respect in Hesa (slangfor Helsinki) ... The basic ideas of my life were reflected in the Stadi (slang for the town, meaning Helsinki) and its variety. A bilingual town, where the mastery of the second national language was approximately centered where the "gentlefolks" lived. Also, the strong division by the Pitkasilta bridge, where this north side was the workers ' 31 ople themselves (cf. Astrom 1994). There are also mentions of more recent festivities and festivals. Since the 1980s there have been new mass events, such as rock concerts, the Night of the Arts, the Ladies' Ten Kilometres, and the Helsinki Marathon; and also a number of other happenings for the townspeople, which have been accepted and appropriated, and this comes out in the narratives of the informants. That things happen in town is generally regarded as something positive and new: old borders are transgressed. safe as children, places where there is a strong feeling of the continuity of the family, places where the town can be viewed and embraced, and places where one can be quietly alone. For those who move out from the centre of the city, there is one way of maintaining contact with that which is felt to be impor- tant: When I long for the proximity of the sea, we drive along the shore lane in Kaivopuisto, or walk there. Symbols of social situations and everyday life The wondeiful autumn storms in particular will take us to the shore of the Kaivari (slang for Kai- vopuisto), and there, the wind tears at us and blows our lungs full of oxygen, and the waves break and splash us with salty dro- ps. We visit the herring market almost every autumn, and on Mayday we will naturally walk in the throng of people and go to the Esplanaadi Kappeli restaurant for a beer. The Stockmann department store is the main place where I have always bought clothes and special items. Researcher born in 1938, IV:5, 7 To sum up, I will quote a somewhat more critical and ironic analysis of Helsinki: A small city or a big town. Helsinki. Sometimes the soul is bigger than the body, sometimes it gets pompous. Something of Engel and of Aalto stills hovers over the town, there is some megalomania, something of a banana republic. The museums are oversized in relation to the population, the operas, churches and culture centres are oversized. Maybe this is a feature typical of a young country figthing for its place, its identity. But Helsinki is no Paris, no Rome, no Vienna, and will never be. Nor is this what I am looking/or or have sought. In Helsinki there is something else, difficult to define, difficult to catch, volatile. Perhaps it is the proximity to the sea, or the short distance to St. Petersburg, or perhaps it is ourselves. IV:28, 3 There is a noticeable element of sensations experienced in cultural life, consumption and luxury, but also with nature; this is wholly compatible with the international trend in uniting local identity with the consumption of culture, goods and events (cf. Andersson 1997, 110). Important local aspects such as the traditional department store and the two carnivals of the town, that is, Mayday and the fish market, occur in most of the narra- tives. The carnivals are experienced as rites in the sense that people feel "obliged" to take part; they are also calendary festivities: on Mayday, summer-Helsinki wakes up to life, and with the fish market, the townspeople say farewell to the previous summer. At that time, the tourists have already left the town, which means that the fish market is a festivity for the townspe- The way of reliving your town The sea and the architecture of the imperial period, the parks and the core city, the islands with fortifications, zoo and bathing, the national monuments, e.g., the contrasts between the built and the natural environment is what the local people themselves set value on. The street as a home area and the salty sea breeze, the freezing cold and the intimate cafes, the difficult process of uniting the self- sufficient "original" culture with the previous agrarian lives of 20th century immigrants, the stressed towndweller's dream of a better world are difficult phenomena to outline. It seems however that the ordinary townsperson has been able to catch them while looking nostalgically back at the blocks where he or she lived in the past. y y On the other hand, two frequently occurring words are Stadi (the slang expression for Helsinki) andpaljasjalkainen (barefoot, i.e. born in town), and I will close by discussing them. The word Stadi, from the Swedish stad, is a nickname for Helsinki which has, during the last few years, occured with increasing frequency both in the press and in daily parlance. The use of this word also means that the memory of an urban lifestyle is recalled to mind, a lifestyle that is felt to characterize Helsinki. At the same time, there is an effort to convey the meaning of the word to future townspeople. The word is intimately related to the local slang, which is called Stadin kieli, and consequently to those who used this language, primarily in the workers' districts and in gangs all over the old stone town and the earliest after-war suburbs. The use of slang occurred in a kind of "street boys' society". It also seems as if the different Helsinki informants would not yet be fully conscious of the "split in the urban space and the castration of the post-modem city, or the re-evaluation of old symbolic landscapes" as postmodernist researches define the cities of today (Andersson 1997, 108-109). The built environment with various national and local elements, and the interaction with nature, as well as the cultural fabric created by all the different people in the town, and the collective experiences of the town is still the history or text that all townsmen can continue to build on. The way of reliving your town Different parts of Helsinki also have nicknames in this slang, which is a mixture of Finnish and Swedish and which was, even at an early date, abhorred by the Finnish-speaking and the Swedish-spe- aking bourgeoisie alike. Nobody has denied, or now denies, that this was a genuine Helsinki feature. In the 20th century, interest in this language and in the style of life that it reflects, and the use of the word Stadi in many secondary meanings has increased tremendously. A slang exposition in the city exhibition hall, with The way of reliving your town The ways of finding one's own identity in a small city are manifold, but they all reflect the locality. The message contained in the verbalizations of the feelings of the townspeople quoted here seems to be that the place has put its imprint on the people. These quotes, I think, display part of a positive history of how the informants have experienced their town. A positive answer to the question "Who are we?" can itself 32 be seen in the fact that people want to express theese experiences. The glimpses that we have had here of the full picture delimits the town in accordance with central symbolic areas, and at the same time, the picture forms the background to the personal identities of the informants. The symbolic elements in these pictures have been united with the townspeople's practices. The sea and the architecture of the imperial period, the parks and the core city, the islands with fortifications, zoo and bathing, the national monuments, e.g., the contrasts between the built and the natural environment is what the local people themselves set value on. The street as a home area and the salty sea breeze, the freezing cold and the intimate cafes, the difficult process of uniting the self- sufficient "original" culture with the previous agrarian lives of 20th century immigrants, the stressed towndweller's dream of a better world are difficult phenomena to outline. It seems however that the ordinary townsperson has been able to catch them while looking nostalgically back at the blocks where he or she lived in the past. by means of self-definition, the way we speak of ourselves and of our town in relation to others. But even a positive story about "us and them" will always remain ambivalent. The "home districts" of the informants are often minutely described. We may thus regard the narratives as one way of outlining the informants relationships to their home districts (Heimat). Nevertheless, the writers seldom expressly use theese words - the urban voculabulary shies away from them. be seen in the fact that people want to express theese experiences. The glimpses that we have had here of the full picture delimits the town in accordance with central symbolic areas, and at the same time, the picture forms the background to the personal identities of the informants. The symbolic elements in these pictures have been united with the townspeople's practices. New dichotomies, new ways of dividing the town and the townspeople A way of exposing a positive history is thus 33 The Esplanade Park is an oasis. Helsinki City museum. "belonging somewhere". As I have pointed out in another connection, this town culture, which used to be a marginal one, possesses many features resembling popular culture in general, such as traditions, ties to a locality, a requirement of authenticity, a language of its own, and now even a reconstructed costume (Astrom 1998). The opposition between the Stadi culture and the bourgeois culture is no longer pointed out, but to the contrary, the Stadi culture can have an attraction even for the inheritors of the old bourgeoisie. In the same way as one is happy to appreciate popular culture, one enjoys that one's childhood dialect in the streets is appreciated. The old social stigma of certain parts of town has now largely disappeared, and a gentrification of the inner town in combination with an intensified city and pleasure life is a fact. Stadi nowadays frequently refers to the whole of Helsinki, although it still mainly refers to the more central parts. Stadilainen refers to a person living in Helsinki, but there are still many old Helsinki people who find it important to make a distinction between genuine "barefoot" Helsinki people and people who have moved in later. In this case the "barefoot" people have higher status. old photographs and texts written in Stadin kieli, attracted as many as 72,000 visitors, which is quite considerable. Primarily, old Helsinki people visited the exhibition. It was as if their subjective personal memories were thus reflected in public, so that the joint collective memories opened a channel. The remembering process could then be shared with others (cf. Eriksen 1997, 131). The memories, which are also social in character, are thus based in the social reality where the informants lived. There are many who maintain that the old dichotomy, the division into workers' Helsinki areas and bourgeois Helsinki areas, today no longer exists. The previous sharp limits were so strong that they actually formed a constituting element in all the townspeople's ideas about the town. In this situation, appurtenance to Helsinki becomes something attractive, but also something raising strong feelings. When Helsinki had been selected one of the nine Capitals of European Culture for the year 2000, the special themes of the town were given as science and technology. New dichotomies, new ways of dividing the town and the townspeople And in fact, an enormous input in com- munication technology and science is a marked characteristic in the current development of Finland and Helsinki. In the second half of 1999, Finland held the chairmanship of the European Union. In this international perspective, the old Stadi culture seems to be on a small scale and something genuine, to nostalgically hold on to. But it has also been made attractive and It also seems however that the mental structure of the town would still require a basis of a "we and the others" dichotomy. The concept of the genuine towndweller, the "barefoot" one, could then function as an axis to which to affix one's identity, or as a way of pointing out the differences between us and the others. The fact that those who inherited the old Stadi culture now frequently live in the new suburbs and no longer in the blocks where there roots are, does not lessen the attraction of this 34 great distress, honestly and without any crookedness. To behave as people behave here, modestly and satisfied with little, to work. There are too many immigrants and Russian-speaking returning emigrants who have come dishonestly and by crooked means only to take economic advantage, to live for free at our expense. Their demands for special treatment and special arrangements cannot and must not be accepted at the expense of our citizens and our townspeople ... The lazy ones, the good- f or-nothings, the crooked ones and the profiteers can be sent away from Helsinki and out of the country, no questions asked. Pharmacist, IV:54, 6-7 capable of development for young people, the slang can be taken further, the mundane and tough attitudes of the Stadi culture are well suited as a background to the fast- moving youth culture of today. So everyone can create his or her picture of a Stadi culture which suits the personality, and an identity which matches or stands in opposition to it. However, the situation is not all that simple. Earlier, the Stadi culture was primarily anti-rural, although there was a relatively high urban tolerance level for other differences between those who were regarded as "from Helsinki", and this is naturally an expression of the "us and them" mentality. Today the unanimosity about the concept Stadi is nothing given, but rather, the word evokes polemics in different quarters. This is no marginal voice. New dichotomies, new ways of dividing the town and the townspeople Yet against the background of the not too strenuous circumstances of the informants, the collected narratives often are a defence for an immaterial dimension of existence in the city, and is based on satisfaction with the opportunities to "do things" that the town offers, which both are ways of getting rooted in the urban environment. Consu- merism is one element here, but only part of the total. Therefore, most of the narratives are stories about various experiences of the town. Such an appropriation and use of urban space as takes place at several levels and is based both on personal histories and on the present, works as a combined process, uni- ting the past with the present and fortifying the personalities. There are also positive conscious stories about how newcomers shoot out their roots, and these describe the process as a personal appropriation of space. The fact that the concept "stadilainen" works as a catalyst for many different approaches indicates an ambivalence in the identifications which is more fluctuating than what the town is as an environment. Nevertheless, the catalyst itself is important. With its aid, Helsinki and the people living in Helsinki achieve a social cohesion of another kind than what the town offers as an environment. It also counteracts the internationalization and the strong expected influx into the town. This concept, then, will become a basis for negotiations, providing cohesion between opposite opinions in a matter which per se is not unambiguous. We are discussing different attitudes to what is "genuine" and what it means to "belong", and this also occurs at the level of bickering. Negative attitudes towards other strangers, the "really other", are still relatively inarticulate, but it is possible that they are based on negative self-images, both a general hostility to foreigners, and a stressing of the ethnic-national, the genuinely Finnish. I will give an example: It remains to be seen if the ways of appropriating urban space in a positive way will stay sufficiently strong and open, so that they can counteract aversion against strangers and, instead, continue to introduce and generate positive self-images for various kinds of townspeople. Refugees and returning emigrants can at a cinch be accepted, if they come here in 35 Bausinger, Hermann 1985. Hembygd och identitet. (Home region and identity). In Kulturmoten och kulturell forandring. Red. Anders Gustavsson. Stockholm. Bausinger, Hermann 1985. Hembygd och identitet. (Home region and identity). SOURCES Lefebvre, Henri 1998. The Production of Space. Cornwall: Blackwell. 496 answers to questionnaires relating to Helsinki as a living environment; The Helsinki of my generation; Helsinki - the capital - my town; and What does Helsinki mean to me? To be deposited in the Helsinki city archives. Mohrmann, Ruth 1989. Dingliche Erinnerungskultur im privatem Bereich. In Mohrmann, Ruth 1989. Dingliche Erinnerungskultur im privatem Bereich. In Erinnern und vergessen. Gottingen: Volker Schmerse. Erinnern und vergessen. Gottingen: Volker Schmerse. Poulet, Georges 1997. Proustian space. Baltimore: The John Hopkins Univ. Press. New dichotomies, new ways of dividing the town and the townspeople In the Proustian way the internal appropriation of a bygone space or world is, while recalling it, given a new and completed form, which also strengthens the identity of the narrator. The time and, here, the space long gone, are thus recovered in an "integral reconstitution of place" (Proust 1982; Mohrmann 1991; Poulet 1977, 66). The fact that the narrators themselves state that the experience of telling their story was gratifying, is a sign that such a mechanism was working. In Kulturmoten och kulturell forandring. Red. In Kulturmoten och kulturell forandring. Red. Anders Gustavsson. Stockholm. Anders Gustavsson. Stockholm. Eriksen, Anne 1997. Memory, History, and National Identity. Ethnologia Europaeav o 1 . 27:2, 1997. Gerndt, Helge 1985. Grosstadtvolkskunde - Moglichkeiten und Probleme. I Grosstadt. Aspekte empirischer Kulturforschung. Eds. Theodor Kohlmann and Hermann Bausinger. Berlin. Gerndt, Helge 1985. Grosstadtvolkskunde - Moglichkeiten und Probleme. I Grosstadt. Harvey, David 1989. The condition of Postmodemity. Cambridge. Basil Blackwell. NILS STORA Commerce and Carnival: The Fish Fair as a Meeting-Place wheels of fortune and other entertainments. Various kinds of food are served as well as drinks and these are sold in, for example, beer tents. The archipelago fair in Eastern Nyland, held on the Lokoren boat pier in the Broby village of Pyttis parish at the end of August 1997, is described in the calendar as "an event smelling of fish and tar", with "things going on from morning until late night". The fish fair in Kuopio is said to be "nationally important" and one of "the large fish fairs", which in 1996 attracted about 25,000 visitors. In comparison to this, it may be mentioned that the autumn fish fair in Abo in the same year attracted approximately 170,000 visitors over four days. The prevailing weather conditions, understandably, have an influence on the number of visitors a particular fair will attract. In 1997, a total of 4 7 fish fairs were organised in Finland. In Abo not only was the usual autumn fish fair held, but a fair was also arranged in the spring, too early (because of the ice) for people from the archipelago to be able come into town by means of their own boats. The times and places of the fish fairs over the year are given in the Fish Fair Calendar which is published by Fiskeritidskrift for Finland (Finnish Fishing Journal) (2/1997). Seven of the 4 7 fairs are called herring fairs, and among these is the autumn fair in Abo, which is the one I will particularly focus on in this article. Fish fairs are held both in towns and in the countryside, mostly on the coast, but also inland, where five fairs focus on the freshwater whitefish, or vendace. The fairs naturally mostly take place in market squares and harbours, or, as in Abo, traditionally along the banks of the river, where goods can be sold directly from boats. Today, the fairs are organised by various organisations, in many cases fishing associations and fish businesses in cooperation with municipal bodies, but also by Lions Clubs and other non-profit making organisations. The fish fairs offer much more than fish in various forms and other foodstuffs. The organisers often aim at increasing the visitors' knowledge about fish as food, about the handling, breeding and cooking of fish. In addition, there is music, dancing, lotteries, There is obviously a great interest in fish fairs today. LITTERA TURE Proust, Marcel 1982. Pa spaning efter den tid som flytt 1-7. Stockholm:Bonniers. Andersson, Harri 1997. Kulttuuri ja paikan politiikka kaupunkiuudistuksessa. I Tila, Andersson, Harri 1997. Kulttuuri ja paikan politiikka kaupunkiuudistuksessa. I Tila, Andersson, Harri 1997. Kulttuuri ja paikan politiikka kaupunkiuudistuksessa. I Tila, paikka ja maisema. (Culture and the policy of site in urban reconstruction.). Eds. Tuukka Haami et al. Tampere. Vastapaino. Anttonen, Pertti 1996. Myyttiset uhkakuvatja puolustuseetos Suomen j a suomalaisuuden konstituoinnissa. (Mythical threat images and defence ethos in the constitution of Finland and Finnishness). Elektro/Loristi 2/ 1996. Astrom, Anna-M. 1995. Der Fischermarkt in Helsingfors (Helsinki) als Identitatsmerkmal der Schweden in Finnland. Schweizerisches Archiv fur Volkskunde 91/1995, H.2, 129-142. paikka ja maisema. (Culture and the policy of site in urban reconstruction.). Eds. Tuukka Haami et al. Tampere. Vastapaino. Anttonen, Pertti 1996. Myyttiset uhkakuvatja puolustuseetos Suomen j a Anttonen, Pertti 1996. Myyttiset uhkakuvatja puolustuseetos Suomen j a Anttonen, Pertti 1996. Myyttiset uhkakuvatja puolustuseetos Suomen j a Astrom, Anna-M. 1997. The town as a landscape in the mind- spatial and social dimensions in chi- ldhood remembrances from Helsinki in the 1950s. Ethnologia Fennica vol. 25. 1997, 35-48. suomalaisuuden konstituoinnissa. (Mythical threat images and defence ethos in the constitution of Finland and Finnishness). Elektro/Loristi 2/ 1996. suomalaisuuden konstituoinnissa. (Mythical threat images and defence ethos in the constitution of Finland and Finnishness). Elektro/Loristi 2/ 1996. 36 36 (Lindequist 1928, 463). (Lindequist 1928, 463). and a popular song. More recent ethnological interests are represented by Birgitta Skarin Frykman's historically focussed studies ( 1993 and 1995) of the St Laurentius Day fair (Larsmassemarknaden) in Goteborg, in which she puts special emphasis on social groups, and by Anna- Maria Astrom's article (1995), inspired by Bourdieu, on the fish fair in Helsingfors during the 20 th century as a Finland- Swedish identity marker. Fish fairs in earlier times can be regarded primarily as a place where archipelago dwellers traded their fish for grain and other necessities. Barter still took place to a certain extent at the beginning of the 20th century, mostly at the autumn fair in Uskela, or Salo, where predominantly Swedish- speaking people from the archipelago traded with mainly Finnish-speaking people from inland. There, unlike in Abo, bartering could be carried out without the interference of the third party consisting of town merchants. On the other hand, the circle of paying customers formed by townspeople was missing at the country fair. Gradually, the number of Swedish-speaking townspeople and merchants decreased. Astrom' s point of departure is the strong Finland-Swedish element at the fish fair in Helsingfors, and thus the visibility of Swedish in an official context, as she emphasizes the symbolic meaning of the fair (Astrom 1995, 135ff). However, fishermen from remote Finnish-speaking areas in the archipelago have also regularly attended the fish fairs both in Abo and Helsingfors. The concept of the fair is characterised by the fact that the time and place for trading are officially determined and advertised in advance in the calendar. As a form of trade the fair is annually reoccurring, short and subject to the control of the local authorities. Definitions depend upon which aspects of the fair concept are emphasized in each case, be they the financial or the social, commercial or carnival elements. As an expression of old trade policy that favoured towns and limited and regulated trade in the countryside, fairs have, despite the focus on trading, traditionally presented themselves as annual gatherings of people associated with church festivities or masses, political meetings, court sessions and tax collection (Staf 1935). Studying fairs as meeting-places for different groups of people is a given. Social anthropologist Borje Hanssen, who explored the social significance of fairs, regarded them as "focal points for the inter- regional contacts between strangers" (Hanssen 1952, 270f). (Lindequist 1928, 463). The various groups that met at the fairs were drawn there by different elements within the multilayered fair complex. The fairs constituted meeting- p laces not only for producers and consumers. At fairs both in town and the countryside, the dominating category of visitors were country people. Free fairs were attended by a considerably wider circle of people than the local, separate fairs, and according to a royal charter of 1788, all fairs were to be free fairs NILS STORA Commerce and Carnival: The Fish Fair as a Meeting-Place What attracts people to them? Who are the visitors? What is the significance of the fish fairs today? In order to illuminate these questions a review of the fair as a concept is needed. Ethnologists who have explored issues connected with local fairs include Marti Rasanen, who has studied the fair in Kuopio ( 1970), and Jouko Heinonen, who has dealt particularly with the fair in Rovaniemi, "Rovaniemen markkinat" (1974 and 1979), which has been the theme of both a film 37 Fairs and fair traditions in Abo The two most important fairs in Abo have been the two annual St Henry fairs (Henriksmassmarknaden ), that is to say the winter fair that started on 19 January and the summer fair, the "birch-bark fair" on 18 June. During the Middle Ages, these were the largest and most frequented fairs in the country. The autumn fish fair dates back to the free fair which was established in 1636 and started at "Mother mass", the St Mary mass (Mariamassa) celebrated on 8 September. Originally this fair was allowed to continue for three weeks. When it was introduced, both inhabitants of Finland and foreigners were given the right to "sell their produce from ships and boats", but it had to be done according to organised 38 The herring fair at the eastern river bank in Abo in 1987. Photo: Annika Jokiranta. Department of Ethnology at Abo Akademi University (40776). forms (Bonsdorff 1894, 499f; Qvist 1909, 8). Later a fourth fair was established, in the form of a movable Lent fair in February, when people from the archipelago would usually come into town by horse over the ice. with a bleeding breast. The crowds amused themselves by throwing rings or games of shooting. Alcohol flowed, according to Lindberg, so that the whole town was like one big open public house. Already by the end of the 1600' s, the town's authorities expressed concern about the harmful influence of the fairs on young people (Lindequist 1928, 466). In one of his newspaper columns, Ernst Lindberg provides a lively description of the events of the St Henry fair on 15 January in Abo during the 1870's (Lindberg 1921, 73ff). Local merchants and craftsmen had prepared well in advance for the arrival of people from various parts of the country. "On the spot where the Hotel Phoenix stands today", he wrote in 1921, the merry- go-round constantly turned while music was playing and bells were chiming; in the tent next to it "the handless woman" and "the lifeless violin player" were on display, while a panorama of the Franco-German war created a sombre atmosphere. Fairs and fair traditions in Abo In the pleasure ground between the Market Hall and Kristinegatan, a waxworks cabinet invited viewers in to see Napoleon and Garibaldi, or "the beautiful Galathea with her rocking bosom", and "the dying soldier" Alcohol was one of the products sold by distilleries in and outside the town, and the purveyance of spirits was traditionally connected with trade, as an important round of bargaining was often concluded by a confirmatory toast. This was a widespread custom, which could easily get out of hand, since the toasting was not limited only to the buyer and seller. The fairs attracted large crowds of idle people and trips to the fair were included in the rights of servants. Maids and farmhands could find new employment at the autumn fairs. Skarin Frykman (1995, 27ff) points to the similarities with so-called hiring fairs in England. For young people, their first trip to a fair, like their first trip to 39 country trade was freed from regulation and transport gradually improved, the financial significance of the fairs diminished and instead the entertainments and drinking came to dominate. The variety of amusements was expanded; for example, dancing was introduced. This contributed to the feeling of carnival that came to be associated with fairs. The selection of goods became more variable, as all kinds ofknick- knacks started to appear. The fairs now functioned more as annual festivities for the people, organised at the same place and officially announced in the almanac. town, could mean a significant step towards entering the world of adults ( see Talve 1960 and 1978), an event they impatiently waited for. Here young people could meet and form couples. Lindberg describes the fairs in Abo in the 1870's, during which farmers' sons and their girls gather at the stalls of the goldsmiths Lundgren and Willgren to try out rings or finger brooches and earrings (Lindberg 1921, 75). If the town's merchants and fish-buying bourgeoisie had a positive attitude towards the crowds that the fairs attracted, it must, however, be assumed that there were other townspeople who disliked the tumult, with all the shouting and shrieking that the fair days often brought about. A considerable portion of the visitors had not come for the commerce, but for the entertainments that were on offer. It has been claimed that the fairs had lost their actual significance by the beginning of the 1880's. Fairs and fair traditions in Abo It is, however, clear that they had such varied offerings that they could survive in some places and still attract large crowds. Not even the extensive years of war could completely break the enduring fair tradition. Some fairs were over time substituted by separate market-days, which were attended only by local people. Other fairs were maintained as specialising in certain goods, such as cattle and also fish. The best known fish fairs during the latter half of the 19th century were those in Abo, Salo and Helsingfors, all three of which mainly sold salted herring. Barter - exchanging goods for goods - still existed to a certain extent at the beginning of the 20th century. Fairs and interest in the fairs During the long period of existence of fairs, their nature has varied. It is usually emphasized that in the 18th century they were still largely an important and integrated portion of trade, but that they later gradually lost their financial significance. As can be seen from the calendars published at the time, the number of fairs decreased significantly in Finland during the 19th century. Already by the beginning of the century, many fairs were abolished both on the coast and in towns and the number of days a fair would run was decreased. In 1821, there were only nine fairs left in the countryside, while the number of town fairs amounted to about 60. In 1867, there were only 20 fairs left (Lindequist 1928, 466ff; Heinonen 1979, 43ff). On the other hand, some new fairs were established, and the fact that a fair was abolished did not necessarily mean that people stopped gathering at the fair ground at the traditional time of the fair. During the latter half of the 19th century, when Spot checks in almanacs show that the number of fairs was just over 30 at the tum of the 20th century, while they amounted to around 20 from 1910-1960. In the 1970's, the interest in fairs increased again as the result of a wave of nostalgia that, for example, revived old folk festive customs (Heinonen 1979, 45). In 1980, the number of fairs had grown to 58 and in 1990 the almanac includes more than 150 fairs, which, however, are probably of greatly varying kinds. This number, which has increased immensely since 1970, thus also includes the fish fairs, which, as already 40 Cuddy boats and longboats at the fish fair in Abo, autumn 1922. Photo: Birger Lundsten. The Abo Akademi Maritime Institute (1373). mentioned, amounted to 4 7 in 1997. their main livelihood. Therefore the autumn fair was the actual fish fair. The fact that it was a free fair widened its circle of customers. However, considering the herring trade, the autumn fair in Abo was held somewhat too early, while the important drift net fishing was still going on. From the perspective of herring fishing, the country fair in Salo and the town fair in Helsingfors were more convenient for many, since they were held later in the autumn. Motor and sailing boats at the fish fair in Abo 15 September 1931, some with the sail tent raised. Barrels of various sizes with salted herring are still a visible element. Photo: Birger Lundsten. The Abo Akademi Maritime Institute (I 375). Fish fairs and herring fishing The old winter fair held in January in Abo was oflittle significance as a fish fair, since people from the archipelago were usually unable to come into town at that time of year because the ice situation. The second St Henry fair, known as the "birch-bark fair", held in June shortly before Midsummer, was more important in this respect. Here, the archipelago dwellers could obtain birch bark which was an important roofing material and usually unavailable on the islands. In 1850, more than 700 boats of various sizes arrived at this fair, which was the largest in Abo at that time (Nikula 1973, 275). Most of the herring available during that season was fished with a seine-net and was not of the same quality as herring fished in the autumn. Nevertheless, the seine herring was salted and found a market particularly in Russia through Tallinn and other Baltic ports. The herring fished with drift nets during late summer and autumn was the best (Stora 1979, 14lff). This was the fish that was to give the archipelago dwellers At the autumn fish fairs the archipelago dwellers could sell most of the year's herring haul as salted fish. The buyers were partly townspeople, partly farmers from inland who could give grain and other goods in exchange for the fish. At the country fairs there were also purchasers who sold the herring on inland. At the market, the town dwellers bought fish and other food for a few days' supply, while at the fish fair they bought all the fish they needed for the whole winter. The trade at the fish fair obviously often depended on the return of the same customers year after year. In a newspaper advertisement for the fish fair in Helsingfors in the autumn 1895, 41 herring, even if herring in earlier times was also conserved in other ways, such as drying and souring (for details see Ston11986 and 1988). Fermented herring existed during the 18th century, but it did not survive in Finland as it did in N orrland in Sweden. Grilled and salted "coal herring" is also mentioned in the Abo region as early as the 16th century. Smoking did not become common until the end of the 19th century, at least not warm smoked herring. Fish fairs and herring fishing A publication on the conservation of fish (Reuter 1898, 3f) claims that smoking of fish (herring and whitefish) is done in Finland only in the Helsingfors region, where "the odd archipelago dweller" will warm-smoke fish to sell in town - using the same method as in the herring smokeries of Kiel and Lubeck. In the winter, it was possible to freeze herring when fishing with a winter seine-net. it is said that most of the archipelago dwellers have old customers that they have supplied with fish for many years. "Everywhere one can witness reunions, greetings and invitations". The advertisement also mentioned goods apart from herring sold on a ship from Kokar in the Aland Islands: whitefish, butter, cheese, eggs, seal fat, smoked meat, down, feathers, black bread, nuts, decoys, empty gulls' and other birds' eggs. The most important merchandise, salted herring, was sold in tubs of various sizes, down to a sixteenth of a barrel. The fact that these small-size tubs were now mentioned reveals that the townspeople's interest in this product had decreased. There was no longer a need to store food for a whole year and with an improving standard ofliving, salted herring was no longer such a favoured food. (Hbl 2 October 1895). In 1877, the autumn fair in Abo was held on 19 September, and the salted herring sold so well that by noon very little remained of the total of more than 1,500 barrels brought to the fair by the archipelago dwellers. Over 200 of the 600 boats attending the fair had Salted herring: producers and consumers When discussing the goods sold at the fish fair, it is reasonable to assume that the staple produce at the fair in Abo was salted 42 The fish fair in 1931 (see picture 3). Photo: Birger Lundsten. The AboAkademi Maritime Institute (1374). been loaded with herring. About three quarters of the barrels are said to be measured, while one quarter were unmeasured barrels, where the fish was thrown in and not neatly layered. The newspaper article I quote complains about the fact the archipelago dwellers still (1877) did not realise the advantage of packing their fish in a "customarily measured vessel", which would sell much better (Folkvannen 3 October 1877). another method through the expertise of Ossian Reuter. Method meant that the herring was layered in the barrels. The old way was to toss the herring into tubs and barrels, which took much less time. At fish fairs in the 1890's some ships from Aland advertised their herring by signs saying "We have herring salted according to method" (Hbl 2 October 1895). Already during the latter half of the 19th century, salted herring started to lose its earlier status. There are many reasons for this. The fishermen who sold salted herring traditionally made their own barrels. The making of barrels and tubs was a skill required of all who sold salted herring. It was a craft that sons learnt from their fathers, an example of the tacit knowledge which attracts great interest today pertaining to the learning of cultural knowledge, both within ethnological and anthropological research. A young boy had to learn to handle more than just axe and knife. Tools needed for making tubs, usually of pine, were, among other things, various kinds of planes, the cross axe, various kinds of knife and files. For the hoops, which were made at a special During the times when salted herring also formed a substantial part of the winter food of town dwellers, large amounts of salted herring were brought to the autumn fairs. To the fair in Helsingfors alone, between 15,000 and 20,000 barrels were brought every autumn at the end of the 19th century (Reuter 1898, 9). Usually, for one barrel of herring, one third of a barrel of salt was used, but the amount varied according to the kind of salt used. There are many ways of preparing salted herring. Salted herring: producers and consumers The Alanders, who also sold fish in Stockholm, had learned a good method from Gotland, and Finska Hushallningssallskapet (the Finnish Agricultural Society) spread 43 Selling fish products at the herring fair in .Abo in 1986, at the eastern river bank. Photo: John Hackman. Department of Ethnology at .AboAkademi University (39345). quality of the stave boards. During the end of the 19th century, the barrels became an increasing problem. In the 1890's an industrial company, Brando-Lemsjoholms fiskkarlsfabrik, was founded and they sold ready-made barrels of good wood during a few years. However, the cost of these was too high for the fishermen in the archipelago. The townspeople became choosier about their food. Their standard of living improved and their diet became more varied. Many people preferred fresh herring to salted. When times got harder, the interest in salted herring increased again, as can be seen during the First World War, the years of financial depression in the early 193 0' s and the latest wars. During the years of food shortage the demand for salted herring was so great that even all badly salted and prepared herring was sold. Bad produce contributed to the aversion against salted herring as soon as the shortage of food was over. Salted herring was increasingly regarded as poor people's food, as "shoemaker's salmon" and its role at the fish fairs diminished (Stora 1986). knife-bench, hoop knives and hoop hooks were needed. To manage the many phases of making barrels was regarded as something of a test of manhood by the people in the archipelago. One had to make evenly planed staves, perfectly round bottoms, and split and cut hoops that tied together the staves with well cut joints, in order for the brine not to leak, and kept the bottoms in place in the notch on the inside of the upper and lower parts of the staves. Sworn crowners travelled around with their marking-irons and cans and tested out that the barrels corresponded to their measures: a half-barrel 24 cans ( 60 litres), a quarter-barrel 12 and an eighth-barrel 6 cans. There were models for each size, since the tubs were to have a slightly rounded form and the edges the correct angle so that the tubs could be pressed closely together. Selling fish products at the herring fair in .Abo in 1986, at the eastern river bank. Photo: John Hackman. Department of Ethnology at .AboAkademi University (39345). The fish fair as a meeting-place today Salted herring in plastic vessels and small wooden tubs are sold together with other fish products at the herring fair in 1986 (see picture 5). Photo: John Hackman. Depart- ment of Ethnology at Abo Akademi University (39346). and polluted fishing waters, fish is today regarded as an attractive, healthy food. Advertisements claim that "Clever people eat fish". Town dwellers used to impersonal supermarkets get into direct contact with producers at the fish fair. The concentrated supply of goods is another contrast to the situation in supermarkets. The interest in fish fairs can perhaps thus be seen as somewhat of a reaction against the negative aspects of today's consumerist society. It is also obvious that it is not only the increased interest in fish as food that has contributed to the townspeople's increased interest in the fish fairs. Even if the elements of old fair entertainment have disappeared, there is still space for amusements and knick-knacks, alongside a varied supply of fish for all tastes. There are even fish products that have more or less eliminated the taste of fish, such as sauna smoked herring ham, for those who do not like fish. Herring flavoured with garlic, dill, carrot, pepper and mustard compete with perch balls, pike burgers and salmon pies. At the 1997 herring fair in Abo a competition for the best "delicacy herring" was organised. The awarded products included salted herring tartar and marinated, smoked herring rolls (AU 24 October 1997). Thus also salted herring, usually sold as shoemaker's salmon, is one of the products that have been developed further. There is tough competition from farmed fish. Despite the occasional debates on mercury However, the most important function of fish fairs is perhaps still that of a meeting- place. A distinct feature of today's autumn fish fair in Abo is "maritimely" dressed summer visitors and leisure sailors who after a few weeks in town re-encounter archipelago dwellers that they know or at least have had prior contact with. For them, the autumn fair marks the end of the summer season. It is also clear that today's otherwise somewhat introverted town dwellers meet not only already established friends and acquaintances, but also make new acquaintances from amongst their fellow townspeople at the fish fair. Salted herring in plastic vessels and small wooden tubs are sold together with other fish products at the herring fair in 1986 (see picture 5). Photo: John Hackman. Depart- ment of Ethnology at Abo Akademi University (39346). The fish fair as a meeting-place today One of the big changes that have taken place from the perspective of the archipelago dwellers is that salted herring, which earlier totally dominated the trade at the fairs, gradually lost its significance. Today's concept of the herring fair no longer includes the tubs of salted herring which, loaded onto boats and piled on deck, constituted a distinct feature of the herring fairs in earlier times. The small plastic buckets of salted herring sold today are of no great significance. As long as salted herring was the most important merchandise, the working year of the herring fishermen was largely focused on the autumn fair. The whole year's income was basically dependent on the fish fair. Making barrels was hard work that was carried out periodically from autumn to spring. It took a long time and good wood was required, which was often hard to find in the outer archipelago. The wood issue contributed to the decreasing demand for salted herring. As steam sawmills were established, sawn boards could be bought, but these were not watertight. The old way of making staves for the barrels were to split the pine wood so that the sap rings, where the brine easily leaked through, did not run across the staves. The fact that the timber was rafted to the sawmills additionally impaired the 44 Interviews with archipelago dwellers today indicate that the income from the fish fairs still is of great financial importance for many. Another important function of the fairs is the encounter with other, sometimes previously unknown, fishermen and fish entrepreneurs, people with shared attitudes who have the courage to choose fishing as their livelihood. Many emphasize the feeling of community that the assembled archipelago dwellers and fish sellers experience today at the fish fair, regardless of their language. Here, the fishermen's identity is strengthened. Most of the Swedish-speaking archipelago dwellers today feel that they know Finnish well enough to sell fish. The fish is on display and "speaks for itself'. Finnish-speaking town dwellers, for their part, understand that a relatively large section of the fish sellers are Swedish-speaking. The informal situation and the "fair atmosphere" make it easier to cross the language barrier. Salted herring in plastic vessels and small wooden tubs are sold together with other fish products at the herring fair in 1986 (see picture 5). Photo: John Hackman. Depart- ment of Ethnology at Abo Akademi University (39346). The fish fair as a meeting-place today In the crowds along the river banks people move much more closely to each other than is customary, which contributes to the creation of a specific "fair atmosphere". It is impossible to keep the normal physical distance from other people. Bodily contact, so to say, is more permissible. There is no 45 socialantropologiska sammanhang under 1600- oc h 1700-talen i sydostra Skane. Ystad Heinonen, J.1974, Rovaniemen markkinat.1-2. Pohjalaisten osakuntien aluetutkimustoimikunta B 12 (stencil). Helsinki. need to show the total indifference to strange people that the contemporary person usually displays in, for example, lifts, where one is temporarily forced into a proximity which in a normal situation would feel uncomfortably intimate. The American anthropologist Edward Hall as early as 1959 (according to Crapo 1990, 188f) showed that the distance we place between ourselves and others can be interpreted as a message in non-verbal communication. He distinguishes between four distances of different lengths, where intimate is the shortest and reserved for situations where cuddling and touching are acceptable. The intimate distance is followed by the personal, after that comes the social distance and furthest away is the zone for the public distance. Heinonen, J. 1979. Markkinat. Kalevalaseuran vuosikirja 59. Hufvudstadsbladet (Hbl) 2 October 1895. Nikula, O.1973,Abo stads historia 1809-1856. Abo. Lindberg, E. 1921, I Abo pd 1800-talet. Bilder och minnen av Ernst Lindberg. Abo. Lindequist, K. 0. 1928, Markkinoista Suomessa. Oma Maa V. (2nd rev. ed.) Porvoo. Qvist, J. 1909, Finlands marknader och finska landsbygdens handelsplatser 1614-1772. Helsingfors. Reuter, 0. 1898, Konservering af jisk. Vart land 12. Rasanen, M. 1970, Kuopion markkinat. Aami 13. Using this interpretation, the crowd at the fair is a situation where crossing the border between the personal and intimate distance is accepted behaviour, marked by a festive context of an informal character. Skarin Frykman, B. 1993, Larsmiissemarknaden. En folklig kameval i 1800-talets Goteborg. Viistsvensk kultur- och samhiillsutveckling: Rapport nr 3. Goteborg. Skarin Frykman, B. 1993, Larsmiissemarknaden. En folklig kameval i 1800-talets Goteborg. Viistsvensk kultur- och samhiillsutveckling: Rapport nr 3. Goteborg. The external scene of the fair is today quite different from the times when wooden cuddy boats, longboats and the occasional skotbllt lay close to each other, creating a forest of masts, sometimes with the sails hung out to dry, fluttering in the wind. The number of boats, mostly plastic ones, is markedly smaller than before and the fish trade has moved up onto the river banks. The fish fair as a meeting-place today The inland farmers have disappeared from the market scene, as have the barrels of salted herring. Despite the changes, the diverse fair tradition represented by the fish fairs still contains attractive elements. Skarin Frykman, B.1995, Larsmassemarknaden och VM i fotboll. Om vastsvensk sarart och kunskapens granser. Saga och Sed 1994. Staf, N. 1935, Marknad och mote. Stockholm. Stora, N. 1979, Finlandskt strommingsfiske. Teknisk utveckling och social diflerentiering. Norveg 22. Staf, N. 1935, Marknad och mote. Stockholm. Stora, N.1986, Strommingsfiskets betydelse for skiirgardsbefolkningen. F iskerimuseiforeningens publikationer 3. Hango. Stora, N. 1988, Barrels and bundles of herring. In: A. Fenton & J. Myrdal ( eds.), Food and drink and travelling accessories. Essays in honour of Gosta Berg. Edinburgh. Stora, N. 1988, Barrels and bundles of herring. REFERENCES Talve, I. 1960, Ensimmainen kaupunkimatka Ja siihen liittyvia tapoja. Kalevalaseuran vuosikirja 40. Helsingfors (Helsinki) als Identitatsmerkmal der Schweden in Finnland. Ein Beitrag zur Diskussion iiber symbolisches Kapital. Schweizerisches Archiv fiir Volkskunde 91. Introduction elements of the narrative as feelings, emotions and smell perceptions. This is Baltus, a fabular beast. It is not, however, an animal character in a story, but a visual presentation of a scheme of narratives that are used in classrooms today. In this lecture21 , I shall discuss the functions and significance of narratives and narrating; why it is important to be able to tell stories, and the significance of narration for the individual. The examples presented here are taken from my research on stories of childbirth (Marander-Eklund 2000). These are also the elements of which narration consists according to linguist Mieke Bal (1985), and that according to another linguist, William Labov (1972), constitute the basic structure or schema of a story. Schema refers not merely to a specific form of narration, but equates to an internal model, a general knowledge concerning a structure that helps organise concepts and events in the memory which can then be brought forth and re-presented when necessary. Baltus - teaching how to narrate The fabular beast Baltus is a concrete visual presentation of the basic structure of a narrative. Baltus is used in drama based pedagogy22 (Hagtvet & Palsd6ttir 1992, cf. 0stern 1994) in some comprehensive schools today, to teach pupils what elements to include in a story and the order in which they may be narrated - in other words, how a message is conveyed in narrative form. The children will learn that a narrative includes the following elements: an introduction indicating who is involved, as well as when and where the events of the story take place. After this, the main action of the story is narrated, with a treatment proper of the story's dilemma, followed by its resolution. In addition, an assessment or evaluation of the event itself is presented, along with a description of such integral These elements, which we can see constitute the fabular Baltus, also exist in narrations of personal experience as they are given in an interview situation where the interviewee is able to speak freely about the event. This study explores narrations of childbirth, where the narrations consist of an oral expression of a personal experience of giving birth to a child. It is an event that is narrated from a strictly personal point of view, taking place in a particular time and a space, by specific actors. Almanackan 1810-1997. Almanackan 1810-1997. Bonsdorff, C. von 1894, Abo stads historia under sjuttonde seklet. Helsingfors. Talve, I. 1978, "Oxpojken" i staden. Kring ndgra finska och nordiska traditioner om resvagar och denforsta resan. Norveg 21. Abo Underrattelser (AU) 24 October 1997. Folkvannen 3 October 1877. Astrom, A-M. 1995, Der Fischermarkt in Hanssen, B. 1952, Oster/en. En studie over 46 Helsingfors (Helsinki) als Identitatsmerkmal der Schweden in Finnland. Ein Beitrag zur Diskussion iiber symbolisches Kapital. Schweizerisches Archiv fiir Volkskunde 91. 47 47 Introduction Furthermore, the events are evaluated or assessed in one way or another. The events are narrated in the form of a plot where a problem is solved at a dramatic turning point - in this case the 'problem' is the delivery and the 'solution' is the birth of the child. Today's school children thus learn to narrate by means of engaging with the 48 The fabular beast "Baltus" Where When? What happened? Why? The fabular beast "Baltus" The fabular beast "Baltus" Figure 1. The tabular beast "Baltus"-a visual presentation of a narrative structure (Freely based on Hagtvet & Palsdottir 1992: 90) Where When? What happened? Why? What happened? Why? presented. Narrating stories is a basic cultural and social skill. fabular beast Baltus as a pedagogical tool. Baltus provides a narrative structure, a disposition that helps the pupil remember what the elements of which a story consists are. Baltus exercises stimulate linguistic awareness, develops story telling techniques, and helps the child produce texts of better quality (Hagtvet & Palsd6ttir 1992, 88ff.) fabular beast Baltus as a pedagogical tool. Baltus provides a narrative structure, a disposition that helps the pupil remember what the elements of which a story consists are. Baltus exercises stimulate linguistic awareness, develops story telling techniques, and helps the child produce texts of better quality (Hagtvet & Palsd6ttir 1992, 88ff.) The function of narration It expresses a wish for individual choice in the delivery itself - to be able to choose a safe and painless delivery or a delivery untouched by outside interference, with the facilitation of it as an experience being a key requirement. The narration confirms the narrators' self- identity as birth-givers and as women; it is a heroic telling of a heroic deed. These childbirth narratives become self- presentations, a description of the narrator herself. She places herself in the position of a woman giving birth when narrating the story when it is told directly after the delivery, and mainly as the mother in the narrations given a year later. Th d f ti i t d d b The first, the individualizing function allows the narrator to reinforce her self as an individual. This is also pointed out by another folklorist, Charlotte Linde, who emphasizes the social function of narratives in creating and maintaining personal identity. Narrating is significant for creating our internal, private sense of self and a major resource for conveying that self and negotiating that self with others (Linde 1993, 98). This function is strongly present in the narrations of childbirth that I have studied. They were all narrated in interview situations. I interviewed women who were giving birth for the first time; once before childbirth, once right after childbirth, and once a year after delivery. The interviewees are at the centre of their narrations, both as narrators and as the main characters of the narration itself. The women clearly had a need to speak about the event which was significant for them; something they narrated with feeling, and great enthusiasm. The story literally "bubbled out" of them. They were also pleased to have someone listen to them, and to get the main role as narrator in the interview situation, talking without being interrupted. During the interview, my aim was to create a situation where the interviewee was able to narrate the experience without being interrupted. I considered this strategy essential as a means of gaining narrations that in due course I could compare in form, content and meaning. This was essential also in order to let the women themselves decide what they considered important enough to be highlighted in the narrative, what message they wanted to convey - in what way they chose to present themselves. The function of narration Narration is a basic form of communi- cation between human beings - a way of communicating experience and under- standing one another. According to media and communication researcher, Professor Arthur Asa Berger, narration is an effective way of sharing ideas, because it is a means of learning about life, and of communicating what one has learned (Berger 1997, 9-10). Narratives are also a means by which an individual can arrive at an explanation of the world, giving expression to his or her own view of the world or of shared values (Hyden 1997, 9-10). Narratives can thus be seen as an analytic resource that helps us to develop social and cultural insights (Hymes 1981 ). A question raised by the example of Baltus is: why do stories constitute such a vital form of communication, to the extent that it is taught in schools? Viveca Adelswiird is a linguist who considers the fact that children are taught the "right" way to tell a story, i.e. a way that is recognised by the listener, as a step on the way towards adulthood - becoming a fully functioning member of society (Adelswiird 1996, 24). In her view, the child learns to tell a story by co-narrating with adults. By co-narrating with the child, the adult trains the child to ask questions such as "when did it happen?", "where was it?", which in tum helps the child to understand what a story may be about or how an event might be Albrecht Lehmann, a folklorist, distinguishes three main functions of personal experiential narration: an individualizing function, an identifying function and a comforting function 49 experience bestows structure to the narration, giving it form and content. The narrator, the woman giving birth, draws the listener into the sequence of events in a space with an intensified meaning, a time of particular significance. The events are narrated, dramatized and explained, all according to the narrative patterns she has access to. The narration is based on examples from expert literature, the media, maternity clinics, and narrations by other women. These narrative patterns shape the structure of the narration and, to some extent, its content as well, naturally in interaction with the general opinions of childbirth. The events that are narrated are assessed and reassessed in relation to perceptions of these external viewpoints on the woman's situation in the present moment. The function of narration Contents and themes of the narratives include expectations, pain, lack of control or hopes of controlling the situation, the functioning of the body and its intuitive responses to the situation, as well as a sense of exposure. The "I" of the narrative expresses both happiness and joy in giving birth, as well as feelings of fear and anxiety related to it. It expresses a wish for individual choice in the delivery itself - to be able to choose a safe and painless delivery or a delivery untouched by outside interference, with the facilitation of it as an experience being a key requirement. The narration confirms the narrators' self- identity as birth-givers and as women; it is a heroic telling of a heroic deed. These childbirth narratives become self- presentations, a description of the narrator herself. She places herself in the position of a woman giving birth when narrating the story when it is told directly after the delivery, and mainly as the mother in the narrations given a year later. The second function introduced by Lehmann is the identifying function, where (Lehmann 1978). experience bestows structure to the narration, giving it form and content. The narrator, the woman giving birth, draws the listener into the sequence of events in a space with an intensified meaning, a time of particular significance. The events are narrated, dramatized and explained, all according to the narrative patterns she has access to. The narration is based on examples from expert literature, the media, maternity clinics, and narrations by other women. These narrative patterns shape the structure of the narration and, to some extent, its content as well, naturally in interaction with the general opinions of childbirth. The events that are narrated are assessed and reassessed in relation to perceptions of these external viewpoints on the woman's situation in the present moment. Contents and themes of the narratives include expectations, pain, lack of control or hopes of controlling the situation, the functioning of the body and its intuitive responses to the situation, as well as a sense of exposure. The "I" of the narrative expresses both happiness and joy in giving birth, as well as feelings of fear and anxiety related to it. The function of narration In these personal narratives the women giving birth are at the centre of the experience. They speak about giving birth in terms of their own physical experience, from the point of view ofan empowered "self'. The physical The second function introduced by Lehmann is the identifying function, where the narrator formulates her membership of a group. Alf Arvidsson, a folklorist, 50 describes this as "narrating your group", suggesting that the essential values of the group are expressed and repeated within the group in order to reinforce membership and belonging, creating a feeling of togetherness (Arvidsson 1998). This function was less evident in my research data than the individualizing function, quite possibly because I encouraged the interviewees to take the main role in the producing of the narrative. The narrations can nevertheless be viewed through a sense of a female community and the act of telling the story does bestow upon the narrator membership in the group comprising "we who have given birth", which by definition excludes women who have not given birth. Women take great pride in talking about their performance in giving birth to a child. At the same time, the role of a mother is experienced as both surprising and bewildering. Their narration gives expression to those values which may be shared by women who have given birth, and the features ofreal, normal delivery. It also reflects the concept of a "real" woman who gives birth to babies. narrating it (Lehmann 1978, cf. Virtanen 1982). Narrating an event is a way of making sense of experiences, structuring the events in a meaningful way, and in a way in which the narrator and the recipient may resolve a dilemma (Robinson 1981 ). As I have already pointed out, the women giving birth felt compelled to narrate this significant event in their lives. Thus, in my view this kind of narration can also be seen as therapeutic: creating order by verbalising the event, analysing what has happened, and making sense of one's own role in it. This also involves opening up sore points, revealing disappointments and difficult experiences. By making sense of the actual event and one's role in a sequence of events, the narrator is also empowered in the situation. The narrator gratifies her own wish to tell the story and perhaps only rarely takes into consideration that her description may inspire aversion, anxiety or apprehension in the recipient. The function of narration One function of narration is 'glossing over'. The narrators transform what did not go so well into a happy end version by making changes they can live with. With time, they become more critical about their own input as well as that of the hospital staff. The woman giving birth excels as the parturient. Narrating the event also involves dramatization and the story is amplified by evaluating the described events. This is done by repeating the descriptions several times with a sense of situation comedy and a comic tone of voice, using laughter as a means to ease a trying experience. · Outside of interview situations, narrations of childbirth also have a pedagogical function, where the narrator gives her personal view of what may happen during childbirth. Narratives on childbirth can also have an entertaining function, despite the fact that some people consider childbirth narrations unpleasant to listen to, and despite the fact that anyone who has not given birth is excluded from the narrative community. Narration is a way of conveying a message. The message may involve the experience of giving birth as such, with positive or negative comments concerning the day in the maternity ward, or the narration experience as such. According to Lehmann, the third function of a narrative is to gain comfort. By telling the story the narrator aims at achieving an inner balance, at times even to reconcile herself to an unpleasant reality. This is often done by turning a negative event into something positive, as if to adjust something that has happened so as to come to a happy end version of the event by NOTES i Lectio praecursoria (introductory lecture at doctoral disputation) 14 April 2000. ii I would like to thank MEd Petra Orn who introduced Baltus as a pedagogic tool for my children and also helped me get more information about this type of pedagogy. i Lectio praecursoria (introductory lecture at doctoral disputation) 14 April 2000. ii I would like to thank MEd Petra Orn who introduced Baltus as a pedagogic tool for my children and also helped me get more information about this type of pedagogy. Message and identity - the power of narration Let's go back to the question as to why 51 it is so important to be able to narrate; what makes knowledge of the structure of narratives so important that it is included in teaching today. There are two reasons for this. Firstly, narrations are a very effective way to convey a message, more efficient than non-narrations, such as descriptions of different kinds, and lists, including chronologies and tables - texts that lack a dramatic turning point. Secondly, stories are vital in forming our self-identity. (Giddens 1991, cf. Lyotard 1984). The individual becomes "a somebody" through stories and narration, as opposed to being a nobody, "a zero". In her study oflife-stories of criminals, ethnologist Birgitta Svensson has been able to draw the conclusion that her subjects preferred their criminal identity to that of being "a nobody" (Svensson 1997). The spirit of the times insists that we all should be placed in a context. Everyone must continuously strengthen their identity in relation to their environment. Narratives are a way to form this identity, a way of saying who you are. We are subjected to a stream of narratives. Narrations conveying a message, biblical metaphors, stories such as Peter Struwwelpeter, or today's news. In the view ofUlf Palmenfelt, a folklorist, TV news has much in common with folklore. For example, the language used is very "down to earth"; events are linked in time and place, with names given to the persons involved. Events or incidents are narrated in a compressed form and with a very clear turning point, often as a battle between good and evil. Stereotyped roles are given to the 'characters' in the news; a person in power, the debater or the opposition to the person in power, the entertainer and the hero. The newsreader's introductions have their parallels in the introductory "Once upon a time ... " (Palmenfelt 1995, 39ff.). It is no coincidence that TV news is structured in this way - it is an effective way to trigger emotions, be it aversion or sympathy, and to convey the message compellingly. REFERENCES Adelsward, Viveca. 1996. Att forsta en berattelse- eller historien am a/gen. Stockholm: Bromberg. Adelsward, Viveca. 1996. Att forsta en berattelse- eller historien am a/gen. Stockholm: Bromberg. Arvidsson, Alf. 1998: Livet som berattelse. Studier i levnadshistoriska intervjuer. Lund: Studentlitteratur. (Etnologiska skrifter 10.) Studentlitteratur. (Etnologiska skrifter 10.) Berger, Arthur Asa. 1997. Narratives in Popular Culture, Media and Everyday Life. London: Sage.Hagtvet, Bente & Palsd6ttir, Herdis. 1992. Lek med spraket! Oslo: Universitetsforlaget. Why is it so important to tell stories about yourself and your personal experience? According to sociologist Anthony Giddens, autobiographical narratives and the formation of self-identity share a lot of common ground. In today's post-modem world, it is no longer the great social and political narratives that our lives are structured by. Interest in self-narratives can be seen as an expression of self-reflection and modernity, the kind of self-reflection that is a prerequisite for identity forming Universitetsforlaget. Hyden, Lars-Christer. 1997. De otaliga berattelserna. In Lars-Christer Hyden & Margareta Hyden ( eds.): Att studera berattelser. Samhallsvetenskapliga och medicinska perspektiv. Stockholm: Liber. 9-29. Hymes, Dell. 1981. "Jn vain I tried to tell you". Essays in Native American Ethnopoetics. Philadelphia: University of Pennsylvania Press. Giddens, Anthony. 1991. Modernity and Self- Hyden, Lars-Christer. 1997. De otaliga berattelserna. In Lars-Christer Hyden & Margareta Hyden ( eds.): Att studera berattelser. Samhallsvetenskapliga och medicinska perspektiv. Stockholm: Liber. 9-29. Hymes, Dell. 1981. "Jn vain I tried to tell you". Essays in Native American Ethnopoetics. Philadelphia: University of Pennsylvania Press. Giddens, Anthony. 1991. Modernity and Self- Hymes, Dell. 1981. "Jn vain I tried to tell you". Essays in Native American Ethnopoetics. Philadelphia: University of Pennsylvania Press. Giddens, Anthony. 1991. Modernity and Self- Hymes, Dell. 1981. "Jn vain I tried to tell you". Essays in Native American Ethnopoetics. Philadelphia: University of Pennsylvania Press. Giddens, Anthony. 1991. Modernity and Self- 52 identity: Self and Society in the Late Modern Age. Cambridge: Polity. identity: Self and Society in the Late Modern Age. Cambridge: Polity. pa mediekulturen. 39-68. pa mediekulturen. 39-68. Robinson, John. 1981. 'Personal narratives reconsidered'. Journal of American Folklore 94. 58-85. Lehmann, Albrecht. 1978. Erzahlen eigener Erlebnisse im Alltag. Tatbestande, Situationen, Funktionen. Zeitschrift far Volkskunde II. 198- 215. Svensson, Birgitta. 1997. 'The power of biography. Criminal politics, prison life, and the formation of criminal identitites in the Swedish welfare state.' Autoethnography. Rewriting the self and the social. New York/Oxford: Berg. 71-104. Linde, Charlotte. 1993. Life Stories. The Creation of Coherence. REFERENCES New York: Oxford University Press. Lyotard, Jean-Franc;ois. 1994. The Postmodern Condition. A report on knowledge. Virtanen, Leea. 1982. Henkilokohtainen kerronta. In Irma-Riitta Jarvinen & Seppo Knuuttila (eds.): Kertomusperinne. Kirjoituksia proosaperinteen lajeista Ja tutkimuksesta. Helsinki: Suomalaisen Kirjallisuuden Seura (Tietolipas 90). 171-205. Minneapolis: University of Minnesota Press. Marander-Eklund, Lena. 2000. Berattelser am barnafodande. Form, innehdll och betydelse i kvinnors muntliga skildring av fodsel. Abo: Abo Akademis forlag. Palmenfelt, Ulf. 1995. Den folkloriserade verkligheten. Tva svenska kvalltidningar som historieberattare. In Torunn Selberg (ed.): Nostalgi og sensasjoner. F olkloristisk perspektiv 0stern, Anna-Lena. 1994. "Sol, sol! - Jag viii va' solen/ ". Kontextbyggande och perspektivbyten i barns berattande och drama. Vasa: Abo Akademi. 53 53 Introduction twenty years. Nowadays, men are also perceived as objects. Masculinity has become both sexualised and feminised by the media. It is now acceptable for men to be soft, passive and body-conscious in advertisements, although they remain strong, in control and dominating (Ekman 1995,68£). Making the right choices in life is increasingly important in our present time, which is characterised by reflexive thought. Previously, magazines for men have been mainly focussed on one issue, for example, hobbies, pornography and comics. Many men have primarily read newspapers. Today, there is a relatively large offering of men's magazines - magazines that partly resemble the women's magazines that have been on the market for quite some time - and they contain articles about most aspects of life. Men's magazines started to appear on the Finnish market in the mid-1990's, but they have existed much longer in, for example, the USA, where they originated. These new men's magazines are intended for men of various ages and lifestyles. Contrary to the hobby magazines, these men's magazines contain articles on a wide range of subjects, in order that all readers may find something they are interested in. The magazines deal with relationships, music, film, sex, technology, women, fashion and everything men are assumed to be interested in. My aim in this article is to explore today's male roles and ideals by analysing the new men's magazines. I have primarily focussed on two magazines. I have also chosen to interview men in order to gain an understanding of how they perceive the masculine ideals prevalent in today's society as well as what they think of the new magazines. Does the everyday ideal correspond to that presented in the magazines? As my article deals with men's magazines, I find it relevant to explain concepts such as masculinity, gender systems and constructed gender. I agree with the theory that gender is socially constructed, primarily on the basis of the nature of my research problem: I suspect that these men's magazines are attempting to convey a way in which a man can achieve masculinity through the acquisition of certain attributes. Many media researchers presume that gender behaviour is increasingly moulded by our contact with the images, concepts and values communicated by the mass media. In today's society, mass communication represents the major part of all communication. The concept of masculinity has changed over the last Problem specification The main problem of my study is the masculine ideal in the new men's magazines. I explore what kind of masculine ideal the reader is encouraged to aspire to. I ask whether these magazines for men are entering into a "search for the new man". I will therefore analyse men's magazines and interview readers and non- readers of these magazines. Furthermore, I will ask what masculinity represents in today's society and the culture in which we live. What norms should a man follow in his life? What is masculinity? There are two basic principles operating within the gender system. Firstly, the sexes must not be mixed, and secondly, man is the norm. These principles can either both be accepted, or both can be rejected. In addition, one might be accepted while the other is rejected. Various strategies have been used during different stages of the women's struggle. It may be asked, whether men in their struggle to maintain traditional masculinity only use strategies that directly or indirectly aim at preserving male dominance. Could it, however, be in the interest of men to diminish the difference in power between men and women (Cox 2000)? The idea of what is masculine varies considerably between cultures, contexts and historical periods. Within some cultures men are allowed to cry, while this is a taboo in others. Nevertheless, we know that gender is fundamental for our individual existence. This is one of the basic principles of social life, according to M. Kimmel, researcher in men's studies (Ekman 1995, 136). Since I use, amongst others, theories on the construction of gender, I will also ask how a man becomes masculine and what the men's magazines present as being masculine. Are the magazines trying to teach their readers to become men? In addition, I wonder whether masculinity is being redefined. Will the currently prevailing masculine ideal disappear? Do the readers experience a feeling of male fellowship? And further, do the magazines communicate a certain beauty ideal for men? Is there a masculine jargon in the magazines? How do the magazines differ from each other, depending on their target group? Problems and theories I have chosen to divide my introductory sections into firstly a specification of problems and then a presentation of theories. I will start by specifying my area 54 gender can be studied at three different levels; that is, at the individual and the conceptual level, and at the level of social organisation. The gender system is an overall description of these levels in a certain society (Astrom 1990, 16f.). The gender system could thus be said to be the mutual social and cultural organisation of the sexes. The gender system permeates all forms of cultural expression in a fundamental way. The interest in problematising gender has increased within ethnology during the last decade. Within this discipline, gender is usually studied with a focus on how it affects the overall cultural reality. In this article, I will study gender at the individual and the conceptual level. of research, identifying the main questions and the subsidiary questions. After that, I will discuss theories and explain concepts that will be used in the article. I have opted to give the theories and concepts ample space, since men's studies is a relatively new area of research, and my definitions of the concepts are fundamental to the argument. The gender system The concepts of gender and the gender system are frequently used within ethnology. Gender can be defined as the socio-cultural aspects of the division of the human race into two sexes. The concept of Masculinity, manliness In Swedish dictionaries we often find that the word 'masculine' (Swedish: manlig) is defined as the opposite of feminine. Then 55 Slitz, for men who are allowed to kiss beautiful girls. used. These concepts facilitate an understanding of the fact that a man undergoes a process of development. Masculinity is thus something that is formed within the male individual in relation to everything that exists outside him. Primarily, masculinity is to be regarded as a social and cultural construction. The term masculinity simply refers to those stereotypes, ideals and expectations aimed at and associated with men in a given society (Ekman 1995, lOlff.). Our society is imbued with an enormous quantity of invisible rules. Usually the point of departure referred to in discussions on masculinity is the cultural patterns for men's expected or assumed behaviour. Masculinity is seen as both a mental and a social structure, depending on the prevailing patriarchal circumstances. There is a stereotypical image of man in today's society. We must, however, keep in mind that masculinities irreconcilably differ from each other, depending on the position of the men in the contractual power hierarchy. Besides, the masculinity of an individual man is based on his personal situation in life and his unique experiences (Haddad 1993, 313). During the late 1970's, men considered masculinity to stand accused for several crimes against women, children and other men, primarily homosexuals. Since the 1980's, men's groups have attempted to counter these crimes by, for example, starting men's movements (Haddad 1993, 37ff.). Men have become aware that masculinity is a problematic concept not only for men themselves, but also for women, homosexuals and other men who do not conform to the standard dominant masculine ideal. Men have therefore realised that masculinity must be reconstructed. Masculinity would then instead form a valuable source for men (Miehyyden tiella 1993, 13ff.). follows a list of characteristics such as brave, noble, strong, courageous, virile and fearless (Svenska Akademien and Sprakdata 2001). In a dictionary published by the Swedish Academy, masculinity stands for bravery, courage and capability (Ekman 1995, 40). Society is still, to a great extent, a male society. Man and his interpretations subconsciously saturate all cultural values. Previously, it has been almost impossible to pose questions about men, and it has been too complex to describe masculinity. Partly this is, of course, due to the fact that men form a heterogeneous group, partly that men have been invisible. Man is invisible because he is the norm. Man has been taken for granted (Ekman 1995, 133f.). Conceptually, both masculinity and the male role are perceived as static. This does not reflect the fact that they are processes, and that masculinity is constructed. Such concepts do not take into account that men are different from each other. Instead of masculinity, then, such concepts as masculinities and male identity may be Sports comprise several stereotypical 56 (Haddad 1993, 37ff.). Researchers have claimed that if men are aware that their masculinity is a social construct, they may then choose to reconstruct their masculinity according to a more ethical line. Men would understand that neither they nor any other person has the right to use violence and power (Maclnnes 1998, 83). According to Freud, human sexuality is not a biological product, but primarily a cultural and symbolic product. Thus, sexuality is a product constructed by society, and therefore something learnt (Ekenstam 1998, 12). In addition, there is the view of essentialist constructivism. According to this school, a child is formed during his or her early years (Bengtsson & Frykman 1987, 9). In this article, I will primarily use the theory that gender is socially constructed. elements of masculinity: comradeship, competition, hierarchy, legitimate emotions, control over injuries, limitations, codes, anxiety and distinction (Badinter 1994, 106ff.). Team sports are often more important for boys than for girls. Pre-school girls and boys are prone to play with children of their own sex. The tendency towards grouping according to sex is strengthened from the age of 6-7 and up to puberty, when subcultures with both male and female emphasis start to arise. Sports involving competition, aggression and violence are regarded as the best way to foster masculinity, particularly in the USA, but also to varying degrees in other cultures (Ekman 1995, 44). Constructed gender and essen- tialism What are the reasons for man being what he is? The most common view is that a combination of biological and envi- ronmental factors influence the way in which a boy is formed into a man (Ekenstam 1998, 11 ). Generally speaking, there are two ways of regarding the institution of gender. One talks about constructed gender, while the other talks about essentialism. Essentialists seek a genuine and authentic masculinity. The interest in essentialism increased in the early 1990's. According to this view, the human psyche is not formed only according to the individual's own biography, but primarily by the collective unconscious and archetypes embedded in the human spiritual and genetic heritage (Ekenstam 1998, 12). What are the reasons for man being what he is? The most common view is that a combination of biological and envi- ronmental factors influence the way in which a boy is formed into a man (Ekenstam 1998, 11 ). Generally speaking, there are two ways of regarding the institution of gender. One talks about constructed gender, while the other talks about essentialism. Essentialists seek a genuine and authentic masculinity. The interest in essentialism increased in the early 1990's. According to this view, the human psyche is not formed only according to the individual's own biography, but primarily by the collective unconscious and archetypes embedded in the human spiritual and genetic heritage (Ekenstam 1998, 12). Constructivist researchers claim that the focus should be on exploring the social construction of various types of masculinity. The construction is influenced by several dimensions (Haddad 1993, 43f.). According to pure constructivism, masculinities are only constructed ideas of what men should be like. Masculinities do not reflect what men are actually like The male role and male ideals Research on gender roles was a growing field within cultural and social studies at the end of the 20th century (Bengtsson & Frykman 1987, 14). The most fruitful approach seems to be to explore how male identity is formed by cultural and social factors. However, this presupposes that the definitions of roles within a culture are relatively uniform. And, as we know, male roles have varied according to class and age, power positions and in relation to female roles (Mannen iforandring 1983, 34ff.). In order to really understand the male role, we must therefore look at how the role or the various roles have been formed. We can ask what the prevailing norms are for the behaviour of boys. How are the norms developed and conveyed? Expectations and influences from his environment are, of course, crucial for a boy's development into a man. Boys experience demands to be men very early in life. The demands not to be like girls trigger dependence in boys, primarily in relation to their friends. It seems to be part of the upbringing of boys Constructivist researchers claim that the focus should be on exploring the social construction of various types of masculinity. The construction is influenced by several dimensions (Haddad 1993, 43f.). According to pure constructivism, masculinities are only constructed ideas of what men should be like. Masculinities do not reflect what men are actually like 57 to learn to be suspicious and guard oneself. There are also strong taboos. Men do not dare appear unguarded in the way women do. Men often have the attitude that everything might be used against them. Therefore they cannot talk about feelings with other men. A pattern in relations between teenagers is to appear "the least interested". Researchers claim that in this way youngsters engage in games with each other. The point is to gain power in the relation by caring the least. Revealing emotions also means revealing that one cares about the relation and thus losing power. It means appearing to be weak in front of others (Bengtsson & Frykman 1987, 18). Nordic culture today is still that boys should be manly. It is true that a greater tolerance of femininity has slowly grown, but the male norm does still not allow men to carry out certain more feminine jobs, such as childminding and cleaning. The attitude towards masculine characteristics can vary depending on age. The male role and male ideals According to, for example, men's studies researcher Daniel Ekman, important masculine characteristics at a young age, around 24 years, are the ability to function in a family, responsibility and foresight (Badinter 1994, 152). I also asked my informants what kind of male ideal they have perceived, and I will present their answers further on in this article. I think the attitude to masculine characteristics to a large extent depends on a person's situation in life. For example, 24-year-olds exist in a wide variety of situations and have different lifestyles. The male liberation during the 1970's tended to describe the male role as defective. Deep down, men, like women, were good at heart but culture and society had bound them in the straitjacket of the role. It was assumed that the traditional male role made men do nasty things. Men became aware of this, primarily through the women's movement, and those showing most insight wanted to come to terms with the role. The most sensible thing to do was to let men take care of female chores at home. Some researchers think that if today's men had grown up with their fathers taking care of them at home, they would be better equipped emotionally. Thus, the new man lives in a clear contrast to an old reprehensible masculine ideal. The physical, challenging macho man, who had not realised the value of softness, differentiation and the will to talk about his problems, was seen as the stereotypical image ofworking- class men (Ortman 1970, 14). Apart from the characteristics mentioned above, the general view is that men should experience strength, control, potency and success according to the norm for the ideal male role. By contributing to the upholding of this unattainable image of masculinity, one heightens the painful feeling of inadequacy among today's men (Badinter 1994, 208). At the moment, it seems as if young men can feel comfortable neither in the soft nor the hard masculinity. They are first generation heirs of a transformed masculinity. Young men appear to find it difficult to identify with their fathers, since young men today are sons of masculine mothers and feminine fathers (Mannen i forandring 1983, 24ff.). At the same time, society is dominated by men and the opinions, values and perceptions of men are prevalent. The present assignment of roles is thus problematic for both men and women. The male role and male ideals Despite the cultural change that is taking place, and which has been taking place over the last century, it is still a common assumption that boys should be active, tough and sporty. An effeminate boy is not as appreciated as a masculine boy (Ekman 1995, 123). In my opinion, the view within However, all men do not hold power in society. The powerless are situated in a hierarchical structure which is the actual 58 condition for the power and dominance of men in the crucial higher positions in society. Most men have been brought up not to break free from these hierarchical structures (Cox 2000). Besides, men today manifest their masculinity primarily through success in working life. Today's economy is global. Companies invest in specialised production and move factories between countries (Mannen i forandring 1983, 24 ff.). This can result in it being both more difficult and more attractive to manifest one's masculinity. Masculinity is something that men can lean back on even if they have to move from country to country in order to find work. In this way, they strengthen their position in society and the hierarchical structure. immensely from the everyday life of most men, there is always a connection between fiction and reality. Masculinity has become a popular subject during the last few years in all industrialized countries. Some of those who have worked with the subject for a long time have shown some dismay concerning what catches the media's interest in men. According to many men's studies researchers, the most popular books on men are full of vague thoughts (Maclnnes 1998, lf.). But why is writing on masculinity so popular among men? One theory is that modernity has systematically weakened the patriarchal society. Men have seen a great deal of diminishing of their power over women. Gender is, in a way, an ideology that is used by people in modem societies in order to perceive differences between men and women based on their sex. The perceptions help individuals understand that they live in a society which is unequal even ifit is formally equal (Ortman 1970, 130f). A modem man must split his personality as he moves from one place, group or activity to another. His various sub-selves need have no connection with each other. The labour market is based on principles of profitability and efficiency. The increasingly bureaucratic system demands impersonality and emotional control. Death of the old male image Lately great technical changes have happened very quickly. More information reaches us at a rising speed. Mass media increases our knowledge and we get an insight into a growing number of incidents. The world is shrinking. All this has impelled us to change our attitudes. For example, we realise that conventional and traditional behaviour and prevalent ways of thinking are not always the best. Since many old values have lost their significance and been replaced by new, many people have become insecure and confused. Some strive for freedom to begin with, while others want to live even more freely than they already do. We become unsure of our gender roles. For example, a small boy still encounters the old masculine image in his childhood. He is thus unprepared for his future family role. Demands on masculinity arise everywhere (Salonen 1995, 187ff.). In The male role and male ideals The male role thus requires that men possess self-control. This is a prerequisite for being able to act rationally, compete and achieve results. This pertains to all men, but takes on various expressions depending on social position. The privileged man is focussed on self-realization and success. Most other men are focussed on fulfilling their duties, for example as breadwinners for their families (Conell 1999, 9). Superman and the four impera- tives The definition of the male role formulated by two American university teachers is, to a large extent, prevalent in Western culture. Robert Brannon and Deborah S. Davies claim that a man who obeys four imperatives is superman. An example of this superman whom the masses have dreamt of for a long time is the so-called Marlboro Man. Most cultures have adopted this masculine ideal and created their own versions of it. The four imperatives are the following: "no sissy stuff', "the big wheel", "the sturdy oalc' and "give 'em hell". By "no sissy stuff' Brannon and Davies refer to the expectation that men should not do anything feminine or effeminate. Even if it is known today that men have the same emotional needs as women, the stereotypical male role forces men to sacrifice a part of their humanity (Cox 2000). Women are aware of the significance of their gender identity, while men continue to live according to the first commandment of masculinity: "no sissy stuff'. The image of man: men in the popular press During the 1930's the strong man was the ideal. However, much has happened since then, from furriness and reflectiveness, to the 1980' s pinstripe suit fashion and aggressive stubble. The man of the 1990's is portrayed in advertisements as at least as vain as his female counterparts. Even if the image of men in the media differs 59 addition, men live in two worlds. One world is inherited from their fathers, and is a world which no longer functions coherently. The other world, on the other hand, advocates a masculinity which is not yet developed enough to function. The new man should defend, but not attack. Man is vulnerable and needs care, but he should also protect others. He should be both strong and weak. Men should be perfect, but no individual can ever be perfect (Ekenstam 1998, 242f.). Cultural liberation, self-reflection and individualization are signs of modernity. This does not, however, pertain to all in the same way and to the same extent. Social and cultural gender are factors influencing this (Badinter 1994, 149). Thus today's male image can not totally die, only gradually change. learn to feel safe, secure and self-confident in their masculinity - and not follow the stereotypical male ideal. Those who are not securely rooted in their masculine identity continuously feel their masculinity being challenged and are forced to always prove to themselves and others that they are real men and no sissies (Badinter 1994, 129f.). "The big wheel" means that man is a very important person. He must be better than others. The scale for masculinity is thus success, power and corresponding admiration. "The sturdy oak" refers to the fact that men should be independent and only trust themselves. "Give' em hell" is an expression for men having to be stronger than everybody else - even by using violence if necessary (Cox 2000). These masculinity rules are passed on from generation to generation. There is one man who, still today, is a symbol for these ideals. He has been the most admired masculine hero in the USA for many decades: John Wayne. Clint Eastwood could also be called a superman of our times. Presentation of the informants and the source material As has been mentioned above, my informants are men between 23 and 25 years ofage. All of them are students. Two read men's magazines, while two do not read them. One of the readers has been exchange student in the USA and the other reader has done a work placement in the USA. This is interesting, since men's magazines originated in the USA. Both non-readers have only spent shorter periods abroad as tourists. One of the infonnants studies at Sydvast Polytechnic, while all others study at Abo Akademi University. I discern no differences between the hobbies of the readers and non-readers; these include music, TV, books, shares, socialising with friends and weight training. Geographically the readers come from Ostrobothnia and the non-readers from Nyland. All informants currently live in Abo. I don 't know, it's the same as for girls and women. It is like the same kind of magazine but for the other sex, more like that. It's not like, it's not so to say any kind of porno magazine in that sense ... there is a clear limit there. The magazine Slitz is intended for younger men, while MG' s target group is the mature man. This is obvious from the headlines, covers and contents of the magazines. In addition to the two men's magazines that I have analysed, I have also read a few issues of other men's magazines. I would have liked to analyse these, too, but I chose to only get a quick overview of other men's magazines, primarily in order to confirm that the two magazines I had chosen were also representative of the rest of what is available. Most men's magazines do have their individual profiles which differ somewhat from each other, depending on what group of men and also what age group they are intended for. Since all my informants are about 24 years old, I have assumed that men of this age might be in slightly different life situations; some might study while others are in working life, some My first object of analysis is the Swedish magazine Slitz. A Finnish-language edition of Slitz is published in Finland, which primarily contains translated articles from the Swedish magazine. I have chosen to only analyse the Swedish Slitz, since there is virtually no difference between the Swedish and the Finnish editions. Methods, source material and choice of method I will present my material and methods in two phases. First, I will explain my choice of working method. After that follows a presentation of both informants and the men's magazines I have analysed. I have chosen to focus my analysis on two men's magazines, Slitz and MG. Both of these are relatively new on the market. I have read all the issues published in 2000 in order to get a clear view of the contents of the magazines and their attitude towards man. I will primarily analyse recurring subjects of the magazines' articles. In addition, I have interviewed four 23- 25 year-old men, who are all students. The very limited age group of my informants needs to be explained. I have chosen this age group because the magazines I have analysed are primarily intended for this Similarly, today's young men should 60 group. Two of these men read men's magazines, while the two other are non- readers. I have interviewed non-readers in order to find out what their opinion of the magazines is, and whether their male image differs from that of the readers. I used slightly different sets of questions for interviewing the two groups. I also quickly realised that it is easiest to find men who do not read men's magazines or at least do not admit to doing so. It seems that reading magazines is something shameful. I assume this might be explained by the countless women's magazines that have existed for a long time. Men are possibly afraid of being labelled feminine. I get the impression that reading pornographic magazines is better tolerated among men than reading men's magazines, probably because this is regarded as masculine. It was, however, interesting that one informant who reads men's magazines said that: might have a family of their own while others are single. I think the lifestyle and life situation of the men influence their choice of magazine. Presentation of the informants and the source material Besides, all my informants are Swedish-speaking, which makes me assume that they also read the Swedish edition of the magazine. All the editors at Slitz are men. At the top of the cover, there is always a sentence starting with the words: "For men who ... ", for 61 on the other hand, presents, for example, new, expensive telephones, cameras and dressing-cases. Style icons are older successful men, who present some of their more valuable possessions. Bistro deals with drinks and gastro with food. "Your place or mine" consists of pictures of a scantily dressed young woman. In addition to these recurring subjects, the magazine contains columns, articles on famous men, fashion, cars, extreme sports, and similar subjects. Every issue also has a column on the subject of men. This is always written by a well-known woman. The pictures in the magazine often show men of the ages of25-35 years. The message of MG seems to be that even at a young age men can become "dynamic, successful salesmen, that is, heroes of our times" (MG 12/2000). The cover usually features a middle-aged well-known Finnish politician, businessman or sportsman. Sometimes the cover model is a Finnish musician, actor or a famous woman. example, "For men who get to kiss beautiful girls." or "For men who only live twice." The target group of the magazine are men aged 18 to 3 5. The magazine contains articles on sports, film, computers, politics, music, women, gadgets, clothes, sex, living together, food, training, drinks and entertainment. Apart from the above themes Stitz also contains articles on everyday life, usually something associated with the military. In addition, the magazine has a column for readers' questions, where a woman answers men's questions, and interviews with well- known men, as well as a section called "Manual", that is, "the ultimate style guide for men". "Manual" uses sub-headings such as "Style news", "Pure style", "Object", "Expert", "Training", "Motor", "Technics" and "Materia". As these headings suggest, "Manual" contains tips for men on clothes, fashion items and gadgets. In "Pure style" men are taught manners and the art of being a gentleman. In "Expert" experts on religion & spirituality, food & training, skin & body care, sex & and co-habiting and economy answer readers' questions on virtually anything. "Training" consists of, for example, tips on how to train a certain body part, and sometimes also introduces new training equipment. Analysis of the men's magazines y g I have divided this chapter into sections on images of masculinity, male fellowship, male roles and male ideals in the magazines, and male jargon. Under all these headings I take into account both my informants' answers and the contents of the magazines, as well as theories of masculinity. I have chosen here not to refer to my theoretical sources, primarily as I have presented them in detail above. Neither do I use references to particular issues or articles when I discuss the men's magazines. My analysis of the magazines is of a more general character, since I have used so many issues of the two magazines. MG is a Finnish magazine. Most of the editors are men. The target group is somewhat older than that of Slitz, that is 25-45 years. The magazine always contains a section called "Men's world" with articles on film, media, music, books, events, gadgets, style icons, bistro, gastro, health, "your place or mine", and "what you must get yourself'. The book reviews usually present so-called male books. By this I mean that the books are mostly written by men or deal with fatherhood and related matters. The gadgets are small things, funny presents and the like. "What you must get", Presentation of the informants and the source material The cover of Slitz always features a scantily dressed, famous woman. Images of masculinity The pictures in the new men's magazines do not seem to take into account that men can have different experiences and a private life situation. Rather, the men's magazines keep to stereotypical images of men. The stereotypical man must own certain items in order to be a man according to the magazines. Masculinity is constructed through clothes, cars, telephones and the like. Slitz says that the reader must to tum to its "Manual" if he wants to dress like a man. Thus Slitz claims that clothes make a man. Both MG and Slitz contain a lot on sports. The area of sport, including competition, aggression and violence are, according to W estem culture, the best way to achieve masculinity (Badinter 1994, 106ff). This attitude appears clearly when reading and analysing the magazines. Men's magazines were originally created in the USA, where many masculine sports are given priority, such as American football and ice-hockey. The Nordic men's magazines that I have analyzed also primarily present traditionally male sports. Jorma Klilirllilnen lasVegasln kautta lavoille hardly existed before. So in that way the image has changed. I also asked the informants what they think masculinity is. I did not get any explicit answers; instead, most informants said that they could not define the concept. I was somewhat surprised by this, while I also realised that a change in the image of man is actually happening. I was expecting answers that would have emphasized strength and control. One of the informants said that being polite to women is masculine. Such an answer suggests a traditional image of man. One of the informants who read men's magazine answered as follows to my question on whether the norms for a man are changing: Well, I don't know if there is anything particular that I think is masculine in that sense. Of course, society has certain ideals for what a man should be like. The man should provide for his family, the man should work and the woman stay at home, but I don 't know if this is so intrinsic any more. It does still exist to some extent in society. But that too is changing, as paternity leave is introduced, for example, Yes, they are changing. As I said earlier, paternity leave has come about and such things. Images of masculinity In advertisements, the man of the 1990' s is depicted as at least as vain as his female counterpart. Many mass media researchers assume that gender behaviour is 62 Harkhnon Y SISRPIIRI Kowta p;;/16 Mo11tU'ollssa SAKU KOIVU Rfstlriltafn«n RAIMO SUMMANEN KAKSI PITl<M JA KAUNISTA Morita Hokolo Jo Son/" Cotant TIGER WOODS mosentoa muut golfml/Jon66tlt . , Jorma Klilirllilnen lasVegasln kautta lavoille hardly existed before. So in that way the image has changed. MG, for traditional and luxury men. increasingly formed by our exposure to the images, concepts and values communicated by the mass media. Men in advertisements are nowadays allowed to be soft, passive and body-conscious (Ekman 1995, 93f.). This is also obvious in the pictures in advertisements and articles in Slitz and MG. The masculinity of a specific man is also based on his personal life situation and unique experiences (Ekman 1995, 102). The pictures in the new men's magazines do not seem to take into account that men can have different experiences and a private life situation. Rather, the men's magazines keep to stereotypical images of men. The stereotypical man must own certain items in order to be a man according to the magazines. Masculinity is constructed through clothes, cars, telephones and the like. Slitz says that the reader must to tum to its "Manual" if he wants to dress like a man. Thus Slitz claims that clothes make a man. Both MG and Slitz contain a lot on sports. The area of sport, including competition, aggression and violence are, according to W estem culture, the best way to achieve masculinity (Badinter 1994, 106ff). This attitude appears clearly when reading and analysing the magazines. Men's magazines were originally created in the USA, where many masculine sports are given priority, such as American football and ice-hockey. The Nordic men's magazines that I have analyzed also primarily present traditionally male sports. O f h i f h d ' increasingly formed by our exposure to the images, concepts and values communicated by the mass media. Men in advertisements are nowadays allowed to be soft, passive and body-conscious (Ekman 1995, 93f.). This is also obvious in the pictures in advertisements and articles in Slitz and MG. The masculinity of a specific man is also based on his personal life situation and unique experiences (Ekman 1995, 102). so then the man can stay at home with the kids and the woman can go to work. (Reader, b. 1976) so then the man can stay at home with the kids and the woman can go to work. (Reader, b. 1976) least one can be just as involved in these things, I don 't know if it must necessarily be a group of the same sex. (Non-reader, b. 1975) I also asked the informants how a man becomes masculine. According to what I have read and seen in the men's magazines, men become masculine through clothes and other attributes. However, none of the informants mentioned this. Most of them said that men become men while doing their military service. They explained that men mature there. One informant said that there is nothing special one can do to become masculine: This can also suggest that the statement that men seldom have a close male friend is true. Exposing his soul to another man would reveal a man's underlying insecurity and thus all men would be potential enemies and rivals (Ekman 1995, 181). But the answer can also be interpreted in a way which implies that men are not constant rivals. It is also possible that the informant answered in this way because he misunderstood the question, or did not want to hurt me as a woman by directly saying that he always has a better time in male company. I also asked my informants who read men's magazines whether they felt a fellowship when reading the magazines. One informant said he felt no fellowship in that situation, while the other answered: If he is a normal man, then I suppose he is masculine. There is nothing special one can do, one should not be ... gay at least. (Non-reader, b. 1975). Images of masculinity But men on the whole have perhaps become more fixated on their bodies and therefore there are many men who undergo plastic surgery. And that has 63 Male fellowship g Slitz conveys an image of boyishness. In this way the magazine appeals to a certain type of man, or it might suggest that Slitz is a haven for all men, a free zone where women have no access. In my opinion, Slitz can be seen as a reaction to changed gender roles at a time when it is uncertain what a man actually is. Many probably read Slitz for entertainment and do not take its contents that seriously. However, I think that men are indirectly influenced by the image of both men and women communicated by the magazine. I assume that those who read Slitz as a Bible and take the magazine seriously form a marginal group. Both informants who read men's magazines said that they do so mainly as a pastime. One informant thought that mostly socially active men read men's magazines, that is, men who either study or work, since the magazines are relatively expensive. One informant who does not himself read men's magazines thought that it is primarily men who are unsure of themselves who read these magazines. In the magazine MG women are also presented as sexual beings, but not exclusively so. Women also appear in articles on, for example, motherhood. So, the male fellowship in contrast to women is not as strong in MG as it is in Slitz. Women are also given an equal position as creative subjects. In MG, I nevertheless discern a male fellowship or a male territory when it comes to the book reviews. All books presented in the magazine, except one in issue 12/2000, are written by and about men. The one book written by and about a woman is a detective story. Three of the other 11 books deal with male politicians, one presents the best sportsmen of the century, one is about the new financial gurus (all men), one is about a Finnish rock group consisting of four men, and the remaining four are fiction with a male main character. In fact, MG' s recurring section "Men's world" is a totally male territory. g Stitz gives the reader a relatively stereotypical image of man and also a stereotypical image of the reader. According to this magazine, men should be interested in sport, alcohol, women, technology and they should think it important to dress in the right way. Male fellowship I think that, I think one needs it at the same time, for example this socialising, nice socialising with other men. Let's say because well, I think one should, that one has the right to it. There's nothing wrong with it. So one does really feel that one is part of a fellowship. (Reader, b. 1977) Well perhaps sometimes. It depends on what one is reading. Well yes, for example these different stories that people tell in there about something or another, then maybe/feel afellowship. Yes really, that's what it's like, sort of There are general perceptions of what men are like together with women and children. A world less known and less explored is men's relations to other men, that is, male fellowship. It is, however, possible to describe what one sees in the team of builders, military service platoon, football team, or in a gang of friends (Ekman 1995, 175). All informants mentioned one of these groups as teams within which they had experienced male fellowship. One informant gave the following answer to the question on where he experiences male fellowship: The magazine Slitz conveys the notion that sports are a male territory, that is, a place for male fellowship. Sports are holy and this is a male area where women are usually not invited. Women are not portrayed as active within sports, but as fans, or they are there for decoration. In Slitz, women and sex are virtually always mentioned in the same article. When women are presented, they are usually portrayed in the role of sexual beings. I therefore asked myself whether the fact that women are portrayed as sexual beings creates fellowship among the men. However, there is one exception: the questions column where men ask a woman Perhaps sometimes at a sauna evening, or actually even if one is with a group of women or girls it can be just as fun, or at 64 of man read the magazines. about relationships, sex, body matters and so on. In this column, on the other hand, the woman is presented as an oracle since she is assumed to have the answer to everything. Nevertheless, one can ask whether this actually is an exception, because on the same page as the questions column there is a picture of a very attractive and scantily dressed woman, who possibly is supposed to depict the oracle. Male fellowship These aspects of masculinity recur throughout the magazine and Slitz thus becomes a producer of a stereotypical image of men. The texts are often instructive and provide the reader with tips and pronouncements on what is right and what is wrong. A man must learn to behave like a gentleman, to satisfy his woman and cook delicious food. Delicious food according to Slitz consists of very exclusive and culinary dishes. A man should not stand by the stove every day, but only cook about once a month. Slitz's idea of men and cooking is comparable to the 1950's notion of man and the barbecue. Male roles and male ideals in the magazines A modem man must split his personality in order to move from one place, group or activity to another. I asked myself what male roles are presented in the two men's magazines that I have analysed. I primarily used pictures, articles and the list of contents of the magazines in order to find male images or male roles. I further asked the informants what they thought about the male roles communicated in the magazines and also if they thought that a certain type 65 well. You 're supposed to be a real superman. (Non-reader, b. 1975) In addition, men should buy the right clothes and fashion items. Men are increasingly interested in clothes and looks, which is also noted by Stitz. Just like women, men also have to change and adapt their appearance in order to be a real man, and Slitz provides the ways and attributes needed to do this. It may be argued whether men have become interested in clothes and looks because of these new men's magazines and other ideal images set up of men, or because of other phenomena in our culture. The informant here describes the superman. The four imperatives no sissy stuff, the big wheel, the sturdy oak and give' em hell to a large extent fit the informant's description of today's male ideal. Admitting weakness, frailty or infirmity makes one seem like a sissy and unmanly. This is obvious also in Slitz. Men should not, for example, do feminine sports. The only exception is if the men are at the top. By feminine sports I mean ballet and other non-aggressive physical activities. Even if men are usually not allowed to do feminine sports, they may make good and finer food, and are encouraged to do this by the magazines. The informants, too, mention the feminine household chores as something that men should help with at home, which is something the traditional man would not do. It can thus be seen that the norms for a man are perhaps changing. The second basic rule or imperative, the big wheel, is also reflected in Slitz and MG. In the former, the rule appears in the section "Power & Freedom". Here, powerful men get to show what characteristics are desirable and thus function as examples for the readers. It could be said that powerful men as role models characterises the entire contents of MG. Male roles and male ideals in the magazines The interest of many men today in their appearance and their spending quite a lot of time in trying to change their bodies in a desirable direction do not necessarily reflect actual changes in gender identities. I would rather talk about certain shifts in what is regarded as feminine and as masculine. The narcissistic man is not necessarily a new man, but perhaps rather a traditional man in a new packaging. This argument has clear relevance with regard to Slitz and MG. The fact that a man is interested in the contents of, for example, the clothes section, does not need to bear any deeper significance. When the whole society is body fixated, it is no wonder that men, too, show an interest in these areas. Even if outer factors might change, the internal can remain more or less the same. One informant said: People who hold a high and secure position in their group or class can easily take on a certain nonchalant attitude towards the cultural rules. Those who know best and are clearly aware of the rules can afford to be a bit careless and bohemian (Ehn and Lofgren 1986, 94 ). Stitz is dripping with irony, which, apart from its function to amuse, can also serve as a shield against criticism. Stitz is always right and can make the critics feel inferior by pointing out that what they attacked was ironic and intended to be a joke. On the other hand, male language stands for rationality, order and objectivity (Dyer 1989, 68). This statement is largely true of the magazine MG and its language. Both pictures and texts radiate rationality, order and objectivity. The jargon is very matter- of-fact, as is the rest of the magazine. It might be asked whether MG is more mature and more masculine than Stitz, or more mature and less masculine. However, I would rather define the magazines as conveying different kinds of masculinities, and therefore their languages also differ from each other. Both magazines try to teach their readers to become men, but different kinds of men. It depends more on personality and behaviour and not necessarily on what he looks like in the magazine. There is a beauty ideal in the men's magazine, but it is possible that men do not perceive this ideal or follow it. Male roles and male ideals in the magazines The magazine MG is primarily characterised by a male image which can be seen as traditional. Men should be successful, the head of the family and wealthy. When comparing Slitz and MG, I notice that MG seems to take the male role more seriously. The articles exclusively deal with successful and career-oriented men. Slitz can be said to speak for the younger macho man, while MG advocates the older businessman. MG 12/2000 contains a fashion article which includes the following statement: Freedom and individuality are in, but there are limits. Your work place defines your dress code - intentionally or unintentionally. The MG man has discretion and style, and he follows strict rules on what a real MG man should be like. MG usually also contains an article presenting a man doing an extreme sport, but in daily life this man is a businessman, director or the like. The non-readers also have an idea of this male model: Body fixation has increased since the beginning of the 1980's and I think that the body has gained a more central role. This can be seen not least on the cover of Stitz where the producers use half-naked beautiful bodies in their attempts to sell the magazine in true commercial spirit. The media has gained increasing power in setting the norms for what is desirable and beautiful. The pin-up girls of both Slitz and MG have very similar forms, which is an example of the media's normative tendency. Also the men featured in the You 're supposed to have one of those muscular bodies and do many different sports and work, have one of those 'suit jobs'. And then you 're supposed to help with the household chores and then you 're supposed to do all sorts of other things as 66 uses unusual words, such as 'esoteric', 'infamous', 'gamine' and 'magisterial', mixed with slang and swearwords. English expressions are 'Swedofied', translated or used as they are. The whole magazine is characterised by ironic, sexual and easy- going language. This is most noticeable in headlines, introductions and captions, where the angle of the media is most apparent. pictures in the magazines have perfect bodies, stylish hair and fashionable beards. The fact that beauty is not something objective is forgotten, and the more pictures of "perfect" bodies we see, the more we perhaps think that they really are beautiful. Male roles and male ideals in the magazines Perhaps beauty is not everything that matters, after all, at least according to my informants. Unprinted sources Haddad, Tony, ed. Men and Masculinities. A Critical Anthology. Canada, 1993. Macinnes, John. The End of Masculinity. Great Britain, 1998. Mannen i forandring. Ideprogram frdn Arbetsgruppen om mansrollen. Stockholm: Tiden, 1983. Male jargon The form of a message is very important as to what kind of concept of reality it conveys and how the readers receive the message. Actually, the focus should not only be on what is said, but also on how it is said. Stitz has created its own jargon which is used in all sections of the magazine with only a few exceptions. The jargon used by the editors is so clear and strong that it even dominates the style of the letters from readers. Slitz The language also separates the two magazines in another way. As I said above, Stitz in a way holds a high position in its group and can therefore be nonchalant. This is true also of MG, but in a contrary fashion. MG also holds a secure position among its readers and thus they can afford not to appear nonchalant. By this I mean that MG 67 by its objective, matter-of-fact and rational language signals that the magazine is so esteemed among its readers that they can let the reader join it at the same level. After all, the readers admire the magazine. By its language the magazine even seems to want the reader to be on the same level. This of course is due to the fact that MG is intended for successful men, or men who are on their way towards success. 1990. Badinter, Elisabeth. XY om mannens identitet. Juva, 1994. Badinter, Elisabeth. XY om mannens identitet. Juva, 1994. Bengtsson, Margot, and Jonas Frykman. Om maskuliniteter. Stockholm, 1987. Conell, R.W. Maskuliniteter. Uddevalla, 1999. Dyer, R., et al. What's that got to do with my gender? Abo, 1989. Ehn, B., and 0. Lofgren. Kulturanalys. Lund, 1986. Ekenstam, C., et al. Radd att falla. Studier i manlighet. Sodertalje, 1998. SOURCES AND LITERATURE SOURCES AND LITERATURE Unprinted sources Personal interview. 6 December 2000. Personal interview. 14 December 2000. Personal interview. 15 December 2000. Personal interview. 15 December 2000. Printed sources MG. All issues from 2000 (12 issues in all) Slitz (Swedish edition). All issues from 2000 (11 issues in all) Literature Astrom, Lissie. Fader och saner. Stockholm, Ekman, Daniel. En mans bok. Falun, 1995. Printed sources Miehyyden tiella. Publication 39. Jyvaskyla: Jyvaskylan yliopisto. Nykykulttuurin tutkimusyksikko, 1993. Miehyyden tiella. Publication 39. Jyvaskyla: Jyvaskylan yliopisto. Nykykulttuurin tutkimusyksikko, 1993. Ortman, Sinikka. Mannen i manssamhallet. Stockholm, 1970. Salonen, Ilpo. Ihan hyva mies. Helsinki, 1995. 68 68 Formula 1 and its great popular- ity in Finland how the ordinary TV audience experiences and describes it. Today only men drive Fl cars, and therefore I think that Formula 1 might also reveal something about the current concept of masculinity. Formula 1 has attracted a great deal of interest in Finland among an increasing number of people. This is primarily explained by the success of the Finnish driver Mika Hakkinen. 23 Finland is also represented by another Formula 1 driver, Mika Salo, and a third, Kimi Raikkonen, has recently arrived on the scene. Formula 1 is a large, international car racing sport, but the overall number of drivers is nonetheless relatively small. Therefore it is quite remarkable that there are three Finnish men among them. Interviews with students The material of this study consists of interviews with nine Swedish-speaking students in Abo. The interviewees were born between 1972 and 1979. Three of them are studying at Polytechnic Sydvast and six at Abo Akademi University. The fact that all my informants are students and of about the same age naturally typifies my material. This article should therefore be regarded as a study specifically of students' opinions of Formula 1. When I started a course in English, the teacher explained that we were all to give a presentation on a chosen subject, but he did not want to hear another one on F 1 and Mika Hakkinen! This shows that there is not only an interest in watching Fl on TV, but also in exploring the subject and talking about and explaining facts about the sport. I also seem to discern a current debate on the meaningfulness versus the meaninglessness of Formula 1. I myself have found Fl fairly meaningless. But why is all this discussed? What values does the debate express? What opinions are there of Formula 1 and what do they tell us about today's society? My aim is to analyse Formula 1 as a modern/late- modern phenomenon; partly as a phenomenon as such and partly in terms of of Formula 1. My friends suggested suitable interviewees to me, whom I then contacted. I also personally know some of the interviewees quite well. I chose these particular persons, since I knew they had a lot to say about Formula 1. As F 1 has gained such popularity in Finland, and as watching F 1 is very widespread in the context I have chosen, this subject can be said to pertain to most persons in one way or another: everybody has an opinion on Formula 1. Therefore, I wanted to interview both persons who are interested in F 1 and persons who are not. However, I permitted more space to those who are interested in My friends suggested suitable interviewees to me, whom I then contacted. I also personally know some of the interviewees quite well. I chose these particular persons, since I knew they had a lot to say about Formula 1. Interviews with students As F 1 has gained such popularity in Finland, and as watching F 1 is very widespread in the context I have chosen, this subject can be said to pertain to most persons in one way or another: everybody has an opinion on Formula 1. Therefore, I wanted to interview both persons who are interested in F 1 and persons who are not. However, I permitted more space to those who are interested in 69 Playing computer games gives you a feeling what it is like to drive Formula 1. Photo: Ann-Helen Sund Formula 1 races on TV. One can never totally disregard one's own opinion, but being aware of one's own stance helps to avoid being biased. On the other hand, I have not actually known very much about Formula 1. Being a sort of outsider perhaps makes it easier to "exoticise" the subject and to approach it objectively. Formula 1 races on TV. One can never totally disregard one's own opinion, but being aware of one's own stance helps to avoid being biased. On the other hand, I have not actually known very much about Formula 1. Being a sort of outsider perhaps makes it easier to "exoticise" the subject and to approach it objectively. The interviewees and their relation to Fl As mentioned above, my interviewees fall into the two categories of those interested in and those uninterested in Formula 1. The interested ones can be further divided into those who have "always" been interested in F 1, and those who have been uninterested to begin with, but later changed their opinion. Actually only one of my interviewees (a woman) falls into the latter category, but it is nevertheless a very interesting group: on what level does one have to rethink in order to be "converted"? Is this a case of crossing a border, and in that case, what border? Those interested in F 1 can also be categorised into those whose motor racing interest is limited to Formula 1, and those who are interested in various kinds of motor racing sports. This difference might partly be explained by the Finnish F 1 drivers' influence on the interest in their sport. As in many other contexts, the "real" fans have been there "from the start", before a phenomenon (e.g. FI, a band, a dressing style) has become too popular and easily accessible. The "real" fans disapprove of popular features that are regarded as superficial. F 1 is very popular in Finland today and in this case this might mean that some people, to a certain extent, distance themselves from the huge interest surrounding Hakkinen or from the F 1 sport as a whole. This is to simplify the matter, but in times when "everything stable evaporates" and people seek authenticity and originality in something, I think it does signify something. In addition, change is the sport, since it is primarily this interest in Fl car racing that is the subject of my study. I had noticed that it is mostly men who are interested in Formula 1, and I therefore interviewed five men, as well as two women, who are interested in the sport, and two women who are not interested in F 1. Of course, I could have included men in the latter category, but it was not, however, easy to find men uninterested in F 1. Criticism can be raised against me for this choice. If men who are not interested in Formula 1 racing form such a marginal group, it would have been interesting to hear their opinion, too, especially as my article explores masculinity in this context. The interviewees and their relation to Fl The point of departure for my work was the notion that it is mostly men who watch Formula 1 ( which is reflected in my choice of interviewees) and that F 1 is usually seen as a masculine sport, which is what I wanted to explore. My own position in relation to my subject is that I have not been interested in watching 70 Peter L. Berger, an American sociologist, who distinguishes five tendencies in modernity: abstraction, individualisation, liberation, secularisation and future- orientation. needed for a phenomenon to stay interesting. For some, their interest has decreased when the one in focus, namely Hakkinen, has been on top for so long. He has proven to be an excellent driver and the hardest competition is experienced as being resolved. Finally, I want to point out that the discussion throughout the article is a discussion with the interviewees. I refer to individual informants only in direct quotations. Thomas Ziehe, a German researcher on socialisation, talks about late modernity. He regards demystification as its most important feature, which can be studied in terms of three tendencies pertaining to "the subjective aspects of reality". These are reflexivity, possibility and individualisation, which together result in a change of the individual's horizons of opportunities. This means freedom for the individual, but also decreased security. This "doubleness" is an important criterion for late modernity. The lack of a stable base to stand on, of ultimate truths, creates a need to substitute this void with something else. In this "something else", certain attitudes or "attempts at cultural orientation" can be discerned. The most common attitude is claimed to be conventionalism, a denial of late modernity and a wish to return to old truths. Other attitudes are subjectifying, "seeking security in the minimum of social relations and in oneself', where emotions are supposed to create security; ontologising, which includes attempts at new forms of mysticism; seeking security in something original and authentic. This orientation attempt differs from conventionalism in that late modernity is not denied, but instead, a pre-modem security is consciously sought. The last category of orientation is potentialising: "artificially loading something with meaning" - what is sought is "intensity as protection against emptiness". The object of intensification is the lack of an overall context in late modem society (Berg 1992, 185ft). The interviewees and their relation to Fl In this essay, I will seek to explore how these modem or late modem themes fit my material and whether the attitudes towards late modernity presented above can be used to shed light on what the habit of watching F 1 races on TV signifies for the viewers. Masculinity and gender Frykman sees parallels between the previous and the present tum of the century. The period of the tum of the 19th and the 20th centuries entailed a break-up of traditional society: the world was unstable and this led to the construction of a new man. This man was self-confident, had a stable character and aimed at "new bold targets, simple definitions and genuine togetherness". Frykman thinks that many similarities to our times can be discerned here. The difference is, however, that at the previous tum of the century, man stepped out of the private sphere while today he is trying to re-enter it. Today's narratives on masculinity must show how this is to happen (Frykman 1994, 13ff). Is F 1 a masculine sport? All F 1 drivers are men and most of the interviewees think that a majority of those interested in the sport, that is, the audience and the TV viewers, are men. All the informants also regarded F 1 as a masculine sport, in that "traditional" male ideals and characteristics are expressed in it. However, not everybody thought that these ideals and characteristics are specifically male today. All informants were positively inclined to the idea that women will in future enter the sport as drivers. In this context, I find it important to discuss masculinity and gender in more detail. In his article "Manlig myt och vanlig vardag - om manlighetens forvandling" ("Male myths and everyday life - on the transformation of masculinity"), Jonas Frykman explores how the masculine is constructed and filled with content. He thinks that today's masculinity is threatened. The genders are being democratised and there are no preordained patterns any longer. This result in a variety of effects: partly a wish to return to the past, partly in men creating new identities that they themselves can verify. Frykman points out that the myths, the narratives about masculinity, must be such that men can believe in them and feel an affinity with them. The narratives and everyday conditions are linked to each other. Frykman says that the myths are crucial, since belief, empathy and emotion are at stake here. The myths create a symbolic family bond between men and express what men are and what they are not. The concept of modernity as a tool for analysis In the analysis of my material, interviews on F 1, I have chosen to focus an a number of themes that to me seem essential for the understanding of F 1 as a phenomenon, and what this phenomenon, both in itself and especially in terms of the habit of watching the races on TV, signify for the viewers. I have chosen to use modernity as my overall theoretical tool. The other two themes that I explore in more detail are masculinity as a social and cultural gender, and sport. These can be placed in the general theoretical framework of modernity. Since I base my study on interviews that primarily deal with "watching Fl races", the medium of television as a narrator also forms an important element. TV presents F 1 as a story, and I will attempt to explore what this story is about and what is experienced by the fans as being important in it. In what ways is F 1 a modern phenomenon? I have used certain characteristics of modernity presented by Magnus Berg in his book Modernitet - som empirisktfalt, som teoretiskt redskap. Brottningar med begrepp ("Modernity - as an empirical field, as a theoretical tool. Wrestling with concepts"). The overall figure of thought in modernity is rationalism; "claiming the right of reason to critically evaluate and discard unreflecting, self-evident knowledge that is legitimated by tradition". Berg refers to 71 Masculinity and gender The aim is to find a "natural masculine substance", something genuine and original, and therefore the body has taken on such an important role in today's narratives of masculinity. The primitive and the wild have also acquired positive connotations, since these are associated with authenticity, with a past where life was easier to grasp. Sports - motor sports All interviewees watching Formula 1 think of it as a sport, while those interviewees who are not interested in FI do not regard it as a sport. What is "sport" actually, what characterises a sport? I do not attempt to once and for all determine whether Formula 1 is a sport or not, but rather, I am trying to say something about what one does with a phenomenon by calling it a sport. What does sport signify? By calling F 1 a "sport", the importance of human beings over machines is emphasized. Driving a racing car (successfully) requires the driver to be in good physical shape, with an ability to concentrate and with courage/ foolhardiness, depending on the viewpoint. The fact that money (the car) is actually to quite a large extent crucial for gaining the desired results is something which is disliked by most informants. At the same time, the big money and the fast cars are two of the basic factors of the sport, contributing to its "extreme" character. Denying that F 1 is a sport means claiming that everything is agreed on, that money is decisive. It is tantamount to degrading the entire Formula 1 ethos and taking all 72 chauvinistic rhetoric. The sportsmen personify an abstract concept, that is, their native country. Sports have such a great impact because they appeal to the emotions. The viewers' fervour turn the national feeling into a purely physical experience for them: they feel excited, happy or disappointed. They feel that they belong to a nation, including everything which that entails in the forms of community and identity (Ehn 1993, 204ft). meaning out of the sport: the competition, the drama, the excitement disappears, since the significance of the individual is diminished. Here, I make no difference between sports and motor sports, since they are so similar, both regarding their structure and what the viewers prioritise when watching them. In his book Mandighet og sport ("Masculinity and sport"), Hans Bonde writes about the history of men involved in the early Danish sports movement of I 880- 1920, i.e. the creation of modem sport, as his point of departure. Sport is not an isolated phenomenon in society, but one of the strongest and most basic rituals of modem society, where important norms and values are dramatised and expressed. Sports - motor sports However, sports are not only a reflection of society, but also an active cultural force. Bonde also thinks that the focus around the turn of the 20 th century was on the significance of sports for male culture; an issue which is seldom addressed today, but which is, however, still relevant. Many norms survive in new forms: having a strong character and ambition are still seen as desirable features (Bonde 1991, 7ff). Frykman, too, claims that masculinity has always been created through a ritual causing of pain. Sport constitutes such a ritual, he writes (Frykman 1994, 14). The media of television as narra- tor I think the above quote suits Formula 1 very well and perhaps illustrate the significance of Mika Hakkinen, the Finnish winner, in the popularity of Fl in Finland. I think the above quote suits Formula 1 very well and perhaps illustrate the significance of Mika Hakkinen, the Finnish winner, in the popularity of Fl in Finland. Klinkmann refers to the Finnish culture researcher Jussi Orajarvi, who thinks that we have doubted ourselves in the sense that we see ourselves as being free from all big narratives except for capitalism. If this is the case, then perhaps our society is not, after all, that late-modern, at least not Formula 1, which in that case could form an offshoot to the narrative of capitalism, the objective of which is to win. Watching F 1 races is not an everyday activity: on the contrary, many say it might become boring if there were F 1 races on every weekend. Today, races are broadcast every other weekend. But this does not, however, prevent there being a predictable routine to the programme: on the contrary. The F 1 races and their preparation always follow the same pattern. The races cover three days, an "F 1 weekend". On the first day, the cars are trimmed and adjusted, on the second, the drivers race for a starting position in the actual race, and - finally - on the third day the race itself takes place, which, of course, being the most important, attracts most viewers. The race also usually follows a special pattern. All of the interviewees agree that the start is the most important, or one of the most important and exciting parts of the race. That is followed by lap after lap on the course, depot stops, overtakings, possibly crashes. Sometimes a race can tum out to be boring, in cases where nothing happens. The broadcast is rounded off with the prize-giving ceremony and a press conference where the drivers analyse the race. The TV broadcast offers an overview of what is happening, an overview that would not be possible to achieve at the site of the race; but, on the other hand, something of the reality of the race is lost. The media of television as narra- tor In this essay, I mainly base my analysis on narratives from watching Formula 1 on TV. However, the TV broadcasts are narratives which have already been edited, and the viewers are guided by professional narrators, the F 1 commentators, who describe what is happening and why, and what is important. Some of my informants think that the TV commentator plays a very important part; others only find him disturbing, since they know enough about F 1 to understand the action of the races. Most interviewees usually watch the Finnish Fl broadcasts, while a few of them prefer to watch the races on Swedish TV. What is interesting is that both groups present the same argument for their choice of channel: the expertise of the commentators. It is, however, pointed out that the Finnish commentator is very focussed on Mika Hakkinen and very enthusiastic when it comes to him and his successes or losses, to the point were he is verging on being notorious for this bias. To sum up, it can be claimed that sport expresses certain male ideals and norms pertaining to masculinity in society, while sport also creates men as they take part in it. Viewers witness this as they watch sports on television. In her book Nostalgi og sensasjoner ("Nostalgia and sensations"), Torunn Se Iberg talks about the use of television in Norwegian everyday life, analysing, for example, news broadcasts. It has been claimed that the strategy of the television broadcast is to attempt to control reality, and herein lies its popularity. We can ask ourselves whether we actually learn Similar arguments can be applied when discussing sport and national feeling. In his book Forsvenskningen av Sverige ("The Swedification of Sweden"), Billy Ehn claims that "sports have become one of the most emotional and expressive areas of the national". This is particularly obvious in sports broadcasts on TV, where the commentators use a special kind of 73 winners. Those who are invisible and marginalised - the large, grey mass - are not winners, but losers. (Klinkmann 2000) anything new, since the programmes follow as strict a routine as the watching of them. Selberg thinks that the news items are actually constituted ofnew editions of well- known versions (Selberg 1995, 274). Driving in a laboratory The car arrived at the same time as modernity, and it can be regarded as a step in the individualisation process, as a symbol of freedom. The car enabled a new kind of mobility. Fl races can be seen as driving a car under laboratory conditions, in a controlled, but still dangerous sphere. It is a kind of ritualisation and dramatisation of driving, which takes place on tracks specially made for the purpose - which fact in itself constitutes an abstraction of driving. Racetracks are required, since the technology has developed and the cars are increasingly fast. Thus, the way has been paved, literally, for the development of the technology, as was done when railways were built for trains and roads were asphalted for cars- but Formula 1 has taken this one step further. Fl has been created for an audience and the racetracks therefore offer a better overview of the races than, for example, driving on ordinary roads would do. The F 1 races contain several components found also in the "real" world of cars: starting the engine, pumping petrol, changing tyres, overtaking, the occasional crash - simply moving forward. But all this The TV broadcasts focus on the winners; those driving in the lead. Those who are less successful are mostly shown only if they crash. Nowadays only the winners count, we want to see winners, we want to (and are able to) identify with winners. Win or get out is to be taken very literally. Those who are seen and heard in the media are 74 Formula 1 competition in Niirnburgring in Germany. Photo: in private possession Formula 1 competition in Niirnburgring in Germany. Photo: in private possession takes place at a much faster pace and everything is prepared and organised. An F 1 race also has a clear beginning and a clear finish. Specific symbols mark these: the start is shown by lamps going out one after another, and when the first car reaches the finish, a black-and-white chequered flag is waved. A special sphere, with its special significances, is entered and exited. Despite F 1 driving being controlled, there is nevertheless a certain degree of disorder and risk: not everything is foreseeable, and if it were, a lot of the excitement of the race would be lost. Driving in a laboratory The viewers try to understand what is happening and to bring order and coherence to the events displayed on the television screen. This requires some previous knowledge of the rules and actors, but the commentator is also important in his role as "explainer". Part of the excitement lies in figuring out how everything is connected. Therefore, the events must not be too predictable. In fact, the unpredictable elements are experienced as the most interesting, as for example overtakings and crashes. Of course, crashes are not totally unexpected, either, but form an integral part of Fl races. They are, however, treated as exceptions, as they break the order and control which the F 1 strives to maintain. This is comparable to what Torunn Selberg says about news broadcasts: even if they usually deal with injustices and catastrophes, they are still regarded as positive. Since the news items differ from what is considered normal, they confirm that normality (Selberg 1995, 266). on the other tracks, which is why many think that it does not offer particularly interesting viewing. On the other hand, the Monte Carlo racetrack is the favourite of some, since the race is, in a sense, moved out into "reality" and thus more varied and "for real" than when driving around a field of grass, or a patch of wood. The abstraction is not so great here. In addition, Monaco has a certain sound to it, which resonates with the origins, traditions and character of Formula 1. The Monaco race is therefore highly prestigious. Mika Hakkinen in Formula 1 - Finland in the world: contrasts Within ethnology, sport has been compared to war. Nationalist sentiments are allowed in sport. Stereotypes of other countries or regions are used and also stereotypes of one's own country are employed. Who are we, what are Finns like and what distinguishes us from others? Various national characteristics and values concerning positive and negative features are processed. Often the heroes of sports become moral exemplars. As a person comes to symbolise the fatherland, the basis for the patriotic feelings, the collective identity, becomes concrete and emotional. In sport, "the enemy" and one's own country or region are seen as clear entities, which is important in order to have something to feel and fight for. One's own country is contrasted with the other, and the A phenomenon which sits on the boundary between the F 1 laboratory and "normal" driving is the F 1 race on the streets of Monte Carlo, Monaco. It is actually a relic, the only street racetrack left within Formula 1 racing. The racetrack does not fulfil today's security requirements for an F 1 track. The races are therefore slightly slower and the opportunities for overtaking are fewer than 75 national is thus created (Ehn 1993, 213). about how good he is, so that probably suits with Ferrari, although Ferrari would perhaps rather have a, for example an Italian driver and not a German one, but he's a very self-confident person, so he fits with Ferrari (L 1428, female b. 1978). national is thus created (Ehn 1993, 213). Formula 1 is an international, highly organised sport where a "team" usually consists of three components: the manufacturer of the car's engine, the stable and the driver. These can all be of different nationalities. However, even if the teams can be very mixed, nationality is relatively important in explaining what kinds ofunits the teams make up. This thought process also entails a large amount of flexibility, particularly taking into account that many drivers change teams before a new season. There are two Formula 1 stables that are more successful than the rest and these are thus also the most visible ones. Schumacher, Hakkinen' s prime competitor, is contrasted with Hakkinen, and certain values concerning positive and negative characteristics are displayed. Often, Schumacher is described as the self- confi dent, boastful and cheeky type, representing characteristics that are perhaps not that highly esteemed according to Finnish values. Mika Hakkinen in Formula 1 - Finland in the world: contrasts Hakkinen, on the other hand, is seen as humble and therefore likeable. But not all admire Hakkinen' s "personality" that much. Most informants agree that Hakkinen is a good driver, but many think that Schumacher's character fits the sport and world of Fl better. I will discuss perceptions of the character of the sport further in the next chapter. Still, Hakkinen stands for Finnish success and it is not surprising that some think that another great Finnish success, that is, Nokia, should sponsor Hakkinen. Statistical surveys show that Hakkinen is currently the best known Finn in the world. These two big stables and their respective front figures, who, slightly simplified, are those who duel on the course, stand in sharp contrast to each other, at least when seen from a Finnish perspective. The English McLaren team have German Mercedes engines in their cars, and their leading driver, Mika Hakkinen, is Finnish. The McLaren cars are as grey "as the English weather"; they are also called "silver arrows". McLaren has the image of being a "calm" stable, which is run with "German" preciseness; everything is controlled and carefully calculated. Mika Hakkinen, as a "quiet and calm, typical Finn" will suit the stable. Well, he's like a person who likes to brag The Fl pilots in the royal class: prestige and respect The other big stable is Italian Ferrari, which has a particularly strong profile since Ferrari is a combined stable and car manufacturer, also making ordinary cars. Ferrari is well-known for its expensive luxury cars. Ferrari's colour in Formula 1 is red and their logotype a black, rearing horse against a yellow background. Associations can be developed from this: Ferrari is the temperamental, southern European F 1 stable. Their main driver is German Michael Schumacher. But how does a German fit the image of the stable? Prestige is a word that the interviewees often use when talking about Formula 1. Prestige in this context is perhaps explained by the fact that everything that is seen as prestigious in F 1 is that which makes the sport serious and "real" and not just a game. The prestigious aspects are not to be underestimated, since they reflect what is regarded as valuable. Prestige and respect go hand in hand. What then is regarded as so prestigious within the sport? The concept of "the royal class of motor sports" in itself reveals a great deal of this issue. Individualisation 76 The Formula 1 cars are constantly developed, both Nagonstans under rubriken to be faster and safer. Can they still be called "cars"? Formula 1 ... " Photo: John Hackman is a strong tendency within F 1. The drivers have successfully made their way up from the "lower" classes of motor sports and F 1 is the culmination of their careers: there are no classes above it in the world of motor sports. The best drivers - the winners, the fastest cars -high technology and the biggest money bestow prestige upon the sport. All these aspects are regarded as prestigious in our society, and money is the means of wielding power. This is an area of contradictive emotions. The drivers must be skilful and in good physical shape; this is what makes Formula 1 a sport. According to sporting ideals, everybody should compete on equal terms in a sport. In Fl, however, a stable can prioritise a successful driver above a less successful one. The latter is given an order to let the other driver overtake him. This is done because within F 1 the winning stable gets money and thus an opportunity to develop their cars. The Fl pilots in the royal class: prestige and respect All F 1 drivers today are men and most war pilots are also men. All the interviewees agreed that there will, in future, be female F 1 drivers, which shows what we are striving for, that is, equality. However, some doubted whether the stables really dare employ women since so much money is at stake. This illustrates that a gender system basically is a prestige system (Bengtsson & Frykman 1988, 71). advertise within F 1. The IT companies symbolise expansion and success. When it comes to the clothes companies, the drivers also function as models. The sponsors provide them with clothes to wear during their "leisure" when they are "seen", and engage them in various advertising events. The fact that menswear has also become part of the F 1 world, perhaps indicates that men have started to care more about their looks. The prize ceremony is a tradition within Formula 1; a ritual always performed in the same way. Champagne bottles ofa gigantic size, as with everything within F 1, perhaps represent something of the luxury, glamour and prestige which is (traditionally) associated with F 1. The prizes are usually awarded by a representative of those in power - for example the mayor - of the place where the race takes place, which also emphasizes the prestige of the sport. The world of Formula 1 entails material affluence, big money and contacts to the political and financial elite, and, in addition, the traditions of the sport create prestige. A stable that is not very successful, can still possess a certain prestige because of its long-standing tradition. ( g y , ) Within Formula 1, the masculinity that was created around the tum of the century is very apparent: that is to say, in the form of a man who is successful in society by being controlled, self-confident and of firm character. As a driver, it is important to control one's nerves and oneself in order to be able to keep control of the car. Loss of nerve is something to be blamed for; it is a sign of not being a good driver. At the same time, F 1 is an aggressive sport. Competition is hard and it is important to be pushy. The only thing that counts is winning, since the losers easily disappear into an anonymous mass. The Fl pilots in the royal class: prestige and respect This is why two stables have come to dominate the sport, which most viewers find makes the sport more boring. One of the interviewees compared the adjustment of technical details of the cars with doping. There is, nevertheless, a difference in that development of the cars is part ofFl, while doping is common, although forbidden, in other sports. ( at least not in the minds of the informants). Rather, there is an opposite tendency: Fl has become a more "decent" sport in many respects, which can be seen in, for example, the advertisements. The Fl cars and the drivers' overalls abound with adverts. The stables have various sponsors, usually large international companies. The most visible and noticeable sponsors are tobacco companies. Today, several countries where F 1 races take place have banned advertising of tobacco products. Smoking does not currently have a positive reputation, but is rather associated with "losers" than "winners". However, tobacco adverts are something of a tradition within F 1, and, to put it more positively, in this context they can be said to represent a certain kind of dangerous living and risk-taking that are part of this sport and aspects which make it fascinating. Recently, however, companies in, for example, information technology and men's top brand outfitters increasingly p The book Idrottens sjal ("The soul of sports") claims that sports are becoming alienated from the old ideals of amateurism, idealism, fair play, decency and a healthy lifestyle. Today, sports mainly provide a show: entertainment, not moral edification (Schoug 2000, 330). The interviewees also say that watching F 1 is entertainment for them. Sports have also become increasingly commercialised, particularly on an international level. In the case of F 1, however, advertisements have, according to the informants, "always" been there, and they claim that Fl has never stood for either amateurism, decency or a healthy lifestyle 77 Here, the F 1 drivers could also be compared to war pilots, those most prestigious actors within the army. War pilots also form a small, select elite endowed with the dangerous task of controlling fast, expensive highly technological machines. They are sent by their country to fight against an enemy. The Fl pilots are "sent" by their stable. Both the pilots and the drivers communicate with a "tactician" via radio. In F 1 the driver also sits in the "cockpit" and the drivers are called "pilots" in many languages. ... as I said, they risk their lives when driving, so being on bad terms with someone ... they have the power to crash The Fl pilots in the royal class: prestige and respect Calmness and aggression: therein probably lays one of the great paradoxes of Formula 1. This is also visible in the code of behaviour concerning the drivers' manners with each other outside the track. There, the drivers are not allowed to display any aggressive feelings. Of course there are old stables with long traditions and it's a pity that many of them have disappeared ... and it's good that there are such old institutions in this world too, but McLaren and Ferrari also belong to these old, legendary stables, for example. (L 1435, male b. 1978) Thirdly, there lies a certain prestige in what the F 1 drivers do: they are entrusted with the fastest, most expensive cars. Within ethnology, it has been suggested that people must be sacrificed in order for technology to develop. Those sacrificed are young men. Thus, the activity of driving racing cars comes to resemble war, and in F 1 this is perhaps particularly obvious. ... as I said, they risk their lives when driving, so being on bad terms with someone ... they have the power to crash 78 with them in some way ... like "o-oh, accident, I killed you". (L 1432, female b. 1979) or early in the morning. A certain lifestyle, probably most typical for students, can be associated with the habits of watching F 1 : ... then they drive at such times that it's early Sunday morning, perhaps five or so, so then usually one's been to the pub all night for example, otherwise it'd be so hard to get up. (L 1437, male b. 1977) The prize-giving ceremony is perhaps one of the events where the duality ofrival- colleague is most highly charged, as the first, second and third prize winners as supposed to celebrate their achievement together: Many find it nicer to watch the races together with other fans, since they can discuss and comment on the events of the race. In fact, often there is not that much discussion during the actual race, but the viewers share the experience of the race and emotions of excitement, joy or disappointment. The girls might find it a bit boring in cases where their (female) friends are not interested in watching races with them. The following are the thoughts of one female informant, who does not watch Fl, of her boyfriend's and his friends' Fl habits: ... The Fl pilots in the royal class: prestige and respect and then they get a bottle of champagne each that they 're supposed to open and then they spray it on each other, but somehow it feels quite forced since they are such rivals many times there and they can be really annoyed with each other since they've just competed on the course. (L 1428, female b. 1978) F 1 could perhaps be called an "aggressive gentlemanly sport". A concept associated with the ideals of a gentleman and which seems to be important here is respect. The drivers' aim is to be respected, both by other drivers and by the audience. This they strive for by proving that they are good drivers. Respect is something that one earns. Perhaps this can be linked to the above discussion on how sports create men and masculinity being a project of conquest. ... when it was clear that he (Hakkinen) lost, it was like, then they knew that there wouldn 't be anything more like that ... the last fun race with the lads would not happen then either, so they were probably a bit disappointed by that, too. (L 1434, female b. 1977) ... when it was clear that he (Hakkinen) lost, it was like, then they knew that there wouldn 't be anything more like that ... the last fun race with the lads would not happen then either, so they were probably a bit disappointed by that, too. (L 1434, female b. 1977) Formula 1 on TV - variations, habits and togetherness On the other hand, some prefer watching the races alone, so that they can better concentrate on the race and what the commentator says. The commentator becomes a kind of "discussion partner". But the feeling of community pertaining to the interest in F 1 can take on many forms. Since all my interviewees have said that F 1 is a masculine sport, one could perhaps talk about an imagined male comradeship between the men watching F 1 and the F 1 drivers. Since the national interest in F 1 is also great, it can also be claimed that F 1 creates a national feeling of community. During the active season, F 1 races are broadcast every other weekend. Each race is part of the World Championship and success in the individual races gives a certain number of points that are added. As this is the structure of the championship, most viewers find it important to watch every race, even if not all name F 1 as their favourite sport. Most informants want to see the races in live broadcasts, and because of the time difference in the various racing places, this means that TV viewers must sometimes get up in the middle of the night 79 This is sometimes very obvious in the TV broadcasts: when Finland has been successful through Hakkinen, the broadcasts have occasionally contained inserts from Hakkinen' s home town where people are cheering and celebrating. Thus it is underlined how all Finns belong to the same community. In addition, the interest in F 1 might be something that one shares with an otherwise unfamiliar person, a topic to discuss not only with Finns but also with other nationalities: The other showed a car driving very fast and daringly and at the end of the ad a female rally driver stepped out of the car. The objective of the advertisements is, of course, to promote a new car model and the message is underlined by discarding an old order. The following quote can be understood against the background that women and technology are traditionally seen as incompatible: If one doesn't know anything about it it's usually boring ... I suppose I had a bit of this silly attitude, had these prejudices of it all that "everybody's boyfriends just sit and watch it on Sundays and ... " so I suppose I was just like everybody else ... Formula 1 on TV - variations, habits and togetherness (L 1432, female b. 1979) .. .I've been student tutor to these exchange students and there were Italians in my group, so they are of course fans of Ferrari and so on, but when they first came here there wasn't perhaps that much to talk about except to say "well this is Abo and this is where we study and bla, bla - do you watch Formula by the way? ". (L 14 31, male b. 1977). Starting watching Fl as a woman might thus be a way of breaking a tradition, which is what this informant had done. Formula 1 is surrounded by an aura of money and fast cars, but also of beautiful women. Traditionally, woman in the F 1 world has been of the "car show type", that is, a young, more or less scantily clad woman, also called "asphalt rose", who is there only as a "decoration". Her task is to emphasize the prestige of the sport and the masculinity of the drivers, as the contrast between man and woman is thus sharpened. The stables also willingly invite models to the races. Thus, two glamorous worlds meet: the world of models, dominated by femininity and the world of Formula 1, dominated by masculinity. This can easily be interpreted as an instance where the traditional dichotomies of female-passive and male- active are still valued. But, as mentioned above, the drivers have also started acting as models for various clothing companies. Looks have become more important for men, too. Below a quote from an interview with a woman who is not interested in F 1. She answers the question whether a certain The image of man and woman within Formula 1 Women, cars and technology. The traditional perception of the relation between these is that women cannot handle cars and technology- an attitude that today has perhaps somewhat modified to the view that women, or most women, are not interested in technology and cars. However, the conclusion is the same: claiming that women in general are not interested in technology makes this appear as a personal choice, which can be associated with the trend of individualisation in modern society. But since this is a categorisation based on gender it is not, after all, a personal lack of interest but a gendered one. This, however, is perhaps changing. Two different advertisements for cars shown on television recently touch upon this; both played with the existing stereotype concerning women and cars. One of them parodied the image of a scantily dressed woman flouncing by a car in a car show. 80 kind of male ideal is discernible in F 1 as follows: project for men (Knutsen 1998, 50). Thus, the question is whether a man can be an F 1 driver and parent at the same time. The interview with Mika Hakkinen in an F 1 magazine on the web ends as follows: No-o (laughter), no I don't think so, not if compared with like other sports, where the men usually are good-looking, have nice bodies that many perhaps watch in order to try to look like them in some way, but I don't think that Formula 1 drivers ... they haven 't really been that good-looking. (L 1438, female b. 1979) Despite wife Eija waiting the couple's first child, Hakkinen responds to talk of retirement with a firm shake of the head. There is life in the old dog yet. He is an angry man, and the determination to avenge Schumacher's success this year will drive him on in 2001. (http://www.atlasfl.com/ 2000/nov08/collings.html) Perhaps pressure by women also make men care more about the way they look, given that one thinks looking good is an ideal to strive for. One of the reasons for the change in men is the change in the situation of women. The traditional relation between men and women is therefore also changing and gender identity is subject to negotiation to a larger extent. Man has also become an object (Knutsen 1998, 52). The image of man and woman within Formula 1 Two ideals collide, and in this passage the "aggressive" F 1 model for masculinity is given a positive charge and filled with vitality, even if the reporter first expresses some doubt. The clash of different male ideals is also problematic in society at large. In the Finland-Swedish newspaper Vasabladet, an article with the headline "New father roles are confusing" was published on 28 October 2000. In it, men's studies researcher Arto Tiihonen says that "men have ended up in a difficult situation where they are expected to be bachelors, have a relationship, make a career and be fathers at the same time". The problem is that all these male roles have different life values. The women who most frequently appear on the TV screen during F 1 broadcasts are the girlfriends and wives of the drivers. My informants find that they represent different roles. On the one hand, they can be seen as a new type of the decorous woman; they are there only "to be seen". On the other hand, they can be regarded as devoted supporters, which is to assign them a more active role. In any case, they represent the private sphere of the drivers and underline the drivers' status as spouses or boyfriends. There may be a conflict inherent in this, too. F 1 has perhaps traditionally been something of a "bachelor sport", while the two presently most successful drivers both have a wife and children. If today's myths of masculinity are supposed to direct the men back into the private sphere, this is what might have happened within F 1. Still, the drivers put their highly dangerous job first, which creates a conflict. Arild Knutsen, who has studied the construction of today's man in the tabloid press, says that being a father has become a new moral The fact that the lifestyle of F 1 drivers seems to have changed is something that some of my male informants find regrettable. The drivers are now more "politically correct": The old image of an F 1 driver is this that he sits at a casino and throws away money like hell (laughter) and like being a playboy... well, as I said, it's a pity in a way that these don't exist any more, since they 're so politically correct... and like Hakkinen now, Formula 1 is like his life, he's never like ... The image of man and woman within Formula 1 Perhaps play is not very highly valued and therefore it can be somewhat embarrassing to play as adult, something "to be hidden in the closet". On the other hand, some of the interviewees did talk in quite a serious tone about the games. So, is it actually a game when they play F 1 games on the computer, or something else? The image of man and woman within Formula 1 no scandals, out partying and rampaging, in the 70 's it was more 81 - Well, do your friends play? - ... yeah, I have some that do (laughter) - Oh, do they have this complete kit with steering wheels and pedals and ... - Yes, they actually do, at least in high school there were some that did ... but I don't know, I don't think that ... well one can 't really know what they've got in their closets (laughter) (L 1430, male b. 1977) common that ... even during the Grand Prix weekends they were out partying on the Saturday night before the race ... (L 1435, male b. 1978) common that ... even during the Grand Prix weekends they were out partying on the Saturday night before the race ... (L 1435, male b. 1978) So F 1 has not only become safer regarding the cars and the courses, but the drivers have also adapted a "safer" lifestyle. The risk-taking has been reduced. "In the old days" the sport was more extreme and therefore more "whole", but now shifts from the "original" have taken place on many levels. If the drivers today are seen as being politically correct, the thought is that they were more rebellious earlier, that they challenged the established order and took risks. Thus, the un-ordered and wild is what is associated with the original and gets a positive quality, while today's more "tame" drivers are seen as having a more ordered and "boring" life. F 1 computer games with steering wheels and accelerator pedals can be regarded as toys. The interviewees have pointed out that F 1 is something serious and real, or possibly a serious game, but never pure play. Perhaps play is not very highly valued and therefore it can be somewhat embarrassing to play as adult, something "to be hidden in the closet". On the other hand, some of the interviewees did talk in quite a serious tone about the games. So, is it actually a game when they play F 1 games on the computer, or something else? F 1 computer games with steering wheels and accelerator pedals can be regarded as toys. The interviewees have pointed out that F 1 is something serious and real, or possibly a serious game, but never pure play. - Do you play these Formula games on the computer? -No (laughter) - Well, do your friends play? - ... yeah, I have some that do (laughter) - Oh, do they have this complete kit with steering wheels and pedals and ... - Yes, they actually do, at least in high school there were some that did ... but I don't know, I don't think that ... well one can 't really know what they've got in their closets (laughter) (L 1430, male b. 1977) Formula 1 - a boy's dream. Toy racetracks and computer games Various toy cars and racetracks have been and still, to a large extent, are boys' toys. Many boys dream of becoming racing drivers and perhaps they never stop dreaming? However, there is a difference between what is objectively realisable and an individual's wishes. To actually become a racing driver is very difficult and, in the end, very few enter that career. Only a tiny elite get the opportunity to drive F 1 cars. Playing F 1 computer games provides a way of experiencing the feeling of driving an F 1 car - and probably the closest most get to actually doing it. There seems to be something childish or perhaps boyish in playing these games. Most (male) interviewees reacted in an embarrassed/amused way when asked about this: The computer games are increasingly technologically advanced. In order for the experience of driving an F 1 car to be as real as possible, it is important that the games are as realistic as possible and that all the details are up-to-date. For a maximal experience, one should have a kit with a steering wheel and accelerator and brake pedals. Views on whether it is a good or a bad thing that the games are difficult vary. Some think that the games are supposed to be difficult, since it is also difficult to drive Formula 1 in reality, whilst others do not have the patience to play if the game is too difficult. There are also varying views on whether more concentration is needed to play or to watch Formula 1: Of course one has to be more concentrated when playing these games, since it is as easy to crash as if one was driving for real since they are so realistic these days. (L 1437, male b. 1977) - Do you play these Formula games on the computer? -No (laughter) 82 It is possible to engage strongly in the games, since they are so realistic, which is rather paradoxical. Driving a car on a computer is an example of the abstraction tendency that characterises modem society. But the purpose of it, that is, seeking experiences, is perhaps more typical oflate modernity. Driving is lifted out of its context, but linked to reality by the games being made as realistic as possible, and by the players own knowledge of F 1. A step into "reality" All my interviewees have a perception of what it would be like to see a race "in reality". The overview of the events would be lost, but one would get a stronger audiovisual experience of the cars' actual speed and not least of the atmosphere, which, to a large extent, cannot be conveyed by TV broadcasts. Thus, watching TV is a version of following F 1 where the viewer perhaps has the strongest experience of being in control of the events. Witnessing a Formula 1 race on the spot is a totally different experience from watching it on TV. One informant gives a lively description of this experience: Formula 1 - a boy's dream. Toy racetracks and computer games so extraordinary about it and one didn't see a thing and it seemed as if they drove really slowly" - it might get more linked with reality, but then again it's nice when it floats there on some unimaginable level. (L 1433, female b. 1979) There is something late-modern in travelling to see an F 1 race and not least in dreaming about going to see a race, to go into "reality". There is a wish to be in the centre of events, to be part of a larger community and enter a reality that promises a penetrating, intense experience. It is something one can dream about, but at the same time fear that once actually being there it would not be that exciting, that the only feeling would be one of emptiness: "is this all?"; the dream would be shattered. This shows the duality in the individual's changing horizons of opportunity, which is a typical late-modem feature. At the same time perhaps I'd like to leave it being like "oh, imagine if one could, if one could ... " instead of when one's seen it it's like "now I've seen it and well, what's Excitement and/or meaningless- ness Formula 1 is not meaningless. It is filled with several meanings and values concerning, among other things, masculinity and femininity, national feeling, prestige, morality and order, which are not isolated concepts, but change with the society that surrounds them. I have tried to show how this dialogue about and negotiation of meaning takes place by focussing on how students watching Formula 1 on TV experience these changes. But how does Formula 1 become interesting and exciting to watch? It is, after all, simply cars driving around racetracks. The attitude to modernity called potentialising, making the late-modem lack of context intensive and exciting, is a possible explanation. However, it is rather modernity being intensified and not late-modernity. The modem belief in development and progress, and the capitalism dominating Formula 1 are perhaps not compatible with late- ... at least what I noticed was, well they are utterly skilled ... it's the speed, the performance, like they never brake at all ... it 's like from 350 to 80 just like that ... it's the noise, it's carnival, it's like the happening around it and there were like a hundred thousand, a hundred and fifty thousand people at the Nurnburgring when I was there... everybody is happy and friends with each other. (L 1435, male b. 1978) Another interviewee talks about her ideas about what an F 1 race might be like: 83 modernity where "everything" can be questioned and changed. TV viewers reflect and take a stance on many issues pertaining to F 1, and this attitude is often of a late- modem kind: Printed sources and literature Berg, Magnus. Modernitet- som empiriskt felt, som teoretiskt redskap. Brottningar med begrepp. Goteborg: Skrifter fran etnologiska foreningen i Vastsverige 11, 1992. Bengtsson, Margot & Jonas Frykman. Om Maskulinitet - Mannen som forskningsprojekt. JAMFO rapport nr 11, 1987. Bonde, Hans. Mandighed og sport. Odense Bengtsson, Margot & Jonas Frykman. Om Maskulinitet - Mannen som forskningsprojekt. JAMFO rapport nr 11, 1987. I'm a bit fascinated by this, that their fascination with speed and excitement ... can be so huge and that they really, since of course they know that ... any time might be their last. (L 1432, female b. 1979) Bonde, Hans. Mandighed og sport. Odense Universitetsforlag, 1991. Ehn, Billy. "Kropp och kansla". F orsvenskningen av Sverige - det nationellas forvandlingar.Billy Ehn, Billy. "Kropp och kansla". SOURCES AND LITERATURE Selberg, Torunn. "Fjemsynsvirkelighet og hvardagsvirkelighet. Orn bruk av fjernsyn i den norske hverdagen". Nostalgi og sensasjoner - folkloristisk perspektiv pa mediekulturen. Torunn Selberg (ed.). NIF Publications, No. 29, 1995. SOURCES AND LITERATURE Unprinted sources The Department of Ethnology at Abo Akademi University Tape Archives KIV A L 1428 female b. 1978, student L 1429 male b. 1972, student L 1430 male b. 1977, student L 1431 male b. 1977, student L 1432, L 1433 female b. 1979, student L 1434 female b. 1977, student L 1435, L 1436 male b. 1978, student L 1437 male b. 1977, student L 1438 female b. 1979, student Vasabladet 28.10.2000. "Nya papparoller ar forvirrande". Vasabladet 28.10.2000. "Nya papparoller ar forvirrande". Excitement and/or meaningless- ness F orsvenskningen av Sverige - det nationellas forvandlingar.Billy Ehn, Orvar Lofgren, Jonas Frykman (eds.). Stockholm: Natur och Kultur, 1993. Fataburen 2000. Idrottens sjal. Bo G. Nilsson I find the above quote a good illustration of late-modern society's contradictory reaction to other people's total devotion to something. While seeing the meaninglessness in somebody risking their life for something like Formula 1, speed and excitement, one can also be fascinated by the fact that the drivers are so totally dedicated to what they believe in. Ehn, Orvar Lofgren, Jonas Frykman (eds.). Stockholm: Natur och Kultur, 1993. Fataburen 2000. Idrottens sjal. Bo G. Nilsson (ed.). Stockholm: Nordiska museets forlag, 2000. Fataburen 2000. Idrottens sjal. Bo G. Nilsson Frykman, Jonas. "Manlig myt och vanlig vardag". Hverdag: Festskrift till Brynjulf Alver. Arild Stomsvag & Torunn Selberg (eds.). Stabekk: Yett & Vitten, 1994. Frykman, Jonas. "Manlig myt och vanlig vardag". Hverdag: Festskrift till Brynjulf Alver. Arild Stomsvag & Torunn Selberg (eds.). Stabekk: Yett & Vitten, 1994. Klinkmann, Sven-Erik. "Cathy Freeman och symbolkedjoma". Vasabladet 4.11.2000. This essay was originally written in the autumn o/2000, and the situation in Formula 1 has changed since then. For example, Heikkinen no longer drives Formula 1. Knutsen, Arild. "Nar mannligheten far trykksverte - om tabliodpressens konstruksjon av 1990-tallets mann". Dugnad 4, Vol. 24, 1998. SVEN-ERIK KLINKMANN Elvis, "Coolness" and Masculinity in the 50s USA online dictionary "cool" has the following meanings: "1. Neither warm nor very co Id; moderately cold" as in: ''fresh, cool water; a cool autumn evening." Its second meaning is "giving or suggesting relief from heat: a cool breeze; a cool blouse." The third meaning given by the dictionary is something "marked by calm self-control: a cool negotiator, and the fourth "marked by indifference, disdain, or dislike; unfriendly or unresponsive: a cool greeting; was cool to the idea of higher taxes." Fifthly, the word can be "of, relating to, or characteristic of colors, such as blue and green, that produce the impression of coolness." Sixthly, cool can, in slang, mean "a. Excellent; first-rate: has a cool sports car; had a cool time at the party." or "b. Acceptable; satisfactory: It's cool if you don't want to talk about it." Lastly, the word can be used in slang as expressing something "entire; full: worth a cool million." This article discusses Elvis Presley in relation to the concepts of coolness and masculinity in the 1950's in the USA. One point of departure is that words can be used in a number of different ways; for example, as carrying either a more literal, or more metaphorical or figurative meaning. An example of the way in which words are given figurative meanings, without us even giving it further thought, is the song by the American country singer and songwriter Tom T. Hall. The song "Old Dogs, Children and Watermelon Wine" tells the story of the singer's encounter with, as the song goes, "an old gray black gentleman". The two adjectives signifying colours, that is, grey and black, here very clearly carry different meanings and are formed on different bases. The word "gray" metonymically, primarily based on hair colour, refers to the man's age, that is, how the man is categorised agewise as an elderly gentleman. Also the word "black" is used metaphorically, but in a different way: as signifying that the man in question is ethnically Afro- American, thus also referring to the problem of colour, which is still highly topical in present-day USA. The concepts of "cool" and "coolness" are also, in my opinion, directly connected with issues of colour in a metaphorical sense. So, it is clear already at this stage that the adjective "cool" has a number of diverging meanings, from chilliness to indifference, from cool colours to calm self- contro 1. Electronic references Interview with Mika Hakkinen: http:// www.atlasfl.com/2000/nov08/collings.html 84 The pre-history of the concept of "cool" In order to trace the pre-history of the concept cool, I think we must assume that the USA is a strongly colour-fixated society, where, in the 1950's black artists, such celebrated jazz stars as Miles Davies or Charles Mingus could still, when stepping out onto the street from a jazz club in New York, be harassed by the police and thrown into "the nick" without any valid reason. Descriptions of this racist culture are found in, for example, Charles Mingus' autobiography Beneath the Underdog, or in Blues People, a history of jazz written by the black author and cultural critic Le Roi Jones, or Amira Baraka, as he later called himself. g Thompson discovered the "cool" character of Yoruba art during a field trip in West Africa in 1963. In what follows I absolutely do not want to belittle the significance of Thompson's observations. The connections between Afro-American and West African cultural expressions are well established within, for example, jazz music. Therefore I find it an obvious fact that a general attitude such as coolness can also possess deep historical roots. However, the timing of Thompson's study of the traditional West African "cool" art might prompt a certain apprehension. In thus connecting a specifically American set of issues from the 1950's and 1960's with a totally different cultural environment, the West African one, or vice versa, there is a risk of a "transferred" interpretation of a cultural phenomenon from a mainly sacred and traditional context in West Africa to a secular and modern setting in North America. Where do the roots of the concepts "cool" and "coolness" lie? The American art historian and folklorist Robert Farris Thompson ( 1973) traces the earliest history of"cool" to West Africa. According to him, the concept in its traditional West African context expresses a social and aesthetic ideal characterised by cultural control, individual restraint and social stability. The concept is also associated with the ritual use of water and chalk, or other substances saturated with meanings of coolness and purity. In a traditional West African environment, coolness pertains to a transcendental balance, Thompson notes. Based on his studies of coolness in tropical Africa and also in black America he has defined what he calls "an aesthetic of the cool". SVEN-ERIK KLINKMANN Elvis, "Coolness" and Masculinity in the 50s USA Here, we can also add another meaning for the word "cool" which the online dictionary does not include, that is, "cool" as a noun signifying a certain musical style within jazz, as created in the 1950's by musicians such as Miles Davis, Bill Evans and Gerry Mulligan. In fact, the word "cool" came to virtually explode in But, starting my study purely linguistically: what does the word "cool" actually mean? According to Bartleby's 85 50's America, not just as a name for a specific style of jazz, but also as a much more fundamental pose and attitude associated with both a rock'n'roll artist such as Elvis, within rock'n'roll music, and, last but not least, within the entire teenage culture, which in the 50's was connected with rock'n'roll and jazz. intertwined elements of sincerity and pleasure, of responsibility and play (Ibid.), which thus also can be described as a complex, artistic and even spiritual form of coolness. Being cool in the West African cultures means being calm, serene, composed, balanced, expressing atonement and offering sacrifices. According to Thompson (Ibid.), this West African form of coolness also expresses 1. discretion, 2. healing, 3. rebirth and 4. novelty, or purity. A central metaphor for people's "cool" qualities is the image of the strong, clean flowing waters of a river. The pre-history of the concept of "cool" This is characterised by fundamental, intricately motivated, artistically aware Perhaps the roots of the concepts of cool and coolness should after all - at least as pertaining to a more limited time framework - be sought in the USA, and there particularly in the black music scene in the New York of the 1930's and 1940's. In his study of the concept of cool as a sign of adolescence in the America of the 1950's (Cool: The Signs and Meanings of Adolescence, 1994), semiotician Marcel 86 1930's and it was called hay, tea, shuzzit, muggles, muta, grefa, gunja, reefer, gauge or weed. Danesi claims that the origins of the concept are to be found in the American jazz scene of the 1930's in the heat and smoky air of the jazz and night clubs (Ibid. 37ft). In order to get some fresh air to breathe in the clubs, windows and doors were opened to let in "cool air". By an analogy, according to Danesi, the type of jazz, slow and polished, played in these clubs was therefore began to be called cool, and the musicians playing this music - black musicians, but also others, including white "experts" of the music, were termed cool, too. The spread of heroin in the jazz circles in the 1940' s often resulted in the musicians being described as cool or withdrawn, emotionally indifferent. This, according to jazz historians, stood in direct relation to the intolerable racial situation of Afro- American artists and intellectuals after World War II. Older jazz artists, such as Louis Armstrong, Cab Calloway or Ella Fitzgerald, had been able to hide behind an attitude of a laughing jester, by carrying a nice and friendly mask (for a description of this defensive attitude of, for example, Armstrong, see Steams 1956/1977:317- 320). As this attitude was no longer adequate among the young black artists in the 1940 's, they adapted a coo 1 stance instead: a protective wall against racial harassment, personified not least by Miles Davies and the attitude he came to represent musically and culturally. I am not quite convinced by the analogy and metaphor of the opened windows. Rather, I am inclined to look for an explanation of the origin of the word and attitude to cultural phenomena such as drugs and the use of drugs. The pre-history of the concept of "cool" This is supported by the fact that the jazz scene in, for example, New York during the 1930's, displayed both a number of drugs and a number of metaphorical expressions poetically renaming the drugs in order to keep the white audience, including the police authorities, ignorant and outside of the entire phenomenon and culture. In his book on the American swing era in the 1930's (The Swing Era, 1989:332f.), jazz historian Gunther Schuller presents a thorough description of the armoury of slang expressions relating to drugs. Here, we should perhaps rather talk about argot instead of slang, which is to say, a language used by a specific subculture, in this case jazz culture. Words and expressions like "viper" or "kickin' the gong around" referred to marijuana or smoking opium. A secret language was created, which also left its mark in the titles of many of the popular jazz songs of the time - for example, ''I'se a Muggin', You'se a Viper", "Viper's Drag", "Minnie the Moocher", "Kickin' the Gong around", "Texas Tea Party", "Chant of the Weed", etc. - and in a number of song texts. Marijuana was the dominant drug on the jazz scene of the Characteristics of coolness George Elliot Clarke lists a number of typical characteristics of coolness in black America: (. . .) coolness involves a willingness to engage in violence(. .. ), to risk death ( .. .), to suppress emotion (in interactions with friends, family members, lovers, spouses, and children), to value spontaneity, expressiveness, and stylishness(. .. ), and to prize verbal dexterity (. . .). These qualities of cool render it an essential survival mechanism in a society in which 'except for people over age eighty-five, black males are dying at a higher rate than any other group at any age ' (. . .). Given this vicious context, any moral code that signals meaning, community, and purposefulness, that is to say, that combats anomie, is potentially irresistible. Coolness is one such code. (Clarke 1998) 87 could be called the new, cool heroes of film, such as Marlon Brando, James Dean, Paul Newman and, to a somewhat lesser degree, also heroines such as Eve Marie Saint. Method acting has been described as a method where the actors try to base their acting on personal and emotional memories and use improvisation and spontaneity to develop the character of their role (for a more detailed description of method acting, see e.g. an online interview with Richard Herskowitz 1998: Cool. The 1 P" Annual Virginia Film Festival). The question of a possible connection between coolness in black America and coolness in West Africa can hardly be answered adequately, based on the short descriptions above. The brief material points in different directions. There are both clear similarities and considerable differences between the ways in which the concepts are used in their respective contexts. Anthropologist Mary Douglas has taught us that something socially or geographically marginal, as for example the predominantly black, hip New York jazz scene in the 1940 's ( which also gave rise to the bebop style), symbolically speaking often might seem very central to its surrounding society. This is also the case with the black musicians who attracted admiration when playing in various joints and clubs on 52nd Street in New York, but could get beaten up by brutal policemen as soon as they came out into the street. The unknown, the marginal, the Other becomes something attractive, exciting and desirable at a symbolic level, through cultural transformation. Characteristics of coolness Alongside film, parts of American contemporary literature adapted an attitude characterised by coolness, particularly so in works by the beat writers Jack Kerouac, Allen Ginsberg, and also LeRoi Jones. The link between cool jazz and the beat writers is very obvious and well documented. The beat writers borrowed a lot of expressions from the 1940 's jazz/hipster slang, such as square, cats, blow your top, dig. And, of course, cool. But the influence from jazz was deeper than just words: the beat writers attempted to transfer the central ideas of the bebop aesthetics and ways of playing to literature. This pertains to both prose and poetry; for example, Jack Kerouac's beat poetry has been described as bop prosody ( see Jones 1963 :23 7). The beat writers, like the black musicians and the radical theatre people, came to stand for a mental revolution against the established American society, particularly perhaps against the backdrop of the experiences from the Korean War at the beginning of the 1950's. LeRoi Jones says he is "almost certain that the 50's got its own special ominous character by the sinister influence from the Korean War and the emotional chaos following it" (Ibid. :219). As LeRoi Jones writes in Blues People (1963:234-238), quite frequent crossings of the so called racial boarder also took place in the 1940' s New York artist and jazz circles. The black and white markings of American racism were no longer valid; the watertight bulkhead of the racist politics here leaked like a sieve. As a consequence, the black musicians' cool, emotionally seemingly indifferent attitude became adaptable also for others. This pertained, for example, to the New York based theatre school the Actor's Studio. When Miles Davis' album, Birth of Cool was released in 1957, the culture of the cool had already begun to penetrate American society to an increasing extent. The Actor's Studio was headed by Stanislavsky's former student Lee Strasberg and, using the so called method acting, the school trained what The step from Miles Davies, Lee Strasberg and Jack Kerouac, who can well be called examples of coolness within an artistic highbrow culture, to popular culture is very short. Just think of the classic 88 composer Leonard Bernstein's American version of Romeo and Juliet, West Side Story, which first opened in New York in 1957 and was filmed in 1961. Characteristics of coolness West Side Story is a love story set against the background of gang wars in New York between the white Jets and the Puerto Rican Sharks. We all remember the chorus in "The Jets' Song": "Just play it cool, boys!". Here, we meet not only Marlon Brando and James Dean as angry young men, but also, and above all, Elvis Presley as representing a new social stereotype, the criminal teenager, the juvenile delinquent, or, to put it more nicely: "the rebel without a cause", to quote the famous 1955 film in which James Dean starred. Suddenly, the concept of cool was present nearly everywhere in American society. And one who was cooler than most was Elvis Presley. of the brightest students in the school system. This group publicly adapted adult values inAmericaofthe 1950's. However, the general cultural strategy of the group also included a manipulative ability to use breaches against these values for personal purposes. As Erling Bjurstrom points out (1997:44f.), Coleman's view of the American youth in the 1950's is based on two primary premises, or figures of thought. One figure of thought pertains to the gap between the adult and the adolescent world; the other, which is connected with the first, to the world of youth being a deviation from the world of adulthood. Bjurstrom draws the conclusion that both premises are static, because the development, values and norms of the adult world are never problematised but are regarded historically. One figure of thought that Bjurstrom does not explicitly mention, but which also seems central and which I will discuss in more detail further below, is that the strongly male dominated discourse of the music form of rock'n'roll has, from the very beginning, been a central expression for this teenage culture. 1 The "dangerous" youth in the USA of the 1950's At the same time, rock'n'roll could also find its most important symbolic frame of reference, its own symbolism, in this form of teenage culture with its (real or assumed) background in perceptions of juvenile delinquents, dangerous motorcycle gangs and hooligans. won nationwide popularity. The novel Rebel without a Cause was published in 1944, but became widely read and admired only about a decade later, in 1955, after the book was filmed with young film stars James Dean and Natalie Wood in the leading roles. Another film that mirrored many of the perceptions of the dangerous and subversive teenage and adolescent world of the time was The Wild One (1954) with its threateningly rebellious lead role played by Marlon Brando, donning black leather, as the leader of a motorcycle gang. The film Blackboard Jungle (1955), with Bill Haley's "Rock around the Clock" playing as background music to the credits in the beginning, also presented an image of adolescents as dangerous. The portrayal of young people as a socially and culturally dangerous category was emphasized in this film by the teachers appearing as an example of adults totally lacking in authority, terrorised by violent and criminal gangs of adolescents. In this case, the habit of regarding adolescents in negatively defined terms emanates from an American petty bourgeois of middle class horizon of understanding. This categorising of young people as potentially dangerous, and thus as objects for regulation by schools, teachers, supervisors, psychologists and criminologists, culminated in the 50's in a number of debates and contributions to the discussion on the dangerous teenager, the adolescent, the juvenile delinquent. g g Adolescence thus became a legal and social status that had to be disciplined, protected and accorded its own institutions. Young people represented a danger both to themselves and to society. At a symbolic level, this discourse of adolescence came to function in two totally contradictory ways, depending on the position from which it was viewed. For those who embraced the dangerousness in the discourse - either in its fictive form, as in Elvis and rock'n'roll, or in a mimetic- realistic form as in the criminal youth gangs - the danger turned into a positive value, something to identify with and obtain strength from. The "dangerous" youth in the USA of the 1950's I the USA, the 1950's can be characterised as the era of the teenager. The difference compared to earlier periods pertained, for example, to the number of teenagers, their higher level of financial and other resources, and their group awareness. p g The rough crowd consisted of a number of teenagers in different kinds of distressed situation. The group was replenished with other marginal groups, which, in the American 1950's, when teenagers and adolescents became known as being dangerous and unlawful, were defined within the frames of something called "subterranean traditions" in an oppositional triad of delinquency, radicalism and bohemianism -that is to say, the things that make up the roots of the different sub and anti cultures of the 1960 's within the area of youth culture. Another sociologist, Philip H. Ennis (1992:24 7), has noted that it is as front figure for, above all, the sociologically regarded "lowest" group, that is, the rough crowd, of high school students that Elvis and rock'n'roll offered a solution to many of the teenagers' problems. While the adolescents could still "love their mother According to James S. Coleman ( 1961 :205f. ), sociologist of adolescence, the American teenage world of the 1950's consisted of three relatively clearly distinguishable groups of adolescents. The connecting feature of the three groups was that their natural environment, above all, was high school, the drug store, the car and, to a certain extent, also the family. Coleman divides his teenage world, which was later portrayed in fictional form in the TV series Happy Days and romanticised and idealised in teenage films such as American Graffiti, Back to the Future, Peggy Sue Got Married and Pleasantville, into three groups; "the leading crowd", "the rough crowd" and "the middle majority". The leading crowd of the teenage world and in high school consisted 89 and God", they could also hunger for the wild promises of love and sex as conveyed by Elvis androck'n'roll. Ennis furthernotes that various artefacts with a strong symbolic significance - such as the black leather jacket, pomaded hair, the cool gaze, but also, and above all, a general resistance to middle class values - found a home in rock'n'roll. The "dangerous" youth in the USA of the 1950's j q The cultural researcher Luisa Passerini ( 1997: 318) has shown that the construction of the dangerous juvenile delinquent reached a clear peak in time at the end of the 1950's, that is, just after Elvis and rock'n'roll entered the scene. However, the roots of the dangerousness constructed as a double or combined image of the parental generation's fears and worries and of the cultural industry's codification of a dangerous adolescent behaviour, existed already in the USA of the 40's. Important books that crystallized the dangerous, subversive adolescent were beat writer Jack Kerouac' s novel On the Road and Robert Lindner's novel Rebel without a Cause. Kerouac wrote his novel as early as 1941, then with the title The Beat Generation, but the book was refused publication and came out only in 1956, the same year as Elvis On the other hand, in the eyes and minds of the "nice" adolescents - those listening to Pat Boone and Connie Francis and not to Elvis - and the well-adapted parents, concepts such as juvenile delinquency and rock'n'roll represented a dangerous Other, 90 an unknown that had to be rejected. For these latter groups, rock and adolescence thus appeared as symbolically charged issues that they strived to discard using all possible means. However, as cultural researchers Peter Stallybrass and Allon White (1986:193) have shown, this dangerous, so- called 'first grotesque' will, on a subconscious level and through what they call the political unconscious, invade the identity formations that have taken place on a conscious and political level. Thus, the second grotesque is born, of which the dangerous youth culture, rock and Elvis can be interpreted as clear examples. thus come to function within their society as experimental laboratories; pools through which various cultural influences have flowed, and giving rise to new hybrid genes which have spread and also become popular outside their original environment. A similar proletarian and marginal feature is also implicated in the birth of rock'n'roll. Or, as Judd Phillips ( older brother of Sam Phillips, the man who recorded Elvis at Sun Records) states: "It was the poor white seeking the soul expression. Not the uptown white, but the poor white" (Bane 1982/92: 125). The "dangerous" youth in the USA of the 1950's p Something of the hesitation between an insider and an outsider perspective which seems to characterise these marginal groups is also, in my opinion, included in the adolescents' use of the concept of cool both as a style marker and a symbolic sign. According to Danesi, the central elements of coolness have not changed significantly since the 1950's. Thus coolness can still, as in the 50's, be signalled by activities such as smoking, drinking alcohol, using drugs, slow movements, the art of wearing "the right" clothes, listening to "the right" music and adapting "the right" postures; that is, signalling what is regarded as right and important within the group one belongs to (Danesi 1994:40, 60-67). The basic assumptions in Danesi' s definition of coolness as an adolescent attribute is, on the one hand, that it is localised to the sub-group, the gang, the "tribe", or sub- culture that the adolescents belong to, and, on the other, that it is gender specific, that is, the expressions of coolness among boys and among girls are clearly different from each other. As Passerini notes, terms such as "tribe" or "sub-culture" are taken from ethnographic studies of persons or peoples who were "different", people who did not share a number of those perceptions that were central for a Westerner (Passerini 1997 :320). The Coolness as a mask - Elvis in action As I have said above, in his study of coolness as the central significant sign for youth in, for example, the American 1950's, Danesi notes that the concept of cool originates in the jazz scene of the 1930's. Accordingly, I find that there is good reason to suggest that coolness, as a quality in youth, is related to the entire set of unique types of cultural formation, which, beginning in 19th century Europe and America, has had a very specific and powerful effect on the development of popular culture. As the anthropologist Victor Turner (1974:233, 252-259) and musicologist Peter Manuel ( 1988: 18f, 60f, 77, 107, 127) have shown, the development of genres of popular music is connected to phenomena such as marginalisation and liminality. Turner describes those marginal groups which have a significant function in forming culture as being part of a petty proletariat. Important historical musical genres within popular culture, such as fado, blues, ragtime, jazz, tango, Paris chanson, flamenco and rebetika, have been developed by forms of anomalistic, marginal grouping, often situated on the outskirts of big cities. These groups have 91 in the audience because the girls took a shine to him and the guys were getting a little jealous". Nonetheless, while pulling out all the stops and unfettered by slickness, the lurid Presley's instinctive control kept the mob just short of open riot - though females continued to shriek and faint in defiance of their boyfriends' sporadic heckling. By the close, all had tuned into the situation's epic vulgarity. (Clayson 1989:21) concept of coolness thus functions as an attribute linked both to liminality and to reflexivity; two phenomena that, according to folklorist Barbara Babcock, are mutually dependent, in that the one presupposes the other (Babcock 1980:5). As an integration of unknown and dangerous symbolic material, transported from one discursive domain to another, coolness constitutes a root metaphor of the type that Stallybrass and White call a secondary grotesque (Stallybrass & White 1986: 193f.). The vulgar, but also obviously irresistible creature that Elvis, according to Roy Orbison, constituted became, as youth researcher James Coleman notes, the favourite singer of that very rough crowd described above. In that group, practically everybody smoked and drank. However, their favourite singer neither smoked nor drank. The logic of the Elvis mask: vulgarity, rebellion, sexuality Elvis's first rock'n'roll mask - that of the 1950's - appears, compared with, for example, his Las Vegas mask of the 1970's, as a mainly realistic one, with its origins in forms of low and popular culture. Its models, particularly pertaining to clothes and choice of colour (predominantly dark and bright colours such as black and pink), are the black hipster and/or pimp. Other background figures for his early rock'n'roll mask are the young rebel and the truck driver. The mask also implies a way of moving (hip, leg and arm movements) that originates in black culture (blues, gospel). These various cultural signs in Elvis's rock'n'roll mask can be said to comprise the stereotypical rock'n'roll rebel; a figure with pronounced low cultural, rebellious, "bodily" roots, and a person who constitutes a threat to the puritan and middle-class view of life and its cultural ideals during the 1950's. Key words that emerge from these short descriptions of Elvis as a symbol for youth and teenagers are "vulgarity", "rebellion" and "sexuality". The texts also contain contradictions or oxymorons such as "He told jokes that weren't funny", "the situa- tion's epic vulgarity" and "His rebellion was masked in southern manners". Thus, both Elvis and rock'n'roll stood, from the very beginning, for a transgression of various borders. Philip Ennis talks about the transformative symbolic process which Elvis and other rock artists started and maintained with a "capacity to transcend race, religion, gender or region in the name of music". The rebellious and challenging trace in Elvis's masks, in his public personae or the impersonations of a rockabilly and rock'n'roll star that he constructed (in 1954-58) is based on borrowing a number of disparate elements from both the white hillbilly and country cultures, from the black street culture, and black and white popular culture. This reflexivity is a strong feature in his early rock mask. Elvis absorbed influences like a sponge. His reflexivity thus also meant that he, consci- ously or unconsciously, placed himself in a liminal position. Even at this early stage he is already both a transgressor of borders and a border guard. While his fans see him as an emblem of opposition and renewal against their parental generation and authorities, his opponents - who form a very heterogeneous group consisting of the "nice" adolescents and above all of somewhat older Americans - regard him as dangerously subversive. Coolness as a mask - Elvis in action Dressed in black rock'n'rolljackets as a symbol of an orientation towards entertainment, cars, music and roller-skates, this group had chosen Elvis as their favourite singer because of the implicit deviance and rebelliousness of Presley (Coleman 1961:205f.). In order to understand what the teenage idol and rock'n'roll star Elvis represented in terms of a concept such as coolness, the public image, the dramatic mask that Elvis constructed must be deconstructed. This mask is laden with many semantic and symbolic significances. A representative image of the various transgressions that the Elvis mask stood for in the USA of the 50's is provided by Elvis's rockabilly colleague Roy Orbison in his description of their first encounter when he was 19, at a country & western jamboree in Dallas on 16 April 1955. The description, included in a biography of Orbison, is a mixture of "direct quotes" of Orbison's feelings and a more reflexive meditation on what was special about the Elvis mask. It thus illustrates the complex web of significances attributed to incidents when they are recounted and, in a way, re-formed by the human imagination: Ennis points out that Elvis meant trouble from the very beginning, since he personified the contradictions "inherent" within the American family concerning the view on parental authority, the independence and sexuality of young people: His rebellion was masked in southern manners bordering on obsequiousness. Even the restraint religion imposed went awry in Elvis's case. His church training and gospel music experience did not prevent the incipient excesses of sexuality from living beside (being, in fact, augmented by) the religiosity that fired the most explosive emotional yearnings in southern Protestant life, white and black. Those themes of rebellion, religiosity, and ungovernable sexuality were emblazoned It was like nothing the bespectacled Wink Westerner [Roy Orbison} had ever experienced before. Presley, sneering, didn 't seem to care how badly he behaved up there - breaking guitar strings, spitting out his chewing gum, swivelling his hips in a rude way, doing the splits, knee-dropping and crawling to the very edge of the stage. He told jokes "that weren't funny, and his diction was real coarse like a truck driver's". Roy observed the "pandemonium 92 in all his performances and in his public presence. (Ennis 1992:244) dangerous one ... The gangster of tomorrow is the Elvis Presley type of today" (Passerini 1997:326). The logic of the Elvis mask: vulgarity, rebellion, sexuality A statement by a witness before the Senate's Subcommittee on Delinquency is very telling: "Elvis Presley is a symbol, of course" ... "but a The figure of Elvis becomes liminal and marginal in relation to the 50's general classification system of social control. This means that Elvis in his rock'n'roll appearance represents a threat against the existing order, a threat that somehow has to be integrated into the prevalent system. The rebel image is also clearly connected to Elvis's southern background. An important feature of Elvis's rebellion is that it is not focussed on any particular issue. Instead, this is "a rebel without a cause"; an angry, young rebel, aiming his opposition against the parental generation, just as Elvis's rock'n'roll persona does. However, the most dangerous and subversive feature of the mask and image that Elvis created in the 1950's was the fact that he combined elements from black and white cultures. Elvis was a prime example of what the New York-based author Norman Mailer called "the white Negro". 93 In 1957, the same year that Miles Davies released his record Birth of Cool, the same year that West Side Story opened and Elvis gained his international break-through, Mailer wrote an essay titled "The White Negro" in the left-wing journal Dissent (also published in Mailer's collection of essays Advertisement for Myself, 1961 ). In it, he defends the thesis that a new white social outlaw had been established in the American landscape, a hipster or a philosophical psychopath, whose primary inspiration consisted of the sexuality and music of Afro-Americans. "The hipster has absorbed the existentialist synapses of the Negro, and for all practical purposes could be considered a white Negro", Mailer wrote in his essay. The description could be one of Elvis Presley, but Mailer found his musical sources of inspiration and examples in jazz. culture ofrock'n'roll and the teenage rebel. The other is a phenomenon usually called morn-ism: a misogynist concept coined by the American writer and debater Philip Wylie in his pamphlet Generation of Vipers (1942/1955), which was widely discussed in the mass media at the time. Wylie's aim is to find a scapegoat for the supposed degeneration of American culture caused by materialism, superficial popular culture, soap operas, fashion, TV, radio, sentimental pop songs, Hollywood and department stores. The logic of the Elvis mask: vulgarity, rebellion, sexuality According to Wylie, all these phenomena are expressions of morn-ism, of a female or motherly sensitivity, which undermines the virile element in American culture. As Jacqueline Rose (1992:166ff) notes, the dangers of femininity and of mass culture have thus been linked very intimately together. This link was further developed within rock'n'roll culture where a gendered division between rock and pop was established according to the principles presented by Simon Reynolds and Joy Press in their study The Sex Revolts. Gender, Rebellion and Rock'n 'roll (1995:5): "Here the correct response (male connoisseurship, discerning and discriminating) is opposed to degraded feminine fan-worship (superficial, hysterical, idolatrous, at once fickle and blindly loyal)". Like the concept of cool, the hipster also originates in black jazz and black street culture; more precisely in the hip bebop- jazz scene of the 1940's. The hipster demonstrated, through a style of dress, gestures and a general cool attitude, that he was something very special: an urban young man who dressed fancily in order to impress people around him. In Elvis, teenage culture had found its hipster figure, a "hipster for the millions", as he has also been named. In the case of Elvis, his "rebel without a cause" iconography comes to appear as a restless, uncertain combination of, on the one hand, his way of imitating male black sexuality in "the white Negro" configuration (see above), and, on the other, an affirmation of his extreme dependence on his mother, Gladys (Klinkmann 1998: 121f). Albert Goldman points out that as a young boy, Elvis was constantly in the company of his mother and was as frequently encouraged to speak out and reveal all his thoughts and feelings to her. Goldman notes that Elvis became so accustomed to sharing his inner life with his mother, so dependent on her opinion and What are the features in these cool rebels - "rebels without a cause" as they may be called after the film discussed above - that is, what is it in the make up of an Elvis Presley, a James Dean, a Marlon Brando that makes them so central to the decidedly masculine iconography of the USA of the 1950's? In order to find an answer to this question, I think we again need to explore both the sociological and the psychological roots of the phenomenon at issue. The logic of the Elvis mask: vulgarity, rebellion, sexuality This shows that two important socio- psychological phenomena emerge at about the same time in post-war USA. One is the 94 of the Finland-Swedish journal Horisont (1992:66). advice, that he later did not hesitate to wake her up in the middle of the night if he had something on his mind and could not sleep. Thus there cannot, according to Goldman, be any doubt about the reason for the extreme reserve that characterised Elvis during all of his adult life. His utter reluctance to confide in anybody or reveal his inner thoughts can be explained by the fact that, in his mind, there was only one single person in the world that could understand and guide him (Goldman 1981:62). This can be compared to the way in which the black, masculine and cool mask or pose, according to sociologists Richard Majors and Janet Mancini-Billson, functioned. That is, as an adaptive mechanism for handling the reality of the existence of Afro-American men: " ... to enhance social competence, pride, dignity, self-esteem and respect. Cool enhances masculinity. Being cool also expresses bitterness, anger, and distrust towards an oppressive society for many years of hostile mistreatment and discrimination" (Majors & Mancini-Billson 1992:105). Cultural loans and social flex- ibility /inflexibility My point is that, although the masculinity created by the black jazz man at once challenged dominant white discourses of heterosexual 95 so complete that on stage he adopted symbolic fornication blacks had unashamedly brought to American entertainment. Elvis was sexy; not clean- cut, wholesome, white-bread, Hollywood sexy but sexy in the aggressive earthy manner associated by whites with black males. Jn fact, as a young man Presley came closer than any other rock & roll star to capturing the swaggering sexuality projected by so many R & B vocalists. (Ibid.:63) masculinity, with respect to women this same powerful and defiant black masculinity just as often maintained unequal relations of power between men and women.) masculinity, with respect to women this same powerful and defiant black masculinity just as often maintained unequal relations of power between men and women.) Against the backdrop of this complex black masculinity, this black bearing, as described by Gray, Elvis's coolness and masculinity expressed in his public persona of the 50's appears as something relatively playful and superficial. However, in his book Blues People, LeRoi Jones (197 4:226f) presents a surprisingly positive picture ofElvis androck'n'roll. He says that rhythm & blues, the contemporary expression of blues in urban environments, was the source of the new vitalisation of popular music, and that rock'n'roll was the end-product of this: "And it is, as every American 'average mother' 25 thinks, music for 'non-intellectuals'. But an Elvis Presley to me seems like something of greater cultural significance than a Jo Stafford" .26 Nelson George then contrasts this superficial blackness, this mimicry effect that Elvis could attain in his stage performance and his singing, with the singer's lack of real insight into black culture: - Elvis was just a package, a performer with limited musical ambition and no real dedication to the black style that made him so dangerous. Presley, as his life later revealed, never put the time into developing his interpretation of blackness - the most important part in his appeal - in the way Mick Jagger has, for example. (Ibid.) According to LeRoi Jones, rock'n'roll nevertheless mostly constitutes a blatant commercialisation of rhythm & blues. In many cases, however, the music is based on enough material which is so unfamiliar to the average middle class and middle intellectual American culture that it, according to Jones, becomes interesting. Cultural loans and social flex- ibility /inflexibility Blackface minstrel24 researcher Eric Lott links the old black face tradition with the later white representations of "blackness" which are also included in the expression "white nigger" and in the Elvis figure: "To put on the cultural forms of 'blackness' was to engage in a complex affair of manly mimicry" (Lott 1995:52). The "black" cultural element that Elvis "steals" in his persona of "white nigger" emphasizes (ultra) masculinity, adulthood and sexuality. However, Elvis's complex and ambiguous cultural representations also include clear androgynous features (Klinkmann 1998:131f, 334; Garber 1992:367). Thus, the masculinity of Elvis can be connected with the new type of male role also exemplified by film stars such as Marlon Brando, James Dean and Montgomery Clift. According to Fredrik Hertzberg, the common feature of these three film stars was that they revolted against conventional masculinity. The new male role was both more masculine and more feminine than before. It was more physical and less verbal than the role typified by predecessors such as Humphrey Bogart, James Cagney and John Wayne. It demonstrated a new way of being cool, but also openly exposed its vulnerability, writes Hertzberg in a special, body themed issue Herman Gray ( downloaded on 29 March 2001) notes that the image of the cool black jazz man was not lacking in contradictive features: As a "different" sign of the masculine he was policed as much as he was celebrated and exoticized by white men and women alike. Policed as a social threat because he transgressed the social role assigned to him by the dominant culture and celebrated as the "modern primitive" because he embodied and expressed a masculinity that explicitly rejected the reigning codes of propriety and place. Drugs, sexism, pleasure, excess, nihilism, defiance, pride, and the cool pose of disengagement were all a part of the style, personality, vision, and practice of an assertive heterosexual black masculinity that could not be confined within the dominant cultural logic. (The lives and careers of John Coltrane and Miles Davies illustrate the complex and wide-ranging relations of gender at play in the jazz world; Coltrane 's wife was a respected member of his band, while Davies often treated women with derision and abuse. Of course, Elvis 'reverse integration was Cultural loans and social flex- ibility /inflexibility 27 Nelson George calls Presley "a damn lazy student" and describes him as a mediocre performing artist who, during his entire career was transfixed in the unyielding grip of his manager Colonel Parker. If Elvis had kept closer to the blues style of his early Sun recordings, he would, according to George, been able to produce 20 years of music that would qualitatively corresponded to what George calls the myth about Elvis Presley. g g The rhythm & blues historian Nelson George (1988 :62f) says that Elvis's exposure to black culture, both blues and gospel, was as strong as it could ever be expected to be, when seen from his white Mississippi perspective. Elvis listened to the black radio stations WDIA in Memphis and WLAC in Nashville; his dress and hair style revealed an image with a clear background in black culture. George's conclusion of this cross-cultural collision is that Elvis adopted black styles from black people who hade adopted a whiter appearance. And George adds: The superficial and playful adaptation of the black man's bearing that Elvis, according to Nelson George, represents, might perhaps explain the socio-cultural mechanism as a basis for the famous rumour that spread in black cultural circles Of course, Elvis 'reverse integration was 96 possible to see the social need there might be today for such a rumour, which has resulted in several black cultural persons believing in it. Gilbert Rodman (1994:465f) quotes a writer, Dan Heilman31 , who points out that black cultural figures such as Vernon Reid (from the group Living Colour). Chuck D (Carlton Ridenhour) from the rap group Public Enemy and film director Spike Lee have all heard the rumour and believed it. and was obviously believed also by leading black cultural persons. Relatively early in Elvis's career, according to Peter Guralnick (1994:425f) already during the spring and summer of 1957, a rumour spreads in black housing areas in the USA claiming that Elvis had said: "The only thing Negros can do for me is to buy my records and shine my shoes."28 In my doctoral thesis on Elvis Presley as a king of carnival (Klinkmann 1998:284), I have, in accordance with the title, characterised the persona of Elvis as markedly camivalistic. Cultural loans and social flex- ibility /inflexibility In my view, Elvis comes very close to the Harlequin strategy essential to the Renaissance commedia dell 'arte theatre; a strategy that seeks to amuse in a chameleon-like way, incor- porating aesthetics that balance between the high and low, the serious and the humorous. Elvis constantly moves between the sublime and the grotesque, between the cultivated, which, with him is particularly expressed in a form of dandyism and a strong feature of"mummy's boy", and the burlesque, grotes-que, which, however, with Elvis is never directly transferred from black culture, but slightly changed, parodied, or, to use Elvis's own expression, "goosed up". Concluding remarks To summarize, it can be noted that in his public persona, Elvis appeared as a white Negro, but his commitment to black people and black culture do not actually seem to have been more than superficial in nature. While in his persona he included important elements that at this time by many whites were seen as a black, animalistic, strongly sexual image, he continued, as the person Elvis Presley, in all essential aspects, to defer to the culture that he came from, a white southern culture of a lower class character. In this culture, in the kind of racially segregated society that Elvis grew up in, poor white people were a "caste" superior to black people, but also in a situation where the "racial sexualization" of society made relations between white men and black women, and between black men and white women threatening, forbidden and dangerous. Taking all of this into account, it is perhaps somewhat easier to understand why a rumour such as the one described above, claiming that Elvis would value black people only for their ability to buy his records and clean his shoes (that is, to be subservient in all to him, the white master) has started.29 The thought that Elvis had stolen the music of black people and gained fame using feathers he had borrowed from them, or that he, to use the white rock reviewer Greil Marcus' s words about the same phenomenon, like a present-day American Prometheus had stolen the black musical fire 30 , makes it slightly easier to discern certain possible background motives for this bizarre rumour. It is also Despite Elvis's proven lack of racial prejudices (Klinkmann 1998:280-284), the impression remains that the rock artist and entertainer Elvis, notwithstanding the black elements in his persona, consciously chose to maintain a certain distance from black culture and black Americans. Thus he appears as a carnivalistic figure, a Harlequin or trickster figure, who can wear black features - and particularly the cool and masculine ones - as a mask, but who avoids making this black bearing a more integral feature of his personality. Concluding remarks Compared to the 97 grotesque and for black people, strongly degrading, parody of black culture that the blackface comedians performed, Elvis's mask of the 50's (a mask with certain selected black meanings, connotations) appears as a slightly ironic paraphrase of black culture, performed with an entertaining, playful twinkle in his eyes. Thus also the dangerous and subversive element in Elvis's representation of coolness and masculinity is disarmed or neutralised fairly quickly. This happened gradually with his entry into military service; the establishment of Elvis as a film entertainer and, finally, with his Las Vegas persona, in which the cool and masculine elements of Elvis as a "white Negro", a "rebel without a cause" were almost turned to their opposites through the increasingly grotesque and self- parodying aspects of the representation. as famous as Elvis. 5 Thus, Jones' s arguments can be interpreted as a kind of confirmation of Norman Mailer's reasoning about the concept white Negro. 6 Guralnick says that trying to trace a source for this rumour is "like running a gopher to earth". Elvis is supposed to have said this in Boston, a place he had never visited at this stage, or in Edward R. Murrow's nationwide TV show on which he had never appeared (Guralnick 1994:426). 7 The untrustworthy nature of the rumour is revealed by the fact that each time it emerges, it seems to have taken on a new, slightly altered form. The following are some examples of the wordings of the rumour, taken from Rodman (1996:34ff, 43, 54, 56): "All I want from blacks is for them to buy my records and shine my shoes" (source V. S. Naipaul), "The only thing niggers are good for is to shine my shoes" ( source Greil Marcus), "The only thing niggers can do for me is shine my shoes and buy my records" ( source N. M. Zuberi), "The only thing they [blacks] can do is shine my shoes and buy my records (source Eddie Murphy), "The only thing a nigger is good for is to shine my shoes" (source Dave Marsh). Rodman summerizes the collection of quotes by saying "While the statement's general theme of contemptuous prejudice is consistent from one version to the next, the precise words Elvis reportedly said vary an extraordinary amount for a statement that's 'on the record'." This, obviously is a folkloristic legend. NOTES 1 For a discussion on the one-sided gendered construction of rock discourse, se also, for example, Lahteenmaa (1988/1989, 1989) and Swiss, Herman & Sloop (eds.) (1997). 2 Blackface, or blackface comedy, was a sub- genre within the popular American type of stage entertainment, called the minstrel show, where to begin with white and later also black artists imitated black people by painting their faces black with charred cork . The genre was born within English music hall entertainment and artists such as Charles Dibdin and Charles Mathews, but was soon exported to the USA, where it became popular with, among others, George Washington Dixon and Thomas "Daddy" Rice. 2 Blackface, or blackface comedy, was a sub- genre within the popular American type of stage entertainment, called the minstrel show, where to begin with white and later also black artists imitated black people by painting their faces black with charred cork . The genre was born within English music hall entertainment and artists such as Charles Dibdin and Charles Mathews, but was soon exported to the USA, where it became popular with, among others, George Washington Dixon and Thomas "Daddy" Rice. 8 See Marcus's essay "Elvis: A Presliad" (Marcus 1975/90). 9 Heilman's text "Trying to get to you: Greil Marcus chases the ghost of Elvis Presley", is published in Rock & Rol!Disc, March 1992:9ff. 3 Note the clear reference to morn-ism in LeRoi Jones' s wording. REFERENCES 4 Note, however, the clear male chauvinist feature in this comparison: Jo Stafford was a contemporary female white jazz singer. Comparing Elvis to her appears to be unfair, not least considering that Jo Stafford was not nearly REFERENCES Literature Babcock, Barbara A. 'Reflexivity: Definitions and Discriminations.' Semiotica 30-1/2 1980, 1- 14. Literature Babcock, Barbara A. 'Reflexivity: Definitions and Discriminations.' 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Schuller, Gunther. The Swing Era. The Devel- opment of Jazz 1930-1945. New York and Ox- ford: Oxford University Press, 1989. Goldman, Albert. Elvis. Stockholm: N orstedts, 1981. Guralnick, Peter. REFERENCES Last Train to Memphis. The Rise of Elvis Presley. London: Abacus, 1994. Stallybrass, Peter and Allon White. The Poli- tics and Poetics of Transgression. Ithaca, New York: Cornell University Press, 1986. Hertzberg, Fredrik. Kroppens artikulation. Orn Marlon Brando. Horisont No 5, 1992. Stearns, Marshall W. The Story of Jazz. Lon- don: Oxford University Press, 1956/1977. Jones, LeRoi. Blues People. Stockholm: Raben & Sjogren, 1963. Swiss, Thomas, Andrew Herman and John M. Sloop. Mapping the Beat. London: Blackwell, 1997. Klinkmann, Sven-Erik. Elvis Presley - den karnivalistiske kungen. Abo: Abo Akademis Forlag, 1998. Thompson, Robert Farris. 'An Aesthetic of the Cool'. African Arts, Vol 7, No 1, 1973. Lott, Eric. Love & Theft. Blacliface Minstrelsy and the American Working Class. New York and Lott, Eric. Love & Theft. Blacliface Minstrelsy and the American Working Class. New York and Oxford: Oxford University Press, 1993. Turner, Victor. Dramas, Fields, and Meta- phors. Symbolic Action in Human Society. Ithaca and London: Cornell University Press, 1974. Wylie, Philip. Generation of Vipers. New York: Pocket Books, 1942/1955. Oxford: Oxford University Press, 1993. Lahteenmaa, Jaana (ed.). Rockin seksuaalisuus. Helsinki: Gaudeamus, 1989. Lahteenmaa, Jaana. Tytot Ja rock. Helsinki: Kansalaisvalistuksen keskus, 1988/89. Electronic references Bartleby's Online Dictionary http://www.bartleby.com/61/17/C0621700.html Downloaded 29 March 2001 Britannica.corn http://www.britannica.com/eb/artic1e?eu=3659 Electronic references Bartleby's Online Dictionary http://www.bartleby.com/61/17/C0621700.html Downloaded 29 March 2001 Britannica.corn http://www.britannica.com/eb/artic1e?eu=3659 Electronic references Marcus, Greil. Mystery Train. Images of Ameri- can in Rock'n 'Roll Music. London: Omnibus Press, 1975/1990. Majors, Richard and Janet Mancini-Billson. Cool Pose. New York: Touchstone, 1992. Cool Pose. New York: Touchstone, 1992. Britannica.corn Mailer, Norman. Advertisement for Myself. 99 Downloaded 29 March 2001 Clarke, George Elliot 1998: Cool Politics http://152.1.96/jouvert/v2i 1/Clarke.htm Downloaded 29 March 2001 Cool. The 11th Annual Virginia Film Festival http://www.therealestateweekly.com/ Archives/ Edit%20Archives/FilmFestivalCool.html Downloaded 29 March 2001 Gray, Herman: Black masculinity and visual culture. Cultural Politics. Three Essays from the Whitney Museum of American Art's "Black Male" Show. http://jhupress.jhu.edu/quick tour/ 18 .2 gray.h tml Downloaded 29 March 2001 Herskowitz, Richard 1998: Cassavetes' Dance. Virginia Film Festival 1998. http://www.vafilm.com/1998/cool essay.html Downloaded 29 March 2001 Cultural Politics. Three Essays from the Whitney Museum of American Art's "Black Male" Show. http://jhupress.jhu.edu/quick tour/ 18 .2 gray.h tml Downloaded 29 March 2001 Herskowitz, Richard 1998: Cassavetes' Dance. Virginia Film Festival 1998. http://www.vafilm.com/1998/cool essay.html Downloaded 29 March 2001 Downloaded 29 March 2001 Clarke, George Elliot 1998: Cool Politics http://152.1.96/jouvert/v2i 1/Clarke.htm Downloaded 29 March 2001 Cool. The 11th Annual Virginia Film Festival http://www.therealestateweekly.com/ Archives/ Edit%20Archives/FilmFestivalCool.html Downloaded 29 March 2001 Gray, Herman: Black masculinity and visual culture. Herskowitz, Richard 1998: Cassavetes' Dance. Virginia Film Festival 1998. http://www.vafilm.com/1998/cool essay.html Downloaded 29 March 2001 100 100 NICKLAS HAGEN Narrative analysis off ootball reports in a Finland-Swedish daily newspaper In the summer of 2000, I began my career as a freelance sports reporter for the regional newspaper Vasabladet. I was to report the local Second Division matches. It was the first time I had written anything that was to be published, and being nervous, I asked advice from more experienced sport reporters, i.e. my father. The advice was as follows: "Remember that the text should be easy to read, and write about the things your readers are interested in!" I wasn't told exactly how that was to come about, but in order to find out what the readers might be interested in, I flicked through some match reports that had been published earlier in the newspaper. Having read football reports before, I was familiar with the style and terminology involved, with words like "canons" and "last ditch tackle". Reading these texts keeping the question "how" in mind, I recognised certain tendencies I had not previously been aware of. Most striking were the similarities in form and content, which had gone unnoticed earlier. The same basic pattern in nearly all: first, the result, followed by the match details, and finally interviews and future prospects - sometimes overlapping, but always presented in this order. In the summer of 2000, I began my career as a freelance sports reporter for the regional newspaper Vasabladet. I was to report the local Second Division matches. It was the first time I had written anything that was to be published, and being nervous, I asked advice from more experienced sport reporters, i.e. my father. The advice was as follows: "Remember that the text should be easy to read, and write about the things your readers are interested in!" I wasn't told exactly how that was to come about, but in order to find out what the readers might be interested in, I flicked through some match reports that had been published earlier in the newspaper. Having read football reports before, I was familiar with the style and terminology involved, with words like "canons" and "last ditch tackle". Reading these texts keeping the question "how" in mind, I recognised certain tendencies I had not previously been aware of. Most striking were the similarities in form and content, which had gone unnoticed earlier. Narrative analysis Discussion on the definition of a narrative is quite extensive. Arthur Asa Berger, for example, defines a narrative as something that has happened or is happening, and it consists of a sequence of events in a certain place, in a certain space of time. Viveka Adelsward in tum gives a more limited definition of the concept; narratives are verbally narrated events that have taken place, and she describes this process: "time passes, something takes place and the event is evaluated or assessed" (Adelsward 1996: 31 ). She continues by saying that a narrative is different from a report because a narrative gives evaluative information, whereas a report is more like a list in its nature. The evaluative characteristic of a narrative is called the narrative point (ibid: 38). Adelsward refers to three different points in a narrative: "anecdotal point", "underlying point" and "the narrative point". "The anecdotal point" can be found in an amusing story - what the narrator considers the listener may find amusing, unusual or exciting. "The underlying points" are more subconscious, what the narrative deals with on a deeper level: the value systems and themes the narrative expresses. "The narrative point" made by the narrative is in a way "an underlying point". A narrative can be seen as self- presentation, and "the narrative point" refers to how the narrator presents himself or herself with the help of the narrative. (ibid: pp. 46-) The tree articles selected were the fist and last written that season, with one from the middle of the season as Jaro secured its second place in the league. The articles were published on the following dates: 30.4.2000, 8.8.2000 and 29.10.2000. These were conscious selections. In choosing the first article I wanted to include the expectations for the team at the beginning of the season, and the last to show how the team managed to live up to the expectations. Furthermore, these were the extremities of the football season. The mid-season article serves as a bridge between the other two articles. As Jaro had secured the second place, they focussed on to the playoffs. This is emphasized by the fact that the last league match is described as "uninteresting" as the result made no difference to promotion (Sundqvist 2000). NICKLAS HAGEN Narrative analysis off ootball reports in a Finland-Swedish daily newspaper The same basic pattern in nearly all: first, the result, followed by the match details, and finally interviews and future prospects - sometimes overlapping, but always presented in this order. the reports is not as hard and fast as I had thought, but this observation did awaken an interest in the form and content of football reports. The aim of this article is to study the structure of football reports, and by concentrating on the main points of the report, create an understanding of football, by which I refer to what makes football newsworthy; why is football written about in the daily papers? Analysing the structure of these newspaper articles, I indirectly regard the newspaper's role as a narrator. As a method for examining the structure of the articles, I decided upon a narrative analysis of my material. This is done mainly by making a thorough qualitative analysis of three match reports on FC Jaro, published in the local paper Jakobstads Tidning (later referred to as JT) during the summer 2000 football season. Furthermore, I have taken into consideration 24 articles about FC Jaro published during the season in the same newspaper, as background without conducting a thorough analysis of them. I was looking for a team with a strong local connection, with a feeling of the team being "our team" in the reports, which is why I chose JT and FC Jaro. I wanted to analyse a Finland-Swedish newspaper and being born and bred in V asa, the local team and This observation was - hardly surprisingly - a coarse simplification. The structure of 101 football season, and receive their main meaning through that. articles about them in the local paper would have been a natural choice. The only problem being that they were mainly written by a person with the same surname as the author of this article, which would have caused problems with analytical distancing. This is why I chose a different team and a different newspaper, FC Jaro and JT. I wanted to analyse all 27 articles published during the season, but in order to limit the amount of data, I decided to concentrate on three and to use the rest as background. In this study, a match report refers to an article about a match that has already been played. These are more than just summaries or reports of a game of football. NICKLAS HAGEN Narrative analysis off ootball reports in a Finland-Swedish daily newspaper The 27 articles were written by seven different journalists, only one of them female. The three main articles were written by two males. I do not think this selection of data in any way distorts any point made in the analysis. A word or a sentence does not gain in value depending on the number of times it is repeated in various texts, but according to the context in which it is used. 1 Narrative analysis The three main articles in this study are special in that they reflect more directly the team's success during the Adelsward's description of "the points" is useful, but I cannot agree with her definition of a narrative. She seems to view narratives as something linked to the past, which I cannot agree with. For the purposes of this study I use Marander-Eklund's less limited view of narratives as "narrated events, that are somehow interrelated, with 102 main headline. These are not necessary, but they are very common. The result is given before the preface in boldface type, and its aim in principle is to describe what the article is about. Between the preface and the text proper, we see the name of the writer. Alongside the text, there will often be some photographs of the match, or archive photos of a player. At times there is a table with points awarded for individual players' performance. cause and effect that are assessed or evaluated, and make a point, also including self-presentation" (Marander-Eklund 2000: 33). ) Narrative analysis is a theory that is suitable as a method of analysis. According to William Labov, a complete narrative consists of a basic structure that includes six elements I shall use in this study. These are: abstract, orientation, complicating action, evaluation, result or resolution and coda. Narratives often include an abstract where the narrator signposts what is to come to the listener. This is followed by the orientation with a presentation of the actors, the time and place, as well as a description of any other significant circumstances. After the introduction follows the core of the narrative, as the narrator describes the action, the problem, the changes taking place and how the problem is solved. The body of the narrative is where the evaluation takes place, i.e. this is the point of the narrative. The coda is where the narrative is summarized and the narration is brought to its end (Labov 1972: pp. 363-). Each report describes a particular match. Naturally, it is not enough for a local paper like JT to merely publish the score of a match involving FC Jara. The newspaper does not report the match, but gives a kind of a narrative about it. The only First Division team the paper reports is FC Jara. Narrative analysis In my view, this is due to the fact that the team and the paper have the same "home town", and at least some of the readers ( and why not also the editorial staff) feel for the team and football. There must be someone who has some interest in the reports in order for them to be published in the newspaper. The fact that it is "nice", or" good" that J aro scores a lot of goals (Lindholm 2000) and that "unfortunately, the edge was lacking in the finishing" (Furu 2000a), indicates sympathy for FC Jara - it is our team. The current material is analysed by trying to discern the six elements presented by Labov in the three main articles. As I want to analyse the structure of the articles, the method also becomes an aim in this study. Labov's structural elements offer a tool to break down the articles into sections and to analyse them. I begin by giving an overview of the features the articles share, as a basis for my analysis. I then move on to analyse the three main articles in more detail by using Labov's theories on narrative structure. The texts are quite similar in content. The results, the team (actors), and various indications of time and place are used consistently. "Expert commentary" may be given in separate interviews, or at times included in the report itself. Various aspects relating to the game or specific incidents are taken up, e.g. the condition of the pitch, weather conditions, decisions made by the referees, substitutions and goals. The main thing is that they build tension in the narrative, or that they may have influenced the final result of the game. One and the same journalist is not limited to a specific form, there is variation between texts and between journalists' style. The journalist's writing style has not The introduction of the narra- tive For the purposes of this study, I shall begin my analysis in accordance with Labov's structural elements. The introduction consists of an abstract and an orientation. The football reports begin with a clearly marked abstract: the headlines. They appear first, and indicate what comes next in the text. They often include background information and if strictly based on Labov' s structural elements should be viewed as 'orientation'. The headlines do not capture the reader's interest through classic narrative means, for example by saying "it was extraordinary", but instead by exposing the contents of the narratives. For example, the headline in the final article "Defeat for Hango - no promotion for FC J aro" (Furu 2000b) contains information about the place (Hango), the actor (FC Jaro ), and the result (defeat), as well as the consequences (no promotion). While containing the information, the headline signposts the contents of the article and it functions both as abstract and orientation. Thus the headline awakens the reader's attention, giving an opportunity for disinterested readers to ignore the rest of the article. According to Umberto Eco, the writer aims at creating a model reader - who interprets the text in the way the writer wishes. In reality, the reader aims at creating a readable, sensible text by interpreting it from his/her own premises (Eco 1992b: 64). Bearing in mind that the reader's choice to either read or not read the article is based on the headline, one can argue that the model reader of the football reports has at least some previous interest in football. been the focus of my research. Interestingly enough, all articles except one include expert commentary, which is cause enough to draw the conclusion that they are of some importance. These commentaries have two main functions: they strengthen credibility by expert evidence, partly to legitimize emotional reactions or moods. In case the author's voice becomes too conspicuous, if the author's person or feelings are too explicit, the assumed objectivity of the narration is at risk. Objectivity and expertise are "narrative points" in journalism in so far as the author never mentions his or her own person in the narrative, and moods are reflected through other actors. Their presence can also be seen as a "narrative point" - the text is supposed to give evidence of the writer's presence, while, paradoxically remaining invisible. Features the football reports have in common The formats of the narratives have some shared features. The headline comes first, and is printed in the largest font type. This is followed by subheadings that support the 103 The football sea- son's first game is described as a "Football party", which express positive assosiations and expectations. The game did not, however, end so happily for FF Jaro, who did not make it to the league football. JT 30.4.2000 The football sea- son's first game is described as a "Football party", which express positive assosiations and expectations. The game did not, however, end so happily for FF Jaro, who did not make it to the league football. JT 30.4.2000 trainers are silent experts, they are the ones that create the game and are the game. By adding commentaries by them, the writer adds "presence" and competence, legitimising emotions in the narrative. The introduction of the narra- tive The writer should have information that the reader does not have access to, and should be able to get his or her message across in a believable manner. A way of doing this is to present the text as a witness whilst showing one's competence. Players and My choice of the earlier example was 104 The beginning of the main text is often reminiscent of the abstract and orientation. This is to be expected, as this is where the narration itself really starts. The three main articles start by giving a fairly superficial account explaining the match photograph or the result. Jaro won the first match of the season by 5-0 ( an overwhelming result in a football match), and the introductory sentence reads: "Jaro was the better team, no doubt about it." (Lindholm 2000). This sentence explains both the match result and the match as a whole, while also strengthening the headline and the lead. The second article in this study tells about a match won by Jaro 3-1, with the introductory sentence: "FC Musan Salama from Bjomeborg is a football team with skilful players such as Saku Laaksonen, Janne Puputti and Tomi Leivo-Jokimaki, and FC Jaro had great problems in asserting control over the game early on" (Furu 2000a). This does not explain the match result, but it does help the reader understand why the beginning of the match was how it was, and also indicating how the game looked early on. This sentence also strengthens what was already said in the lead: "The reds won in the end quite comfortably" (Furu 2000a). carefully made - not many of the headlines give as much background information. Quite a few of them, however, narrate both where the match was played and which team the article is reporting, and only few of them can be seen as "genuine" abstracts. The first article of the season is an example of the latter type, with the headline "The football feast has started" (Lindholm 2000). This headline does not specify the actors, the time, events, etc, but it says that there is a football feast that has started the moment the word "feast" is read. According to Labov, different kinds of background information may be presented later in the narrative or, as in this case, before the action. The order of the different elements is not fixed (Labov 1972: 364). The main action of the narrative The main action of the narrative deals with incidents that may have had a significant effect on the outcome, or were in some way sensational. These specific incidents are narrated as shorter narratives. According to Alf Arvidsson, narratives are "limited episodes told by the interviewees as narratives with a beginning and an end, built around a narrative structure with a complication - resolution (Arvidsson 1998: 25). Despite the fact that Arvidsson talks about interview situations, the terminology applies to match reports, too. They consist of stories about incidents during the course of the match. The main action of each report - being a narration as such - consists of several narratives that more often than not include the central elements of a narrative: time, space, place and action. The action in the reports is presented in narrative form. The following passage is an example of this: 5. "In the 45 th minute Nicklas Widjeskog took a quick free kick ... FC Jaro were able to go in at half time with a secure 2-0 lead." 6. " ... the same frenetic pace continued for about ten minutes early on in the second half. That was how long it took FC Jaro to extend their lead to 3-0." 7. "After the 3-0 goal, the game fizzled out." 7. "After the 3-0 goal, the game fizzled out." 8. "FC Jaro did nothing until there was only 18 minutes of the match remaining ... " 9. "With only 15 minutes of the match remaining, FC J aro changed their tactics and employing man for man markers." 10. "He came onto the pitch with 20 minutes left, and in the 88th minute he increased their lead to 4-0." 8. "FC Jaro did nothing until there was only 18 minutes of the match remaining ... " 9. "With only 15 minutes of the match remaining, FC J aro changed their tactics and employing man for man markers." 10. "He came onto the pitch with 20 minutes left, and in the 88th minute he increased their lead to 4-0." 11. "In the last minute of normal time, Pelto la set up Borissov for 5-0." (Lindholm 2000. The author's italics) It is obvious that the action is presented in chronological order. Furthermore, the narratives exist in their own separate time frame, as time is expressed in terms of match time. The introduction of the narra- tive "Three minutes after the opening goal, the visitors almost equalized." q 4. "Five minutes before half time FC Jaro put together a fine attack ... " The introduction of the narra- tive Even though it may be difficult to differentiate the elements, it is obvious that the football reports always begin with an abstract and an orientation. The headline, the subheadings and the lead already tell how the match went, and gives a general outline of it. The subheading of the first article reads:" No problems for FC Jaro in the first match". The lead of the same article reads: "FC Kemi Kings hammered in the premier 5-0 ... " These two sentences strongly indicate that FC Jaro is superior, and they enable the reader to read and understand the rest of the article. The final article is different from the previous two in that the headline and the lead do not paint a picture of the match, but instead focuses on the result and its consequences. FC Jaro lost the match described in this article and did not gain automatic promotion. Since FC Atlantis beat FC Jokrut, the result of Jaro' s last match was irrelevant regarding the outcome of the season for the team, subsequently the match photo was not of much interest. The introduction explains FC Jaro' s loss by saying: "the players could hear when anything happened in the Jokrut-Atlantis match ... when the situation was 0-3, the loss of interest showed on their faces ... " (Furu 2000b ). Each individual match has In order to clearly indicate the result, the football report always shows the result between the headline and the lead, where also the sport in question is indicated, as well as the teams, the division, how many goals were scored by each team. This information is emphasized - and emphasizes - information in the text, in the headline and the lead, which also often give the names of the teams and the result. These two combined outline the match and the final result. In football reports the abstract gives quite a lot of background information, not only signposting what is to come in the text proper. 105 to be viewed in the context of the season as a whole. Jaro aimed at gaining promotion and it seems that this aim gave significance to the whole competition. I will return to this later in this study. sharper ... 2. "In the 17th minute of the match the first goal was due." 2. "In the 17th minute of the match the first goal was due." 3. 1. "Early on also the visitors seemed a little The main action of the narrative Places of action are specified in more detail in the narrative that in the introduction, and one writes about "the left touchline", "on the right hand side" and "on the edge of the penalty area" rather than "Jakobstad" and the name of the stadium. The narration moves on to using football terminology in terms of space: the football pitch. the body than in the introduction, and the narration is narrated by using concepts of football time, space and actors. The introduction creates a framework through which the short narratives are interpreted. The narrative is evaluated and assessed by narrating the "surface points" of the action that are mentioned in the narration. They have been selected as points of interest. I recollect the advice I was given: "The text should be easy to read, write about the things your readers are interested in". In other words, the goals and scoring opportunities, exceptional circumstances, incidents that are relevant to the final result, etc. are all "surface points". The development that takes place in the course of the season is another one - individual matches and how they affect the season's ambitions or the league position are commented on frequently. The last article begins: "Because FC Atlantis outclassed Jokrut by a 5-2 score line in Helsingfors, it did not matter how the match between Hango Sports Club and FC Jaro ended" (Furu 2000b, my italics). This means that FC Jaro did not gain promotion. The sentence has both "a superficial point" (FC Jaro did not gain promotion) and "an underlying point" (the individual match gets its significance from the season as a whole). As I mentioned earlier, the crowd and the fans had expectations in regard to the team. The players also had their own ambitions, the goals they expected to score before the season started. The season lasts from the end of April to the end of October, and everyone involved in such a long undertaking will want to see the fruits of their labour. The "value" of the final goal increases with the amount of time and effort put in. Sure, it's always nice to win, but matches certainly gain in value as the season progresses; to reach the long-term objectives is more important than success in an individual match. The main action of the narrative "The final nail in the Bjomeborg-lads' coffin was the last ten minutes of the match. Svanback made a successful interception, arriving before the oncoming MuSa goalkeeper. This time Svanback unselfishly pulled the ball back to Timo Peltola who had an easy job of putting the ball in the net." (Furu 2000a). In the introduction the narrator places match time in the context of ordinary time. Time is stated more precisely in the narratives, than in the abstract and the orientation, in accordance with match time, Borissov did not score at 8.15 pm, but in the last minute of the match. This section of the report narrates an incident in the match, and consists of time, place, actors as well as action. In addition to the time frame, the narratives also name the actors, and often also a place of action. Actors and places are more accurately indicated in narratives of action than in the introduction/ orientation. Rather than merely stating the name of the team, the narration takes place The narratives function as a description of the action and are most often presented chronologically. The action of the first article is presented in this order and with the following definitions of time: 1. "Early on also the visitors seemed a little 106 on a more individual level, calling the players by name, for example: "Ridvan Zeneli and Fredrik 'Mini' Svanback worked a short comer and the resulting cross was headed in by Mathias Kass for 1-0 .... ", rather than, for example "one Jaro player passed the ball to another who crossed the ball to a third player who headed in 1-0." Without the details, the article would be all too uninteresting. As an observation, it may seem absurd, but it makes a point: despite the fact that football is a collective team sport, the articles require individuals as heroes and losers. Rating of each player's performance is done in a separate box at the end of some articles, which supports the above assertion. One should, however, pay attention to the fact that the ( opposing team) players are often described in terms of their role on the pitch, such as "full backs" and "MuSa- defender" (Furu 2000a). The actors are named in football terminology. The same applies to descriptions of space, when certain areas of the pitch are mentioned in more detail. The main action of the narrative In other words the season acts as a frame One of the functions of the article is to explain why the result was what it was. A general principle is that you start from the match result and then go through the whole match, describing the main action chronologically, until you have created a narrative that reaches the end (the result), which was already stated at the beginning). This is achieved by narrating the dramatic, significant and strategic incidents, together with commentaries from the players and the coaches. A text is constructed that affirms the initial image. The action is described in more detail in In other words, the season acts as a frame 107 of reference by which to interpret the match reports. It gives meaning to the individual matches. Each match in tum gives meaning to individual incidents occurring within it. The anecdotal point/superficial point in telling how, when and where a goal was scored, or an opportunity to score that was missed has consequences on the course of the game. The underlying point in these narratives is that these have consequences on the season's success. This functions simultaneously as a superficial point in the narratives of the matches: how the season progresses. think that the imagined reader should also empathise and support FC Jaro. As Anne Eriksen writes, the mass media appeals to us not through its content, but through embellishment, and the way the narrative catches our emotions (Eriksen 1989: 67). The match reports are very emotive - they allow the reader to feel the joy of winning and the disappointment in losing a match. What is the source of this belonging and interest in a football team? What makes people scream and hug each other out of joy when a team scores? Jaana Venkula states in her praise of football "Fair play jalkapallon sieluna Ja kiiytiintonii" ("Fair Play, the soul and the practise of football") that football imitates life, which makes it an emotive activity. The significance of mimetic activities lies in the fact that being emotive they allow the expression of these emotions in a way that is not as "dangerous" as they would be elsewhere (Venkula 1998: 45f). Venkula wants to explain why football is played, but I think it also explains the interest in football of the numerous people who do not actually play themselves. The main action of the narrative The fact is that there is a sense 108 Jaro sakrade andraplatsen the coda to lead the reader away from the text. The conclusion of the narrative The three main match reports are rounded off with different kinds of abstracts. The last sentence of the first article says: " In The appearance of the footbal articles vary. This article cosist of both a picture, grades and a separated interview. The result between the headline and the introduction ap- peared in all the articles studied. JT 8.8.2000 of justice that mitigates the circumstances. The match result is evaluated in the light of the match as a whole. In the match reports this equals the overall picture that is given. It is the "sum total" of the narratives, which become the evidence in evaluating the fairness of the match. The evaluation is often given in direct terms, e.g. "the visitors were good opposition and they deserved to win" (Lindholm 2000) and "the right team won the match with the right result" (Furu 2000a). The season's result is evaluated in relation to the aims of the season - how a team achieves its ambitions. In the quotation I gave earlier J aro' s coach called for an organisation that functions better, that "does not promise what it cannot keep" (Furu 2000b) in order not to disappoint the fans. How things go for "us", and how things should go decides if "we" are the heroes or the losers. Even if it is important to win, the self-image is preserved if the team gives a good fight. The appearance of the footbal articles vary. This article cosist of both a picture, grades and a separated interview. The result between the headline and the introduction ap- peared in all the articles studied. JT 8.8.2000 Jaro sakrade andraplatsen Jaro sakrade andraplatsen the coda to lead the reader away from the text. The conclusion or resolution is more demanding to deal with. As I explained earlier, the obvious objective of the story is to explain why the result was what it was. The narratives do not aim at exposing the result of the match, that information is given in the very beginning in the introduction, and the rest of the narrative supports and explains the initial information. The main action of the narrative According to Labov, the conclusion or the resolution should be placed at the end of the main action of the narrative, but in the articles analysed in this study the result is given very first, and yet the reports follow the development towards the result. The main action reaches the conclusion, and the results are given when the narratives work through to match result. The appearance of the footbal articles vary. This article cosist of both a picture, grades and a separated interview. The result between the headline and the introduction ap- peared in all the articles studied. JT 8.8.2000 The main action of the narrative As my study indicates, football is popular because it is emotive (raises emotions) - at least for some. Football and narratives about it are a battle between winning and losing which gives anyone interested a chance to feel and express the joy of victory and the disappointment of defeat. The battle between winning and losing - between heroes and losers - makes it attractive and exciting. With this in mind, it may not be that difficult to understand a pass as "catastrophic" if it gives the opposing team a scoring opportunity- a goal that may change the narrative of victory of a winning team to a narrative of defeat for a losing team. What is the point of knowing how the season goes? In my view, the "underlying point" of football and football narratives is that they awaken emotions, a feeling of belonging to a winning or a losing community that is created in the team. The relationship between the team and its supporters can be demonstrated by the following quotation: "He calls for an organisation that functions better, that does not promise what it cannot keep. According to Paananen, FC J aro has a responsibility towards its fans ... " (Furu 2000b). This expresses a responsibility the team has, not to disappoint its supporters - by giving the fans a sense of belonging to a losing community. As I pointed out earlier, objectivity and information overflow could be "narrative points". I also found that there has to be an interest in football and FC Jaro that makes them worthy of the space given in the newspaper. There is also an emotional engagement that is recognizable in the way it is talked about. Empathy for the team can be seen as a "narrative point" highlighting the sense of belonging. When writing that it is good to see the team score, the journalist strengthens the sense of belonging with the model reader, who, choosing to read the match report on reading the headline is supposed to be interested in football. I also Is it not then unlikely that one can feel joy by investing emotions in a football team? Only about one in ten to twenty teams can be the best at the end of the season, does that mean that there are many who never win? The conclusion of the narrative The three main match reports are rounded off with different kinds of abstracts. The last sentence of the first article says: " In addition to scoring the team likes to push forward which is promising". Considering that the lead mentions "FC Jara is one of the favourites for promotion", this is very interesting. I think that, too, expresses the significance of the rest of the season. The same happens in the second main article, when FC Jara' s coach comments that the team has secured second place in the regular season. The last sentence says: "I would like to express my gratitude to the players and their performance so far, Paananen said". This sentence expresses that the aim has been partially fulfilled, the ambition has not quite been achieved yet. This is a way of setting the time. Both past ambitions and expectations, and those remaining are expressed here. The first The conclusion or the resolution need not be the match result; it can also be expressed in the description of the final goal or the final whistle. This is where the narrative descriptions of the football incidents or narrative events the narrative moves on to 109 introduces the text, the second leads the reader on. be seen as narratives. They are narratives of interrelated incidents which show cause and effect, evaluation, points, and self- presentation. Labov's six elements of narrative structure correspond to a certain extent with my data. The reports include an introduction describing the match, arousing the ( assumed) readers' attention and interest. The body or the main action of the narrative consists of shorter narratives describing match incidents that underline the description in the introduction. This is, in my opinion, reasonably clear, but how Labov' s elements should be defined is open to interpretation, because it is not always possible to distinguish between the different elements. There are similarities between the structures of the reports, but no particular order can be identified. The ways the reports are concluded vary a lot, some seem to have no coda at all, some have no abstract, they finish with a conclusion (a resolution) or "leftovers". Those finishing with a conclusion put a full stop on the match action, for example by saying: " ... the team were more or less just waiting for the final whistle" (Back 2000). The conclusion of the narrative By "leftovers" I refer to any extraneous material tacked onto the end of the text, which did not fit earlier. A couple of "leftovers" can be found in the data studied here, mentioning, for example, the attendance or the amount of red or yellow cards issued. The third main article is different in that almost the entire latter half is dedicated to explaining why the season's ambitions and aims were not achieved. This is by no means out of the ordinary, the season had come to an end by the time of writing. Expectations have either been achieved or crushed. If all the season's reports were analysed as one long narrative, this report could be called the coda of the season's narrative. The narrative became, in this case, a narrative of losing, and the last sentences express the moral of the long narrative: Evaluations of the match incidents are useful when evaluating the match and the whole season. Football is a battle between victory and defeat, and whether "we" are winners or losers, good or bad - is decided by trials and tribulations of the team. The point of a narrative is created by this battle, and a sense of belonging, but the points vary. The anecdotal point is that the match result is significant. The anecdotal point in commenting on the matches is in tum that they are significant to the outcome of the season. With increasing engagement as the match drags on, the tension between victory and defeat increases, and emotions become involved. The underlying point of the narrative is that "we" FC Jaro supporters are winners or losers, depending on the outcome of the match or the season. Sympathies for a team function as a narrative point, giving expression to the sense of belonging. Judgements in the match reports are, in other words, also judgements of "us". The fairness of the result is judged on the basis of the performance and the passion of the struggle. The performance is connected to your expectations of the team, and the "It is not enough for the team to work towards promotion. Even the organisation has to pull its weight. The conclusion of the narrative Both Tampere United who was promoted last year, and FC KuPS who took a step up this year have a well functioning organisation behind them, concludes Paananen." (Furu 2000b) The reason FC Jaro' s narrative did not become a winner's narrative can be found outside the field of play. The reader is safely guided out of the narrative, the scapegoat is found, and it is neither the team nor the fans, and therefore not "us". Conclusion It is fairly obvious that football reports can 110 relationship between expectations and their realization influence the sense of joy or disappointment. Football offers football fans a chance to share an affinity with those who win or lose. Thus, it is not enough for the newspaper to only report the match or publish match results. Instead, it must make an attempt to describe the excitement or fairness of the match by narrating it. This concurs with Anne Eriksen's view on the mass media addressing us through our emotions. http:/ /www.ffjaro.fi 27 .3.2001 http:/ /www.ffjaro.fi 27 .3.2001 Printed material Adelsward, Viveka 1996: Att forsta en berat- telse - eller historien om algen. Stockholm: Bromberg. Arvidsson, Alf 1998: Livet som berattelse. Stu- dier i levnadshistoriska intervjuer. Lund: Arvidsson, Alf 1998: Livet som berattelse. Stu- dier i levnadshistoriska intervjuer. Lund: Studentlitteratur. (Etnologiska skrifter 10.) dier i levnadshistoriska intervjuer. Lund: Studentlitteratur. (Etnologiska skrifter 10.) Studentlitteratur. (Etnologiska skrifter 10.) Berger, Arthur Asa 1997: Narratives in Popu- lar Culture, Media, and Everyday Life. London: Sage. Back, Soren: Oavgjort rattvist i Tammerfors. Jakobstads Tidning 4.8.2000. Eco, Umberto 1992a: Interpretation and his- tory. Interpretation and overinterpretation, Stefan Collini (ed.). New York: Cambridge University Press. Eco, Umberto 1992a: Interpretation and his- tory. Interpretation and overinterpretation, Stefan Collini (ed.). New York: Cambridge University Press. NOTES 1 There are most often one or two photos that accompany the match reports, but I decided not to include them. I also exclude anything that is not a part of the match review. Commentaries by players or trainers are often included, but there were none in six of the articles. Five of these had an interview with a player alongside the match review/report, but I chose not to include them in my analysis if they were a separate article. Neither have I included articles of cup matches, or smaller articles about FC Jaro. The material in this study is limited to the season in Division 1. In Finland the Finnish Championship league is the top league, followed by divisions starting from 1 downwards. The divisions are divided into regions and districts, and the further down the divisions you go, the number of districts included increases. As the districts become smaller, the more local, less time consuming and less professional it gets. The different divisions have different rules concerning promotion. In the summer of 2000 that I used in my study, in the beginning of the season FC Jaro was forecast a possible candidate to promotion during the season. (Lindholm 2000). However, they were not successful. Eco, Umberto 1992b: Overinterpreting texts. Interpretation and overinterpretation, Stefan Eco, Umberto 1992b: Overinterpreting texts. Interpretation and overinterpretation, Stefan Collini (ed.). New York: Cambridge Univer- sity Press. Collini (ed.). New York: Cambridge Univer- sity Press. Eco, Umberto 1992c: Between author and text. Interpretation and overinterpretation, Stefan Collini (ed.). New York: Cambridge Univer- sity Press. Eriksen, Anne 1989: Massekulturens kommunikationsform. Budkavlen 1989. Furu, Fredrik 2000a: Jaro sakrade andraplatsen. Jakobstads Tidning 8.8.2000. Furu, Fredrik 2000b: Forlust i Hango - inget ligakval for Jaro. Jakobstads Tidning 29.10.2000. Labov, William 1972: Language in the inner city. Studies in black English vernacular. Phila- delphia: University of Pennsylvania Press. delphia: University of Pennsylvania Press. Lindholm, Paul 2000: Fotbollsfesten har borjat. Jakobstads Tidning 30.4.2000. Marander-Eklund, Lena 2000: Berattelser om barnafodande. Form innehall och betydelse i kvinnors muntliga skildring av fodsel. Abo: Abo Akademi. Sundqvist, Patrik 2000: Jaro avslutade med oavgjort. Jakobstads Tidning 22.8.2000. Venkula, Jaana 1998: Fair play jalkapallon sielunaja kaytantona. Helsinki: Atena. http://palloliitto.mork.net/sarjat/ohjelmat.htm 27.3.2001. ULRIKA WOLF-KNUTS Due to historical and political matters, what is, today, called Finland was a part of Sweden until 1809. Thereafter, the region was an autonomous Grand Duchy in the Russian Empire. Beside Latin, for a long time, Swedish was the language of the authorities, of education, and partly of science and scholarship. Not until the Russian time Finnish played an important role as a language outside the private sphere. German and Russian, respectively, were used on special occasions. Finland certainly was a polyglot region. contemporary understanding of historical sources, information valuable to the folklore students of today was stored. Only much later, historians started to question the value of certain historical sources, in order to improve the ability to discern so-called facts from fantasy. This was an advantage to the folklore students. Although neither the word 'folklore', nor the study of folklore existed until the middle of the nineteenth century, the folklore material was still gathered, although with the wrong preconditions as historically reliable sources. Folklore plays a great role in the shaping of the identity not only of single individuals, but also of nations. The Finnish case demonstrates this very clearly. Even during the Middle Ages, the initial perspective was to prove that the Swedes were Gothic descendants. Already in the fourth century, Jordanes had placed the primordial home of the Goths in the island called Scandza, i.e. Scandinavia. The aim was to create a model for courageous behaviour in times of war and crisis. During the seventeenth century, Sweden was one of the most influential and expansive kingdoms of Europe. Again, efforts were made to prove the rich history of the country. In 1666, the Chancellor of Sweden (rikskanslern) founded the Antikvitetskollegium (The Collegium of Antiquities) the task of which was, in a systematic way, to collect reminiscences of historical events and to document historical sites. Due to the The eighteenth century was the time of Enlightenment. Folklore was interesting for two different reasons. On the one hand, the authorities wanted to document the lives of the inhabitants of their country, in order to be able to improve economics. This happened by describing the customs and rites found in the countryside. They also strived to eradicate such cultural traits that they regarded as irrational, but before this was possible the beliefs had to be documented. Unprinted material http://palloliitto.mork.net/sarjat/ohjelmat.htm 27.3.2001. http://palloliitto.mork.net/sarjat/ohjelmat.htm 27.3.2001. 111 ULRIKA WOLF-KNUTS Therefore, the priests and other representatives of the Enlightenment reported on what special kinds of non- Christian forms of religion they had seen in their parochial environment. These collections are extremely valuable. On the other hand, in Europe inspired by the ideas of Montesquieu and Rousseau, an interest grew for the Nordic countries as exotic countries. The people were supposed to live 112 near Nature, their lives were regarded as parts of a Paradisiac way of existing. Again, the old history was accepted as a model for human life. outside the nearest circle. Huge systematic work started to reach this goal. Many a university student, no matter what language he regarded his own one, felt responsible and helped in collecting folk poetry which was considered the best source to start with for this To some extent these ideas also affected Finland. However, folklore as a target for studies for its own sake was not evoked until the end of the seventeenth century. Certainly, there had been some efforts earlier, too, to collect and discuss folklore texts, but the main achievement was that of Henrik Gabriel Porthan (1739-1804), librarian and teacher at the university library in Abo (Turku). He was inspired by the ideas of Enlightenment, but also by the new humanistic thoughts found in Europe that admired classical antiquity with its aesthetic and ethical values. Moreover, contemporary Finnish-minded political currents seem to have had some influence on him. This combination of ideas made Porthan aware of the Finnish folk poetry, which later on was the target of several students at the Abo Akademi and which, during the first three to four decades of the nineteenth century, resulted in huge collections of folk poetry and the Kalevala editions of Elias Lonnrot. During this period in Finland, i.e., the beginning of the nineteenth century, Swedish was still generally spoken. However, the eagerness to create the Finnish language made people take the existence of the Swedish language for granted. This circumstance made the Vice Rector of the school Vasa Lyceum, Johan Oskar Immanuel Rancken ( 1824-95), aware of the risk at losing the corresponding insight into the folk culture of the Swedish speaking population of Finland that the immense collecting work in the Finnish regions brought along. Therefore, he started to gather folklore items specifically in Swedish, but recorded in Finland. ULRIKA WOLF-KNUTS He had quite a lot of problems and disappointments before he saw that the most efficient way to reach his goal was to encourage his pupils to document their own environments. Anyhow, thanks to Rancken's efforts, the folklore archives in Finland contain quite a lot of records in Swedish from the 1860s onwards. The question, what language, Finnish or Swedish, was the main constituent in the identity of these young men is not quite easy to answer. Some of them had a Finnish background, others were from Swedish speaking families in Finland, but Swedish was the main language for educational purposes, which means that all of them were in command of the language. However, political reasons must also be considered. After Finland had become part of Russia in 1809, a growing awareness oflanguage can be spotted. In fact, being the language of the losing part, Swedish could no longer be the official language, and Russian was the language of the conqueror and, therefore, not suitable. The only remaining possibility was to create Finnish as a valid means for any kind of communication, also Largely at the same time in Helsingfors, where the university had moved from Abo after the great fire in 1827 and changed its name, there was a separate awakening for the Swedish folk tradition through one of the teachers at the Imperial Alexander University, namely Axel OlofFreudenthal (1836-1911). He supported a liberal perspective on being an inhabitant of Finland as he maintained that the Swedish population should function as a link between the Finns and the Scandinavians. The Swedish dialects in Finland were his special field of interest and, therefore, he urged his students to collect and discuss dialect items, such as legends, superstition 113 and other folklore. He was the founder of the Svenska Landsmalsforeningen in Finland (The Swedish Dialect Society in Finland) and he inspired a great number of young men to become aware of the conditions ofliving in Finland, but speaking Swedish. constituted a sign of high quality folklore collections. Later on, in accordance with a growing interest in performance and in the process of folklore, this way of presenting the material and this kind of publishing and editing work were questioned. Therefore, the series has not yet been finished, but the recordings are available at the Archives of the Literature Society in Helsingfors. ULRIKA WOLF-KNUTS Rancken was a historian who regarded his folklore collections as a means to the understanding of the Swedish culture in Finland, and Freudenthal was a historically educated philologist. Each of them had a political motive for their folklore interest. Freudenthal succeeded better than Rancken to realise his ideas, perhaps because he stayed in the capital of the country. In 1885, the Swedish Literature Society of Finland was founded by Freudenthal, among others. One of the aims of the Society was to collect and record folklore items. During the last decade of the nineteenth century, a system was created for how to do this work. The Society engaged the teacher Ernst Lagus (1859-1923) to plan this work, but he also gave the collectors instructions for how to conduct their work and criticised the result after they had handed over their collections to the Folklore Commission of the Society. Eventually, the collections grew, but soon the Commission realised that they gave an uneven image of the Swedish folk culture in Finland. Therefore, specially educated experts were engaged in order to collect completing recordings, which they also did. This work was accomplished during the first decade of the twentieth century. This is the explanation for the exceptionally big collection abounding in variants of Swedish folklore from Finland that, today, is kept in the archives of the Swedish Literature Society in Finland and which is also, to a great extent, published in eighteen volumes in Finlands svenska folkdiktning, 1917-. The publication work started when comparative research methods prevailed and therefore a great amount of variants were regarded necessary because they During the nineteenth century, the collecting and recording of folklore items was a scholarly task per se. On the one hand, the aim was from an evolutionist- devolutionist perspective, to gather all kinds of items along the contemporary principles for how to define folklore. Folklore was needed for political reasons, i.e., to demonstrate the identity of the nation. This identity was not one and the same to all inhabitants. The two language groups wanted to profit in different ways from their folklore, correspondingly. The Finns, and their sympathisers wanted to demonstrate the value of the Finnish language as a means of literary, cultural, administrative, and scholarly activities. The Swedes wanted to show their connection to the Scandinavian culture and the Scandinavian peoples. ULRIKA WOLF-KNUTS p p One of the preconditions for this twofold aim was the evolutionist theory with its historical perspective. In 1858, although he had a strong Swedish identity, Carl Gustaf Estlander, called upon the necessity to compare all the variants of the Kalevala. He also emphazised the importance of relating the Kalevala to other epics and he referred to what had been done with The Songs of Ossian when the debate on their authenticity raged. Other scholars worth mentioning here are, for instance, Carl Collan, who investigated Serbian folk songs, and O.A. Toppelius, who was inspired by the Danish folklore student Svend Grundtvig in his study of the songs on Marsk Stig. Estlander's thoughts preceded Julius and Kaarle Krohn' s research. In the 1860s, 114 Julius Krohn pondered over the age and descent of Kalevala. His son, Kaarle Krohn, refined his method for the purpose of folklore studies. The method relies on different kinds of comparison over time and space and it has, therefore, been called the historic-geographic method, or the Finnish method. Its concentration on the will to find out primordial facts on folklore items was a good tool when it came to demonstrate where and when an item had come to be and how it had changed and spread since. To the Swedish folklore students in Finland, in the latter half of the nineteenth century, it became more and more important to prove a western influence to their folklore, due to the above mentioned fennophil ambitions. documentation of folk culture. However, this organisation was not very important for the investigation of folklore. g Otto Andersson (1879-1969) is a prominent figure in Finnish folklore studies. He started as a collector of folk music with the Swedish Literature Society in Finland, but in due course, in 1926, he was appointed professor of musicology and folklore studies at the recently founded Swedish university in Finland, the Abo Akademi in Abo (Turku). The chair was a counterpart both to chairs of the kind in Sweden, and to the folklore chair founded in Helsingfors in 1898, which was one of the very first in the world, and which was held by Kaarle Krohn. Andersson combined his interest in folk music and folklore, he worked eagerly for the Swedish culture in Finland, and eventually, he became the Vice Rector of his university. ULRIKA WOLF-KNUTS He continued to collect folk music items, he introduced the phonograph, he built up a huge archive on folk music recordings, he analysed, edited, and published his material, and he wrote a lot of scholarly articles. He had the most important support from Greta Dahlstrom and Alfhild Forslin, two ladies who were able to record songs and plays just by ear. Alfhild Forslin also published a lot of articles and monographs on Swedish folklore in Finland. The Swedish Literature Society certainly supported the scholarly ventures. The above mentioned Ernst Lagus planned the publication of the monumental collection called Finlands svenska folkdiktning, for he saw the need to make the collections available to comparative scholarly investigations. In their dissertations on charms and the Polypheme legend, respectively, his colleagues O.J. Brummer and 0. Hackman regarded the Swedish folklore as a reflection of the Swedish parts of Finland, mostly situated along the western coast, as a bridge for folk culture to reach the country. However, around the turn of the twentieth century, at the Literature Society, mainly collecting work was conducted, whereas the folklore research was carried out at the university. Since the middle of the 1920s Swedish folklore in Finland has been of interest to the persons connected to the Swedish Literature Society in Finland situated in Helsingfors and to the university people at the Abo Akademi University in Abo. The collecting work went on in the capital, the editions of Finlands svenska folkdiktning were published at the instigation of the Society, but by the work of scholars who were situated both in Helsingfors, at the Society, and in Abo, at the university. The Archives of Folk Tradition expanded. In Abo, at the university, Otto Andersson mainly collected folk music and published In this connection it is also important to mention the Brage society. The founder was Otto Andersson, who had been working with the Literature Society. In this connection his main contribution was on the area of folk music and folk song. The participants at Brage paid tribute to Swedishness, and the aim of the society was to revive folk culture, but also to influence on the gathering of folklore and the 115 his collections. A great amount of scholarly articles and monographs were published, too. and research. During the last decades of the twentieth century the number of folklorists increased again. ULRIKA WOLF-KNUTS Among other things is worth mentioning the archives specialised in Ostrobothnian folk culture in Vasa. The Folk Culture Archives conducted several field expeditions along the Swedish areas in Finland and Estonia. A fair amount of questionnaires and competitions was administered and arranged. Regularly, several hundreds of informants corresponded with the archives on specific topics. A catalog of the collections was published and a lot of interesting books on folklore issues are, nowadays, part of the series published by the Swedish Literature Society in Finland. Cooperation with other folklorists in Finland and the other Nordic countries goes without saying. In the middle of the 1930s an idea came up within the Swedish Literature Society that an institute should be founded in order to prepare for an atlas on Swedish Finn folk culture and for an ethnographic dictionary. The model was brought from the Finnish Sanakirjasaatio (the Dictionary Fund). In 1937 the Folk Culture Archives (Folkkultursarkivet) was founded within the Literature Society. Its aims were defined as collecting folklore, ethnographical material, dialect recordings, and place names. Moreover, at this time the collections of folklore gathered at the archives of the Swedish Literature Society had grown so voluminous that they needed an organisation of their own. In 1939 Valter W. Forsblom, who was in charge of the collections, quit his work and his tasks were taken over by the two employees at the new archive, Ragna and Olav Ahlback. They made inventories of the existing material, they collected new recordings, and they created questionnaires. However, "pure" folklore material, i.e. folk poetry, folk belief, and folk music was not at their focus, Ragna Ahlback being an ethnologist and Olav Ahlback a scholar of dialects. Their perspective was inspired by corresponding archives in Uppsala and Stockholm and comprised all kinds of folk culture. 1939- 44 during the wars, everyday work was made difficult, but soon thereafter it took more or less normal forms again. In Abo the successor of the retired Otto Andersson was John Rosas. He was in charge of the folklore studies at the university, but he was himself more interested in the other part of his chair, i.e. music, than of folklore studies. Still, he managed to find money to pay part time teachers to give lessons on folklore. ULRIKA WOLF-KNUTS It is worth mentioning, among others, Alfhild Forslin and Ann-Mari Haggman from Finland, 10m Pi0 from Denmark, and Britt- Marie Insulander and Jan-Ojvind Swahn from Sweden as inspiring teachers coming for some weeks to give courses on general and special folklore topics. Gradually, a growing interest in folklore studies came to the fore with the students at Abo Akademi University and J an-Ojvind Swahn was appointed a permanent visiting teacher from the 1970s. During that decade, the organisation of the university changed so that folklore studies were administered within the department of ethnology. Swahn spread the knowledge of folklore among the students to such an extent that the Foundation of the Abo Akademi University decided to appoint at half time lecturer of folklore studies from the beginning of the In 1967, the first officer specialised in "pure" folklore was appointed. Since then, folk music, folk songs, folk dances, oral traditional narratives and belief recordings have been gathered in great amounts. During Ann-Mari Haggman' s era collecting and publishing work was in the center of the activities. Later on a balance was found between the main fields of purposes, i.e. collecting and registering, public service, 116 1980s. Since 1985 this lecturership was a full time appointment held by Ulrika Wolf- Knuts. Since 1987 Lena Marander-Eklund is her working partner, today she is an academic lecturer of folklore studies. Society is more far-reaching, due to the greater number of individuals in the Finnish population. Today, the two organisations work parallelly within their language areas, and in a corresponding way the universities share the students and young researchers along their command of Finnish or Swedish, respectively. Rescuing the folklore is no longer an issue within archives or universities, instead contemporary folk tradition and ways of living and narrating are documented and analysed according to the interests of the scholars. The last two decades of the twentieth century were characterised by international cooperation. The Nordic Institute of Folklore, followed by the Nordic Network of Folklore, may be mentioned beside the Coimbra Group of Universities' Culture Task Force which arranges international conferences on various topics of folklore. Arv, Nordic Yearbook of Folklore was published at the Abo Akademi University from 1993 to 2002 BIBLIOGRAPHY During the last twenty years nearly forty students have received their master's degrees and four doctors have defended their dissertations. Although working conditions for folklorists are not very good, the young people have still found their ways of earning their living. In the future there will be more research conducted on Swedish Finn folklore from various aspects adjusted to relevant politically correct currents, but there will also be studies on individual fields of interest, historical and contemporary themes. A number of new doctoral dissertations will be published to complete the existing ones on folk belief, migration, women's narratives, and contemporary popular culture. Andersson, Otto,Finlandskfolklore. Abo, Abo Tidnings- och Tryckeri AB 1967. Mustelin, Olof, Det andra halvseklet. (F orskning och vitterhet. Svenska litteratursallskapet i Finland 1885-1985. 2.) Helsingfors, Svenska litteratursallskapet i Finland 1986. (Skrifter utgivna av Svenska litteratursallskapet i Finland 523, 2. Steinby, Torsten, Det forsta halvseklet. (Forskning och vitterhet. Svenska litteratursallskapet i Finland 1885-1985. 1.) Helsingfors, Svenska litteratursallskapet i Finland 1985. (Skrifter utgivna av Svenska litteratursallskapet i Finland 523, 1. Wikman, K. Rob. V., Finlandssvensk folkminnesforskning. Historia och ideer under ett sekel. Budkavlen 1955, pp. 1-23. Characteristic for Swedish Finn folklore work have been matters of identity. The first decades were marked by a desire to rescue Swedish Finn folklore from being forgotten when Finnish folklore was put in the core of collecting and publishing. The national eagerness to popularize folklore and spread the knowledge of Kalevala had no counterpart in the Swedish population. Instead, scholarly critical editions of folklore were published. To some extent, the Finnish Literature Society and the Swedish Literature Society in Finland were each others' equals, although the Finnish Wolf-Knuts, Ulrika, Folkdikten och dess upptecknare. Finlands svenska litteraturhistoria 1, utg. av Johan Wrede. Helsingfors, Svenska litteratursallskapet i Finland & Stockholm, Atlantis 1999. Pp. 398--403. Wolf-Knuts, Ulrika, Folkloristikens folk. Folket. Studier i olika vetenskapers syn pa begreppetfolk. Red. DerekFewster. Helsingfors, Svenska litteratursallskapet i Finland 2000. (Skrifter utgivna av Svenska litteratursallskapet i Finland 626.) Pp. 125-136. Wolf-Knuts, Ulrika, Folkloristiken vid Abo Akademi. Saga och Sed 2002. Uppsala, Kungl. Gustav Adolfs Akademien 2003. Pp. 154-157. 117 Wolf-Knuts, Ulrika, Den folkloristiska universitetsundervisningen. Etnologi och folkloristikvid universitet, museer och arkiv. Ed.: Anne Bergman & Carola Ekrem. Helsingfors 1988. (SLS arkiv 16.) Pp. 11-14. Wolf-Knuts, Ulrika, A Hundred Years of Folklore Studies. Symposia. Caiete de etnologie si antropologie 2003, pp. 11-15. Wolf-Knots, Ulrika, Johan Oskar Immanuel Rancken - en pionjar inom folkloristiken. ANNA-MARIA ASTROM What is a Swedish Ethnology in Finland? In his article Five ethnologies - the rise of Finnish ethnology from a Finland-Swedish point of view, prof. Nils Stora has characterised the development of Ethnology in Finnish and in Swedish not as dichotomised striving for two unique kinds of ethnology based only on two languages in Finland but as five paths that together make the history more diverse and sometimes overlapping, sometimes not. The article looks for the roots in different spheres: antiquarian interests, languages, regionalism, an interest in cultural history and in anthropology. Prof. Stora takes us up to the 1960s. The starting point is of course language based disciplines where the need for this emphasis was felt very strongly. In Finland almost 5.9% of the population has Swedish as their mother tongue; in the beginning of the 20th century the percentage was 12,9. A result of the fact that the Swedish speaking also under the Russian rule until the late 19th century held important civilian and political posts, was that Finland after the independence 1917 was declared a bilingual state (1919). This meant and means that the Swedish speaking have a strong guarantee when it comes to their linguistic rights. Also strong institutional support make a Swedish or bilingual life especially in the Swedish coastal regions and towns possible. are working and also in which scientific context one can look at the ethnology that is carried out at the Abo Akademi University in Abo (Turku), that is the Swedish university in Finland. But I will also consider the ethnological work at the Folk Culture Archives in Helsinki, since a similar documentation and research work has been made there. A third actor is the Scientific section of the Brage Society also situated in Helsinki. To understand the present one has to have in mind also the outset when the discipline was founded and the chair of ethnology donated (1919). What one then must know is that not only the interest in the culture of ones own, that is the Swedish culture in Finland but also in other cultures, has since the beginning of the 20th century been a distinctive mark of Swedish speaking ethnologists in Finland. The study of ones own culture goes hand in hand with studying others. In this respect the two professors in Abo, Helmer Tegengren (1952-1971) and Nils Stora ( 1972-1997) are exemplary. BIBLIOGRAPHY Norveg 1997. Wolf-Knots, Ulrika Nordisk folkloristik. Abo Akademi 1918-1993. Forskning och institutioner 2. Abo, Abo Akademi 1993. Pp. 209-216. Wolf-Knuts, Ulrika, Nutida folkloristik. En amnespresentation. Saga och sed 1991. Uppsala, Kungl. Gustav Adolfs Akademien 1992. Pp. 65-71. Wolf-Knuts, Ulrika, On the history of comparison in folklore studies. Thick corpus, organic variation and textuality in oral tradition. Ed. Lauri Honko. Helsinki, Suomalaisen Kirjallisuuden Seura 2000. (Studia Fennica Folkloristica 7.) (NNF Publications 7.) Pp. 255-283. Wolf-Knots, Ulrika, Johan Oskar Immanuel Rancken - en pionjar inom folkloristiken. Norveg 1997. Wolf-Knots, Ulrika Nordisk folkloristik. Abo Akademi 1918-1993. Forskning och institutioner 2. Abo, Abo Akademi 1993. Pp. 209-216. 118 118 ANNA-MARIA ASTROM What is a Swedish Ethnology in Finland? It had been the idea of Edward Westermarck that a deliberate direction in ethnology was to be towards the Scandinavian cultures because the Swedes in Finland orientated from the west. The chair was thus founded for the research of the origins and development of this culture. Already in 1885 a learned Society, the Swedish Literary Society in Finland was founded with the same purpose but without the Nordic orientation. Gabriel Nikander engaged himself also in The Swedish Literary Society in Finland and in questions concerning museums and he was also head of the Brage scientific section for the study of folk life. This section gathered also anthropologists and folklorists and their main forum was Budkavlen, which presented their articles in different spheres such as folk architecture, economy of the archipelago, folk customs, folk religion and belief and other different folkloristic genres. Early members were among others YngvarHeikel, K.R.W. Wikman, Gunnar Landtman, Otto Andersson and Fritz Burjam the last ones folklorists. The professors succeeding Nikander can on the other hand be said to come close to the anthropological paradigm of Edward Westermarck (1862-1939), contempory and a colleague of Gabriel Nikander. The ethnologists seem to have been content in researching Northern and Arctic people and did not like W estermarcks pupils expand their interest to other continents like for instance Gunnar Landtman ( Papua New Guinea), Rafael Karsten (Peru, Bolivia) or Hilma Granqvist (Palestinia). An important researcher between these two trends is K. Robert W. Wikman, who did his research on premarital relations with a comparative perspective using material not only from Scandinaiva but also from remote parts in Central Europe (Die Einleitung der Ehe 1937) and concentrating on folk customs, a speciality also for the "westermarckians" (Stora 1992, 98-101) Shortly before the war also a new agent hade come into being as The Swedish Literary Society founded an Archive for Swedish Folk Culture in Helsinki 193 7 with MA and later PhD Ragna Ahlback, the daughter of Gabriel Nikander, as the first ethnological archivist. When after the war the task for Helmer Tegengren since 1952 was to build the Cultural history department and broaden and consolidate the Nordic orientation, the task for Nils Stora was since 1972 to continue this work and keep the Nordic contacts alive at a period when ethnology in Scandinavia changed in a direction not foreseen. ANNA-MARIA ASTROM What is a Swedish Ethnology in Finland? Helmer Tegengrens dissertation concerned a rural parish in Ostrobothnia, but his main scientific work adressed the historically distant culture of the Sarni in the Kemi lapplands. Nils Stora in his work has also been interested in two marginal cultures in Finland, the Finnish Archipelago and the Scolt Lapp society as well as the same parish that his teacher engaged in, that is My intention is to look to what has happened in ethnology since 1960 in this Swedish context and what actors where and 119 Kronoby in Swedish speaking Ostrobothnia. In this they distinguish themselves from their predecessor and teacher, the first professor in ethnology, Gabriel Nikander (1920-1951) who on the whole can be said to have dedicated his scientific life to the culture of the Swedish speaking Finns. His scope was nevertheless very broad as he was interested not only in the folk culture of the peasants and other rural elements, but also in the culture of higher layers exemplified by manor- and mill owners and the urban bourgeois and the surroundings they formed. The area for his studies where thus cultural history and folk life studies, also stipulated in the name of the chair in his time (Stora 1992, 92-98, Lonnqvist 2000, 24-25) Wikman as its head. It had been the idea of Edward Westermarck that a deliberate direction in ethnology was to be towards the Scandinavian cultures because the Swedes in Finland orientated from the west. The chair was thus founded for the research of the origins and development of this culture. Already in 1885 a learned Society, the Swedish Literary Society in Finland was founded with the same purpose but without the Nordic orientation. Gabriel Nikander engaged himself also in The Swedish Literary Society in Finland and in questions concerning museums and he was also head of the Brage scientific section for the study of folk life. This section gathered also anthropologists and folklorists and their main forum was Budkavlen, which presented their articles in different spheres such as folk architecture, economy of the archipelago, folk customs, folk religion and belief and other different folkloristic genres. Early members were among others YngvarHeikel, K.R.W. Wikman, Gunnar Landtman, Otto Andersson and Fritz Burjam the last ones folklorists. Wikman as its head. ANNA-MARIA ASTROM What is a Swedish Ethnology in Finland? Tegengrens interests where directed to cultural contacts and cultural diffusion, hence his interest also in founding the Questionnaire network and the Archives, the material of which was Operating in the ethnological field in the 1920s and 1930s was thus the holder of the chair of Nordic Cultural history and folk life studies at the Abo Akademy University, with intimate relation to the chair of Nordic history in the same building and since 1927 also the Institute for Nordic folk life research with K.R.W. 120 gathered through extensive fieldwork and documentation in the Swedish regions. Tegengren's main areas for study where economic contacts, economy in itsef, colonisation and settlement and his views could widely be labelled as cultural anthropological. Prof. Storas interests are directed to acculturation, technology, innovations and the ecosystem and natural resources of the archipelago. Prof Storas interest also in the history of the discipline has been instructive for this article. not as alive as before, but still courses in cultural history belonged to the curricula. Anthropology as a separate discipline can almost be said to have died away with the last pupils of Edward W estermarck, but fortunately a chair was erected at the university in Helsinki, with Arne Runeberg as its first professor, a rescue operation that soon led to a flourishing tradition again. In Abo both professor Stora and the Finnish professor Talve had mentioned urban ethnology as an important task for the future. In Sweden the direction in ethnology moved towards social anthropological perspectives, later to more hermeneutic approaches and reflexions about the role of the researcher. Also studies of everyday life in the present became a new sphere that again meant that many ethnologist took up fieldwork but now with contemporary problems also in mind. A special focus was now on small commumtles, working culture, subcultures, youth culture and other economic activities than that of farmers and fishermen. In the 1960s the ethnological archivists at the Folk Culture Archives in Helsinki where two and.on to the scene came MA, later PhD and professor Bo Lonnqvist, with studies in Finno-Ugric Ethnography at Helsinki University as his background. In Turku at that time the comparative methods where used with a material collected there, in Helsinki the documentary work was stabilised. ANNA-MARIA ASTROM What is a Swedish Ethnology in Finland? There an Ethnological Atlas over the Swedish folk culture had seen the light already in 1945 with Ragna Ahlback as its editor as the first number in the series of Folklivsstudier (Folk Life Studies), which soon was filled up with research reports also of the Abo School. Nils Arvid Bringeus' Manniskan som kulturvarelse (Manas cultural being) has stayed as the core literature for beginning students as well as Orvar Lofgrens and Billy Ehns Kulturanalys (Cultural Analyses). The move to studies of contemporary society and all layers of society was never fulfilled completely in the ethnology at Abo Akademi and by such a direction also the traditions of the former ethnologies were saved. But the focus of study was since the 1970s new: The rural population was broadened to encompass crofters and different categories of maritime populations as well as workers of different factories and mills. The comparative methods stayed in use, also historical approaches were applied and modernisation theories used. For long the research object had been cultural patterns and life forms. Also an interest in artefacts remained as a speciality for the The 1980s - an era of new theo- retic insights and intensive fieldwork Abo Ethnology. The intensive questionnaire work continued with dedicated informants in both Swedish and Finnish speaking areas all over the country. The intendent John Hackman was one of the first to use information technology in handling the answers. The interests of study were artefacts in rural and maritime surroundings and the economy in these areas. In Abo the challenges of the new Swedish ethnology where taken up in teaching at all stages. This manifested itself in the choice of themes for master's degree thesises. Life modes, life styles, subcultures of different groupings in society as well as more traditional subjects of rural and maritime life where targets of study for the young and eager generation. The move towards documentation of contemporary society was accompanied by new insights in the mechanisms of culture and by intensifying fieldwork in surroundings where the categories of peasant life could no more give clue to an understanding of the manifestations. One could not however talk about a shift of paradigm, since another focus was still on the disappearing forms of traditional life. Concepts such as cultural identity, acculturation, cultural niche, cultural ecology nevertheless gave a deeper insight to the studies accomplished. The habits of summer residents in rural areas, new forms of economic activity in the countryside as well as the traditional way of women's life were focuses of studies among the Abo ethnologists. A keen interest in the material life and artefacts were still very strong. Since 1981 Bo Lonnqvist was also attached to the department as docent and teacher. At the Folk Culture Archives the decade was also full of activity. Bo Lonnqvists dissertation Driikt och mode i ett landsbygdssamhiille 1870-1920 in 1972 had led him to more deliberate research funded by the Academy off inland and the staff now consisted of four ethnologists the tasks of whom where divided into rural ethnology, maritime ethnology, ethnology of customs and documentation (photography and drawing). Ragna Ahlback, Ivar Nordlund and Mary-Ann Elfving and since 1979 Bertil Bonns and the writer of this paper formed the team. Bo Lonnqvist had stipulated the work also to be directed to cultural elements and cultural forms, their vanishing, change and appearance. The Scene in the 1970s The name of the chair in Turku at the Abo Akademi had in 1974 on professor Storas request been changed to Nordic Ethnology and Folkloristics whereas the brother Finnish University in Turku was dedicated to Finnish and comparative Ethnology ( in 1986 Folkloristics was changed to a discpline of its own). The five ethnologies described by professor Stora had changed a lot but new challenges were still ahead. The antiquarian ethnology stayed important as ethnology as a discipline was also directed to museum work, the linguistic base was never forgotten and the regional research was still one outset. The cultural historical trend was 121 The 1980s - an era of new theo- retic insights and intensive fieldwork Inspirations were sought by keen attending to international conferences such as the SIEF conference in Zurich (The Life Cycle) and the German ethnological congresses with City culture, Children's culture, Remembering and forgetting, Culture Contacts-Culture Conflicts and Industrial man as their themes. Orvar Lofgren, Jonas Frykman, Lissie Astrom, Barbro Klein, Karl Olof Arnstberg, Britta Lundgren and others. An interest in modem life was self evident by now and themes of gender end ethnicity came to the fore. This was reflected also in the work of Monica Nerdrum on tradition and modernity among archipelago women and a deliberate study and teaching about the different minorities in Finland. Courses were also held in Nordic ethnology and ethnicity as a field of study. different attitudes and life modes of their own. The extensive fieldwork was reported in a volume of the series Folk life studies: Kaska - kontinuitet och forandring i en smllstad (Kasko continuity and change in a small town, 1985). Fieldwork was always carried out with a historical perspective that sought for answers to the changes of the 20s century. This meant that also historical sources and newspapers were used to clarify the picture of change. Some of the fieldwork took place as joint expeditions to different localities in the Swedish speaking areas with ethnologist from Jyvaskyla and Abo as partners and even Swedish partners from Sweden were invited. The position of the memories that the interviews highly relied on were not problematised in other manners than that the remembrances had to be controlled by other data and with memories from the same epoch. Bo Lonnqvist work was directed to different aspects of the Finland Swedes and the historicity of this population. But the bourgeois culture also attracted the interest of both him and me, with both the urban scene and the manorial as its main focus. Inspirations were sought by keen attending to international conferences such as the SIEF conference in Zurich (The Life Cycle) and the German ethnological congresses with City culture, Children's culture, Remembering and forgetting, Culture Contacts-Culture Conflicts and Industrial man as their themes. y y This was also the most active period of the Brage section for folk life studies with monthly meetings and yearly symposias with titles such as Goodbye to urban history books, Nature and Culture, The Dynamics of Culture, Symposium on Symbols, Ethnicity, Archipelago Culture and the Land of the Regions. The 1980s - an era of new theo- retic insights and intensive fieldwork pp Work was dedicated to fieldwork in different surroundings ( a manorial society in western Nyland, a paper mill near Porvoo, insular communities in the western, south western and southern archipelago etc.) Ragna Ahlbacks work still consisted in analysing the peasant culture in a historical perspective and a move on to handicrafts. The Archive got a printed Catalogue and Bo Lonnqvist published a Bibliography on all ethnological publications in Swedish until 1976. The questionnaires documented school life, the culture of children, reading habits, dress, annual festivities and material culture as well as the habits of drinking, smoking and the habits of taking snuff and the poor in society. The marginal layers of culture where to be covered. At the Folk Culture archives in Helsinki an even heavier weight than before was put on fieldwork with an problem oriented outlook from the start. The archipelago life mode was thought of as moving and changing as a result modernisation in for instance communication, and with intensive contact relations between residents of the islands and summer residents. Peasant life was studied with the concept of time as its outset and a small town (Kaska) was studied as an arena for different urban dwellers and language groups to manifest 122 different attitudes and life modes of their own. The extensive fieldwork was reported in a volume of the series Folk life studies: Kaska - kontinuitet och forandring i en smllstad (Kasko continuity and change in a small town, 1985). Fieldwork was always carried out with a historical perspective that sought for answers to the changes of the 20s century. This meant that also historical sources and newspapers were used to clarify the picture of change. Some of the fieldwork took place as joint expeditions to different localities in the Swedish speaking areas with ethnologist from Jyvaskyla and Abo as partners and even Swedish partners from Sweden were invited. The position of the memories that the interviews highly relied on were not problematised in other manners than that the remembrances had to be controlled by other data and with memories from the same epoch. Bo Lonnqvist work was directed to different aspects of the Finland Swedes and the historicity of this population. But the bourgeois culture also attracted the interest of both him and me, with both the urban scene and the manorial as its main focus. The 1980s - an era of new theo- retic insights and intensive fieldwork The small town urban studies with an ethnic component was continued by a team under the head of professor Matti Rasanen of the Turku University in the formerly Swedish town of Loviisa and at that time the Soviet town ofVoru in Estonia as a joint Finnish-Estonian project. The Swedish part was written by an ethnologist from Abo Akademy, Marina Airo (Everyday life and Ethnicity 1994). This study focussed on the contacts and possible conflicts between Finnish- and Swedish speaking local residents of Loviisa in comparison to those of the Estonians and Russians in Voru. Also new studies were initiated in the capital of Helsinki, where four large ethnological questionnaires were sent to the inhabitants looking for memories of and attitudes in the capital. Already in 1990 a book calledHemma bast ( At home is best) dedicated to childhood memories in bourgeois and worker families in the first three decades of the 20th century written by Swedish speakers was published and sold out in a year. The new material also resulted in a book Elamaa kaupungissa - Att bo i stan (Living in the city) which concentrated on memories of living in the immediate center of Helsinki in the bustle and traffic of the 1950s. The memoirs were published in the language in which they were written, either Finnish or Swedish. honour for Bo Lonnqvist called Kring tiden, Around time, for his 50th birthday, with a deliberate outset in the concept of time. In his teaching docent Lonnqvist had always underlined this concept among with the concepts of things and space. Bo Lonnqvists own large work on children's creative culture Ting rum och barn, Things space and children was published 1992 and won the National prize of information in 1993. The small town urban studies with an ethnic component was continued by a team under the head of professor Matti Rasanen of the Turku University in the formerly Swedish town of Loviisa and at that time the Soviet town ofVoru in Estonia as a joint Finnish-Estonian project. The Swedish part was written by an ethnologist from Abo Akademy, Marina Airo (Everyday life and Ethnicity 1994). This study focussed on the contacts and possible conflicts between Finnish- and Swedish speaking local residents of Loviisa in comparison to those of the Estonians and Russians in Voru. The 1980s - an era of new theo- retic insights and intensive fieldwork Only the last mentioned covered explicitly the Swedish speaking Finns whereas the other were directed to general issues under the headings. As chairman functioned Bo Lonnqvist and as secretary Anna-Maria Astrom. Lesser seminars and meetings were held where tourism, seaside resorts, the Russian culture in Finland and stereotypes where dwelt upon. All of the papers presented were published in the bulletin Laboratorium for folk och kultur that had since the light in 1989. The ethnological series Budkavlen that had been founded by Brage had since 1928 jointly been published by Brage and the Institute of Folk life research in Abo but in 1971 the work had been taken over entirely by the ethnologists and folklorist in Abo. Thus the new circle in Helsinki had decided to take the risk at starting a new series that had the popular stance which was connected to Brage, a society founded 1906 to ensure and preserve the interest in Swedish folk life and folklore in Finland. The 1990s - the era of new re- search in ethnicity, urban ethnol- ogy and modernisation, generational and gendered cul- ture and historical anthropology At the ethnological department in Abo the concept of culture and its consequences for its many-faceted articulation of differences could be dealt with by heavily leaning on the thought of Swedish scholars such as The 1990s - the era of new re- search in ethnicity, urban ethnol- ogy and modernisation, generational and gendered cul- ture and historical anthropology At the ethnological department in Abo the concept of culture and its consequences for its many-faceted articulation of differences could be dealt with by heavily leaning on the thought of Swedish scholars such as The decade of the 1990s proved to be productive in many other ways. The Swedish ethnologists published a book of 123 honour for Bo Lonnqvist called Kring tiden, Around time, for his 50th birthday, with a deliberate outset in the concept of time. In his teaching docent Lonnqvist had always underlined this concept among with the concepts of things and space. Bo Lonnqvists own large work on children's creative culture Ting rum och barn, Things space and children was published 1992 and won the National prize of information in 1993. The 1980s - an era of new theo- retic insights and intensive fieldwork Also new studies were initiated in the capital of Helsinki, where four large ethnological questionnaires were sent to the inhabitants looking for memories of and attitudes in the capital. Already in 1990 a book calledHemma bast ( At home is best) dedicated to childhood memories in bourgeois and worker families in the first three decades of the 20th century written by Swedish speakers was published and sold out in a year. The new material also resulted in a book Elamaa kaupungissa - Att bo i stan (Living in the city) which concentrated on memories of living in the immediate center of Helsinki in the bustle and traffic of the 1950s. The memoirs were published in the language in which they were written, either Finnish or Swedish. The writer of this article had since 1987 been working on the historical cultural historical sources and the historical antropological disseration "Sockenboarne" was presented in 1993, soon followed by Monica N erdrums dissertation Skargardskvinnor Archipelago women ( 1998), where the author discusses the modernisation process among women. An interesting and thorough study on the world view of a female museum creator and curator Irja Sahlberg, Kvinna i museivarlden, was presented by Solveig Sjoberg-Pietarinen (1997). Thus two books dedicated to women studies made the male dominated ethnology change. Already in 1987 a book written by one of Gabriel Nikanders pupils, Hjordis Dahl, could be published although the author was over eighty. The book was named Hogsang och kladbod and it is a grand survey of the textile culture of the Swedish peasantry in Finland. And thus the link to museology was once again intimately connected to the university discipline ethnology. In 1993 professor Nils Stora celebrated his 60th birthday with the Festschrift Resurser, strategier, miljoer and in 1999 the portrait of him was uncovered in the auditorium at the Humanisticum building in Abo, where he sided with professor Nikander, Tegengren and Wikman and the donators of the building councillor of commerce Ernst Dahlstrom and his wife Rosina. Professor Storas many-faceted task was successfully completed and he can be said to have done something impossible: reforming ethnology at the same time as staying true to his own visions. He has since continued his work and in 2002 a new book will appear. The 1980s - an era of new theo- retic insights and intensive fieldwork The Finnish ethnologists on the other hand began the debate by inviting anthropologists and ethnologists to reflect on cultural contacts and cultural conflicts rather than looking at different cultures from inside. The ethnic questions were thus focal and this was reflected also in dissertations about contacts between different segments of the Finnish population. Works in these matters were carried out for instance by Pirkko Sallinen-Gimpl and Outi Tuomi-Nikula. The former studied the interference between evacuated Carelians and Finns after the Second World War and the latter marriages between German men and Finnish women. These topics also activated ethnologists in the Swedish institutions: Anna-Liisa Kuczynski studied the acculturation of Polish immigrants in Finland and later wrote her licentiate thesis on the cultural loyalty in marriages between Finns and Poles. Marjut Anttonen in her tum defended her doctoral thesis on Finnish descendants in Norway and their political awakening in the 1990s. Since the middle of the decade Finland's decision to join the EU also activated the cultural sciences. quest for having to define oneself in terms of ones own had began to appear on the scene. The construction of this image of the Swedish speaking Finns relied on two different pillars, the one consisting of four regions (Ostrobothnia, Aboland, Nyland and the Aland Isles) and the other of a "pact" between the leading layers, the political party and the "people". Our goal was to show how this apparatus worked on the image plane and how confrontations between different language groups took their content from different images and stereotypes about oneself and the other. The result was a monograph with the title "Gransfolkets barn" ( Children of the border) which won a National information prize in 2002. Finland the challenge from the outset was the European integration. Many a book on what Finnishness is appeared in the early 1990s, seeking different and inherent traits but also discussing the term in a constructionist manner. The Finnish ethnologists on the other hand began the debate by inviting anthropologists and ethnologists to reflect on cultural contacts and cultural conflicts rather than looking at different cultures from inside. The ethnic questions were thus focal and this was reflected also in dissertations about contacts between different segments of the Finnish population. Works in these matters were carried out for instance by Pirkko Sallinen-Gimpl and Outi Tuomi-Nikula. The 1980s - an era of new theo- retic insights and intensive fieldwork As the 1980s in Sweden can be said to be historical sources and the historical antropological disseration "Sockenboarne" was presented in 1993, soon followed by Monica N erdrums dissertation Skargardskvinnor Archipelago women ( 1998), where the author discusses the modernisation process among women. An interesting and thorough study on the world view of a female museum creator and curator Irja Sahlberg, Kvinna i museivarlden, was presented by Solveig Sjoberg-Pietarinen (1997). Thus two books dedicated to women studies made the male dominated ethnology change. Already in 1987 a book written by one of Gabriel Nikanders pupils, Hjordis Dahl, could be published although the author was over eighty. The book was named Hogsang och kladbod and it is a grand survey of the textile culture of the Swedish peasantry in Finland. And thus the link to museology was once again intimately connected to the university discipline ethnology. In 1993 professor Nils Stora celebrated his 60th birthday with the Festschrift Resurser, strategier, miljoer and in 1999 the portrait of him was uncovered in the auditorium at the Humanisticum building in Abo, where he sided with professor Nikander, Tegengren and Wikman and the donators of the building councillor of commerce Ernst Dahlstrom and his wife Rosina. Professor Storas many-faceted task was successfully completed and he can be said to have done something impossible: reforming ethnology at the same time as staying true to his own visions. He has since continued his work and in 2002 a new book will appear. As the 1980s in Sweden can be said to be the decade when the Swedish ethnologists woke up with the questions like what is Swedish and how can Swedishness be reliably researched, the decade for such reflexive thoughts were the 1990s in Finland. This new interest was a direct result of the multicultural situation in Sweden, but in The writer of this article had since 1987 been working on the historical cultural arena of manors owned by Swedish officers and officials in the eastern Finnish district of Savo. The work was entirely based on 124 Finland the challenge from the outset was the European integration. Many a book on what Finnishness is appeared in the early 1990s, seeking different and inherent traits but also discussing the term in a constructionist manner. The 1980s - an era of new theo- retic insights and intensive fieldwork Methodological questions were discussed during seminars and the Brage Symposias: on debate were mentalities, topophilia and lived space, symbol theories, ethnicity, stereotypes, representations of different kinds, not the least in the area of museums and tourism, authenticity, and the intricate questions on memories and questionnaire answers. Ethnology had come to a reflective phase. The quest for historical milieus is at the moment under focus on different levels in Abo. Solveig Sjoberg-Pietarinen is completing her doctoral thesis on two open-air museums: The Klosterbacken handicraft museum in Abo and the Amuri workers living museum in Tampere. But her ambitions are higher: to look for what can be represented in open-air museums and how the representation work is done. As ethnology in Abo still is connected to cultural history and courses on European cultural history and historical anthropology is being taught, it is natural to look at contemporary culture with one eye to history. Besides master thesis on Internet and mobil phones many young ethnologist have been fascinated by new forms of history presentation and historically valuable living areas. The former studies have been led under the title of History in our times and encompass studies of how the 18th century is elevated in dramas and historical enactments beside expositions in museums, how the medieval times come to live one week in Abo every summer as the medieval market is put to play, and how children join pedagogical groups at the museums focusing on medieval knights and archaeological times at ecomuseums. Other studies under the head of History in local society, focus on the gentrification processes of urban areas including mostly wooden houses in the centers of the towns or just outside and the awareness of history by the inhabitants and the meanings they give the historical milieus. Other studies focus on the agrarian roots and how living on historical farms is understood and used as a means for living or how old farms only consist of one alternative while modem replicas have higher value for young families. Thus it is well understood that postmodem ideas and images are as easily planted in the rural milieu as in the towns. Th ti i d t t d The 1980s - an era of new theo- retic insights and intensive fieldwork The former studied the interference between evacuated Carelians and Finns after the Second World War and the latter marriages between German men and Finnish women. These topics also activated ethnologists in the Swedish institutions: Anna-Liisa Kuczynski studied the acculturation of Polish immigrants in Finland and later wrote her licentiate thesis on the cultural loyalty in marriages between Finns and Poles. Marjut Anttonen in her tum defended her doctoral thesis on Finnish descendants in Norway and their political awakening in the 1990s. Since the middle of the decade Finland's decision to join the EU also activated the cultural sciences. At the end of the 1990s you could fairly say that ethnology with the Swedish prefix in Abo and Helsinki was well informed with the latest trends in the field. The theoretical points of departure encompassed semiotic perspectives, historical anthropological views, constructionist views on ethnic identity and on modem modes of life in rural and urban settings. What had not been neglected either was the study of modem customs and material culture with new inspiration from symbol theories of different kind, nor where the ecological theories abandoned. The difficult connections between nature and culture were still ahead waiting for answers, not the least because of the population areas of the Finland Swedes: the vulnerable areas at the Baltic Sea. Pollution problems and other problems concerning the environment were in focus also because the Abo Akademi University had declared itself an Environment University. An ethnological answer that focused on the cultural environment of the archipelago appeared 1998 with the name Etnografi pa hemmaplan (Ethnography at home) The position of the Swedish speaking Finns in this new political and scientific context was somewhat blurry. The Academy of Finland launched a great program Ethnicity, Identity and Multiculturalism in 1994 and in this context three scholars, Bo Lonnqvist, Yrsa Lindqvist and myself, got the opportunity to offer our view on the question. The two cultures, the Finnish and Swedish was thought to intersect on each other both by transgression of borders and by upholding borders to keep the cultures as pictures or mental images clear. At the same time a The shift to studies of contemporary society was well under way and new studies 125 of urban life and urban memories were initiated in the Finnish Academy project Town Dwellers and their places, which was a joint project with ethnologists from Abo, Jyvaskyla and Helsinki. Into the new millennium and looking back to historical times The insights in industrial logistics were also thought provoking for ethnologists, and led us to contemplations about consumer society on the whole. New topics are now also found in youth culture, rites the passage and the challenge of the new migrants to Finland. Another area of great interest is also consumption where focus already has been on ecological and reliable consumption and the consumption of young people in different decades. number of students is 82. the 1950 questionnaire (200 answers) two more have been released since 2000: Our Nature (120) and Mill and factory milieus (by now 90 answers). The network being the whole of Finland we have got long narrations from very different and distant localities both in Finnish and in Swedish as the main officials and high officials in the factories used to be Swedish speaking until the end of the 1960s. The last fieldwork in May was also located to a factory milieu, in Varkaus in eastern Finland, where the students could feel as anthropologists in a somewhat alien milieu of a large paper mill and an inland town. We also visited Sorsakoski, where the manufacturing of cutlery last year was moved to France. But the delivery station was still functioning and the Hackman saucepans still produced in this factory that was started as a sawmill in 1787. Thus a vanishing culture is explored, the material of which will be useful for many disciplines in the future. The insights in industrial logistics were also thought provoking for ethnologists, and led us to contemplations about consumer society on the whole. New topics are now also found in youth culture, rites the passage and the challenge of the new migrants to Finland. Another area of great interest is also consumption where focus already has been on ecological and reliable consumption and the consumption of young people in different decades. A new project has started in 2002. It is project initiated by the Swedish Literary Society with the name Faces of the City. It is an interdisciplinary project, that focuses on urban culture in 1880-2000 in many different towns and from different angles. For the first time historians, ethnologists and researchers in literature are working together with the aim at founding new ways in looking at the urban transition and how it has been perceived. Into the new millennium and looking back to historical times International contacts are intimate with Sweden, especially with Gothenburg and Stockholm but also more distant universities as St Petersburg Kiel and Krakow are sites of contacts. Ifwe look at the near future of ethnology at Abo Akademi it can be said that focus is both on contemporary society and its historical roots; applications lie waiting for funding for two large research projects: Tradition and modernity concerning public festivities in cities and The modernisation of the countryside in which the aim is to study the new challenges rural areas and the rural population have to confront. As the interest in anthropology is growing, more than elementary courses has to be offered. Thus ethnology in Abo could once again be connected with the great traditions were Edward W estermarck started. Into the new millennium and looking back to historical times The new millennium meant even more changes and challenges also for the ethnology that can be characterised as Swedish in Finland. The starting point was defined to rely on an examination of what the post- or late modem times have meant for the cultural arena and everyday life in localities in Finland. The late modem times are said to be fragmented and focused on different life styles at the same time as history becomes a fantasy area to explore at the same time as heritage sites become popular places to visit. At the department of Ethnology at Abo Akademi the look for days gone was started by a questionnaire concerning the 1950s. This starting point could reveal both how people remembered this already remote time and how they considered the changes of time. The students made an exhibition of this material that took us the 1950s of Abo and a book Sa minns jag mitt Abo (Thus I remember my Abo). The exhibition in tum proved to be successful, and one masters thesis was dedicated to the source critics and the study of narration, generations, places and things that were mentioned in this context.. This thesis by Katja Hellman was one of many that sought to penetrate the role of History in our lives and in contemporary society. The questionnaire department under John Hackman is as active as ever. Beside 126 the 1950 questionnaire (200 answers) two more have been released since 2000: Our Nature (120) and Mill and factory milieus (by now 90 answers). The network being the whole of Finland we have got long narrations from very different and distant localities both in Finnish and in Swedish as the main officials and high officials in the factories used to be Swedish speaking until the end of the 1960s. The last fieldwork in May was also located to a factory milieu, in Varkaus in eastern Finland, where the students could feel as anthropologists in a somewhat alien milieu of a large paper mill and an inland town. We also visited Sorsakoski, where the manufacturing of cutlery last year was moved to France. But the delivery station was still functioning and the Hackman saucepans still produced in this factory that was started as a sawmill in 1787. Thus a vanishing culture is explored, the material of which will be useful for many disciplines in the future. Astrom, Anna-Maria 1999. Laboratorium for folk och kultur - 10 ar jamte artiklar 1989-1998. Laboratorum for folk och kultur 2/1999. Astrom, Anna-Maria 1996. "Budkavlar och Laboratorier" Brages gamla och nya sektion for folklivsforskning. Laboratorium for folk och kultur 1/1996, 20-23. Solveig Widen. Abo: Abo Akademis forlag, 193-208. BIBLIOGRAHY Kursfordingar i etnologi lasaret 2001-2002. Etnologi, Abo Akademi. Kursfordingar i etnologi lasaret 2001-2002. Etnologi, Abo Akademi. As has been presented, ethnology at Abo Akademi university has engaged in contacts with other ethnological departments in Finland in joint research projects. This is quite natural because the personnel at the department i Abo only consists of the mentioned professor Anna- Maria Astrom (since 2000), the intendent Johan Hackman and the assistant Niklas Hulden. Six researchers are attached to the department with projects of their own and they also take part in the training of the students. For the time being the total Lonnqvist, Bo 2000. Ett paradigmskifte i finlandsvensk etnologi. Laboratorium for folk och kultur 1/2000, 24-28. Lonnqvist, Bo 1976. Folklivsforskning. En bibilografi over Svenskfinland. Meddelanden fran folkkultursarkivet. Helsingfors:SLS. Mustelin, Olof 1986. Forskning och vitterhet. Svenska litteratursallskapet i Finland 1885-1985. Del II Det andra halvseklet. Helsingfors:SLS Stora, Nils, 1992. Five ethnologies. The rise of Finnish ethnology from a Finland-Swedish point of view. In Pioneers. The history of Finnish 127 Astrom, Anna-Maria 1996. "Budkavlar och Laboratorier" Brages gamla och nya sektion for folklivsforskning. Laboratorium for folk och kultur 1/1996, 20-23. Astrom, Anna-Maria 1999. Laboratorium for folk och kultur - 10 ar jamte artiklar 1989-1998. Laboratorum for folk och kultur 2/1999. 128 128
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Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq
Maǧallaẗ al-handasaẗ wa-al-tiknūlūǧiyā
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4,549
Abstract The conventional remotely-sensed satellite imagery is suited for regional investigations in areas of good exposures which are reduced in southern Iraq due to vegetation, water – covered areas and flat terrains. An imagery produced from digitally processed geophysical data is an extremely powerful technique for identification of geological features in such areas of extreme economic importance . By utilizing an appropriate digital processing, the geophysical contour maps have been converted to geophysical images. Images produced from digitally processed geophysical data (gravity and aeromagnetic) have been analyzed by polynomial filtering ( using several degrees ) allow major features to be delineated clearly. An integrated image of gravity and aeromagnetic data sets has been produced using the best of polynomial filtering ( 6th degree residuals ) . The interpretation of final output images shows the association of negative gravity and magnetic residual anomalies with some anticlinal structures , while positive residuals are associated with the others. The positive residuals could be due to basement uplift , and the reversed values could be due to deep- seated light core probably salt beds. Basrah depression exhibits negative residuals. Keywords: GIS; Digital image processing; Polynomial filtering; Geophysical data; Geological features. * Building and Construction Engineering Department, University of Technology / Baghdad ** Ministry of Science and Technology/ Baghdad *** Remote Sensing, Iraq. 236 ﻤﻌﺎﻟﺠﺔ ﺭﻗﻤﻴﺔ ﻟﻠﺒﻴﺎﻨﺎﺕ ﺍﻟﺠﻴﻭﻓﻴﺯﻴﺎﺌﻴﺔ ﻟﺘﺤﺩﻴﺩ ﺍﻟﻅﻭﺍﻫﺭ ﺍﻟﺠﻴﻭﻟﻭﺠﻴﺔ ﻓﻲ ﺠﻨﻭﺏ ﺍﻟﻌﺭﺍﻕ ﺍﻟﺨﻼﺼﺔ ﺍﻥ ﺍﻟﺘﺼﻭﻴﺭ ﺍﻟﻔﻀﺎﺌﻲ ﺍﻟﺘﻘﻠﻴﺩﻱ ﻤﻼﺌﻡ ﻟﻠﺘﺤﺭﻴﺎﺕ ﺍﻻﻗﻠﻴﻤﻴﺔ ﻓﻲ ﺍﻟﻤﻨﺎﻁﻕ ﺫﺍﺕ ﺍﻟﻤﻜﺎﺸﻑ ﺍﻟﺠﻴﻭﻟ ﻭﺠﻴﺔ ﺍﻟﺠﻴﺩﺓ ﻭﺍﻟﺘﻲ ﺘﻜﺎﺩ ﺘﻨﻌﺩﻡ ﻓﻲ ﻤﻨﺎﻁﻕ ﺠﻨﻭﺏ ﺍﻟﻌﺭﺍﻕ ﺒﺴـﺒﺏ ﺍﻟﻨﺒﺎﺘـﺎﺕ ﻭﺍﻟﻤﻨـﺎﻁﻕ ﺍﻟﻤﻐﻁـﺎﺓ ﺒﺎﻟﻤﻴـﺎﻩ ﻭﺍﻟﺘﻀﺎﺭﻴﺱ ﺍﻟﻤﻨﺒﺴﻁﺔ . ﺍﻥ ﺍﻟﺼﻭﺭ ﺍﻟﻨﺎﺘﺠﺔ ﻤﻥ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺍﻟﺭﻗﻤﻴﺔ ﻟﻠﺒﻴﺎﻨﺎﺕ ﺍﻟﺠﻴﻭﻓﻴﺯﻴﺎﺌﻴﺔ ﻫـﻲ ﺘﻘﻨﻴـﺔ ﻓﻌﺎﻟﺔ ﻓﻲ ﺘﺤﻴﺩ ﺍﻟﻅﻭﺍﻫﺭ ﺍﻟﺠﻴﻭﻟﻭﺠﻴﺔ ﻓﻲ ﻤﺜل ﻫﺫﻩ ﺍﻟﻤﻨﺎﻁﻕ ﺫ ﺍﺕ ﺍﻷﻫﻤﻴﺔ ﺍﻻﻗﺘﺼـﺎﺩﻴﺔ . ﺘـﻡ ﺘﺤﻭﻴـل ﺍﻟﺨﺭﺍﺌﻁ ﺍﻟﻜﻨﺘﻭﺭﻴﺔ ﺍﻟﺠﻴﻭﻓﻴﺯﻴﺎﺌﻴﺔ ﺍﻟﻰ ﺼﻭﺭ ﺠﻴﻭﻓﻴﺯﻴﺎﺌﻴﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻤﻌﺎﻟﺠﺔ ﺭﻗﻤﻴﺔ ﻤﻼﺌﻤﺔ . ﺘﻡ ﺘﺤﻠﻴـل ﺍﻟﺼﻭﺭ ﺍ ﻟﻨﺎ ﺘﺠﺔ ﻤﻥ ﺍﻟﺒﻴﺎﻨﺎﺕ ﺍﻟﺠﻴﻭﻓﻴﺯﻴﺎﺌﻴﺔ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺭﻗﻤﻴﺎ ) ﺍﻟﺠﺫﺒﻴﺔ ﻭﺍﻟﻤﻐﻨﺎﻁﻴﺴﻴﺔ ﺍﻟﺠﻭﻴﺔ ( ﺒﺎﺴﺘﺨﺩﺍﻡ ﺘﻘﻨﻴﺔ ﺍﻟﺘﺭﺸﻴﺢ ﻤﺘﻌﺩﺩ ﺍﻟﺴﻁﻭﺡ ) ﻭﺒﺩﺭﺠﺎﺕ ﻤﺨﺘﻠﻔﺔ ( . ﺴـﻤﺤﺕ ﺍﻟﺼـﻭﺭ ﻋﻠـﻰ ﺘﺤﺩﻴـﺩ ﺍﻟﻅـﻭﺍﻫﺭ ﺍﻟﺠﻴﻭﻟﻭﺠﻴﺔ ﺍﻟﺭﺌﻴﺴﺔ ﺒﻭﻀﻭﺡ . ﺃﻨﺘﺠﺕ ﺼﻭﺭﺓ ﻤﺘﻜﺎﻤﻠﺔ ﻟﻠﺒﻴﺎﻨﺎﺕ ﺍﻟﺠﺫﺒﻴﺔ ﻭﺍﻟﻤﻐﻨﺎﻁﻴﺴﻴﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻤﺘﻌﺩﺩ ﺍﻟﺴﻁﻭﺡ ) ﺍﻟﺩﺭﺠﺔ ﺍﻟﺴﺎﺩﺴﺔ ( . ﺃﻅﻬﺭﺘﻔﺴﻴﺭ ﺍﻟﺼﻭﺭ ﺍﻟﻨﺎﺘﺠﺔ ﻤﺼﺎﺤﺒﺔ ﺍﻟﺸـﻭﺍﺫﺍﻟﺠﺫﺒﻴﺔ ﻭﺍﻟﻤﻐﻨﺎﻁﻴﺴـﻴﺔ ﺍﻟﺴﺎﻟﺒﺔ ﻤﻊ ﺍﻟﺘﺭﺍﻜﻴﺏ ﺍﻟﻤﺤﺩﺒﺔ , ﺒﻴﻨﻤﺎ ﺼﺎﺤﺒﺕ ﺍﻟﺸﻭﺍﺫ ﺍﻟﻤﻭﺠﺒﺔ ﺍﻟﺒﻌﺽ ﺍﻷﺨﺭ ﻤﻨﻬﺎ . Eng. & Tech. Journal, Vol.28, No.2, 2010 Eng. & Tech. Journal, Vol.28, No.2, 2010 Introduction 47º- 48º longitudes which is a bout 12,000 km2. This area is generally featureless except the presence of Jabal Sanam uplift , but it lies on alluvial mud laid down by the river and the original ground level varies between minimum 0.0 m and Unlike the common other remote sensing data, geophysical data have traditionally been present as contour maps in order to acquire regional information [1,2]. However, contour plots do not fully express all information available from geophysical data sets. This is because they only show fixed (and arbitrarily defined) intervals rather than the entire continuum of intensity variation. In addition, the human visual system evolves to optimize information extraction from an ‘image’ rather than the analyses of mapped lines .On the other hand, when more than one method are applied the integrated data interpretation will be done as a comparison between two or more separated maps even when these data are plotted at the same sheet [3,4] . Furthermore, most of remote sensing data interpretation programs deal with images rather than contour maps. Moreover, it is easier to study the relationship between geophysical data and satellite data when geophysical data are represented as an image [1]. Therefore this study aimed to apply appropriate digital processing techniques for conversion of geophysical contour maps to geophysical images to overcome the disadvantages of using such contours and to utilize different processing operations for geophysical images and synergistic display of data recorded by different remote sensing systems. G l f th A maximum 4.0 m (GTS). The other main features within the area are Tigris, Euphrates and Shatt Al-Arab rivers , the large marsh ( Hor Al- Hammar ) in the northern part , and Al -Batin fan [5,6]. Geologically , the area is covered mainly by recent late Pleistocene sediments of Al-Batin fan and recent sediments of Tigris – Euphrates rivers . Besides, other sediments of older ages recognized by Dibdibba, Zahra and Injana formations . Dibdibba formation is exposed through extensive flat and slightly wavy terrains of the area . Tectonically , the area is located within the limits of Mesopotamian Zone (Unstable Shelf). It exhibits the presence of gently plunging subsurface structures generally N-S trending anticlines and synclines of different sizes having poor reflection on earth surface . Abstract ﻭﻗﺩ ﺘﻜﻭﻥ ﺍﻟﺸﻭﺍﺫ ﺍﻟﻤﻭﺠﺒﺔ ﻨﺎﺘﺠﺔ ﻋﻥ ﻨﻬ ﻭ ﺽ ﺘﺭﻜﻴﺏ ﺍﻟﻘﺎﻋﺩﺓ , ﺒﻴﻨﻤﺎ ﺍﻟﻘﻴﻡ ﺍﻟﻤﻌﺎﻜﺴﺔ ﻗﺩ ﺘﻜﻭﻥ ﺒﺴﺒﺏ ﺍﻟﻠﺏ ﺍﻟﺨﻔﻴﻑ ) ﺭﺒﻤﺎ https://doi.org/10.30684/etj.28.2.2 2412-0758/University of Technology-Iraq, Baghdad, Iraq This is an open access article under the CC BY 4.0 license http://creativecommons.org/licenses/by/4.0 * Building and Construction Engineering Department, University of Technology / Baghdad ** Ministry of Science and Technology/ Baghdad *** Remote Sensing, Iraq. 236 https://doi.org/10.30684/etj.28.2.2 2412-0758/University of Technology-Iraq, Baghdad, Iraq This is an open access article under the CC BY 4.0 license http://creativecommons.org/licenses/by/4.0 * Building and Construction Engineering Department, University of Technology / Baghdad ** Ministry of Science and Technology/ Baghdad *** Remote Sensing, Iraq. Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq ﻁﺒﻘﺎﺕ ﺍﻟﻤﻠﺢ ( ﺍﻟﻤﻭﺠﻭﺩ ﻋﻠﻰ ﺃﻋﻤﺎﻕ ﻜﺒﻴﺭﺓ . ﻭﻴﺘﻤﻴﺯ ﻤﻨﺨﻔﺽ ﺍﻟﺒﺼﺭﺓ ﺒﻭﺠـﻭﺩ ﺍﻟﻤﺘﺒﻘﻴـﺎﺕ ﺍﻟﺴـﺎﻟﺒﺔ . Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq 47º- 48º longitudes which is a bout 12,000 km2. This area is generally featureless except the presence of Jabal Sanam uplift , but it lies on alluvial mud laid down by the river and the original ground level varies between minimum 0.0 m and maximum 4.0 m (GTS). The other main features within the area are Tigris, Euphrates and Shatt Al-Arab rivers , the large marsh ( Hor Al- Hammar ) in the northern part , and Al -Batin fan [5,6]. Methodology (Geophysical Data Sets and Digital Image Processing) As aforementioned , contour plots do not fully express all the information available from geophysical data sets that is mainly because they only show fixed and arbitrarily defined intervals Geology of the Area Introduction Most of these structures resulted from basement block movements and salt tectonics and are of extreme economic importance for their huge amounts of hydrocarbons [5,7] . Methodology (Geophysical Data Sets and Digital Image Processing) Geology of the Area The area under investigation (Fig.1) is situated in the southern part of Iraq, between 30º - 31º latitudes and 237 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq rather than the entire continuum of intensity variations. It is possible to overcome these disadvantages because the appropriate digital processing the geophysical data can be interpolated to a regular grid of values analogous to an image composed of pixels. Geologists look for geophysical methods to exploit the synoptic view of large regions, besides images of such regions may analyze geological provinces as entities rather than in a piecemeal fashion. Thus imagery was ideally suited for regional investigations in areas of good exposure though results in temperate terrain often proved disappointing because of extensive vegetation cover [8] . images using the following processing steps : 1-The scanning process by which the hard copy contour map is converted to digital image as a matrix of pixels , each pixel holds a reflectance value in the scanned map . p 2-Geometric corrections of the scanned maps with World Geometric System ( WGS84 ) by using ERDAS .The original coordinates of geophysical maps grid intersections, in Clark 1880 ellipsoid and UTM projection, were converted to (WGS 84) ellipsoid and still in UTM projection by using the geographic translator program (GEOTRANS) which is an application that converts geographic coordinates among a wide variety of coordinate systems, map projections, grids and datums. Besides, the digital copy of the scanned map has many geometric errors incoming from hard copy paper shrinkage and from scanner itself. Therefore it needs to be geometrically corrected. This process had been done by using ERDAS package, to correct these maps and change their datum according to the new coordinate of known grid coordinates. Consequently, geologists had to rely on geophysical data which have traditionally been presented as contour maps, in order to acquire regional information. So, this section will deal converting potential field maps ( both gravity band magnetic fields ) to Digital Elevation Model ( DEM ) raster images. These DEMs are processed as images. Different processing operations used for this section will be discussed in detail later. 3-Vectorization process of the geometrically corrected contour maps. Vectorization (or digitization) that attempts to distill points, lines and polygons from a scanned image . 3-Vectorization process of the geometrically corrected contour maps. Vectorization (or digitization) that attempts to distill points, lines and polygons from a scanned image . PDF created with pdfFactory Pro trial version www.pdffactory.com The geophysical data sets are presented as two contour maps. The first is the Bouguer anomaly with scale 1 : 1000 000 compiled by Iraqi Petroleum Company, IPC ( 1960 ) with contour interval of 1 mgal. The second is the aeromagnetic map that carried out by C.G.G.(1974) with contour interval of 0.5 gamma. The original Bouguer map, which incorporated negative and positive values , was 4-Computing geophysical points on regular grids ( 1 km. ) by creating a surface from all contour lines and creating points as (X,Y,Z coordinates ) from that surface . A surface model created from these data, is a three- dimensional geometric representation of the surface of an area of land. These surfaces represent phenomena that have values at every point across their extent. They are usually used to represent terrain and created from initially rescaled to a positive one. The conversion of the geophysical contour maps and their datasets to raster 238 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq 7-Data enhancement and interpretation. Images produced from digitally processed geophysical data ( gravity and aeromagnetic ) ( Figs. 2 and 3 ) have been analyzed by polynomial filtering , using several degrees, to allow major features to be delineated clearly. values sampled at height values for an elevation surface, but here it refers to the geophysical value. A TIN , or triangulated irregular network , is a surface data structure composed of triangular facets defined by nodes and angles. To create TIN lines, Autodesk Land Desktop connects the surface points that are closest together. These TIN lines interpolate surface elevations, filling the gaps where no survey data or contour data is known, to create an approximation of the surface. The g y g ( Figs. 2 and 3 ) have been analyzed by polynomial filtering , using several degrees, to allow major features to be delineated clearly. Results and Discussion ( DEM ), but here the word elevation refers to the geophysical value in the image . This system incorporates the functions of both image processing and GIS . These functions include importing, viewing, altering, and analyzing raster and vector data sets . One of the most flexible analytical techniques for residual –regional separation is the polynomial . A computer program written in MATLAB language was used for polynomial equations. The regional is represented by low order analytic surface. 6-Combining geophysical single band images into one image (multi–band image) with three bands. After creating various geophysical “single band ” images, each three of these images can be combined in one image with three layers “three bands”. Each one of these layers is represented in one color. The resulted image is RGB image. Polynomial filtering in various degrees (from 1st to 6th degrees) have been applied for both gravity and aeromagnetic images in order to 239 PDF created with pdfFactory Pro trial version www.pdffactory.com Results and Discussion The original gravity and aeromagnetic images are shown in figures ( 2 and 3 ). Generally, pixels of original Bouguer gravity images are of negative values which were rescaled to a positive grayscale range 0 - 255. Bouguer values are generally decreased from - 37 mgal in the southeast to - 81 mgal in the northwest .High gravity gradients and low negative Bouguer values are at the southern part, whereas low gravity gradients and relatively strong negative Bouguer values at the northern part. ( geophysical values ). TINs capture the variation in a surface better than do rasters. The spot elevations (geophysical values) can be irregularly distributed to accommodate areas of high variability in the surface and their values and exact positions are retained as nodes in the TIN. This makes TINs well suited to geological and engineering applications. ( geophysical values ). TINs capture the variation in a surface better than do rasters. The spot elevations (geophysical values) can be irregularly distributed to accommodate areas of high variability in the surface and their values and exact positions are retained as nodes in the TIN. This makes TINs well suited to geological and engineering applications. Pixel Values of aeromagnetic image within the area are positive and decrease slightly from NW to SE. the maximum and minimum values are 4920 gammas in NW and 4833 gammas in the SE of the area respectively . The general horizontal gradient of the magnetic field appears high in W and NW portions of the area, while low gradient is presented in E and SE portions of the area. 5-Converting the geophysical points into image using ERDAS IMAGINE package to create a Surface of Digital Elevation Model 5-Converting the geophysical points into image using ERDAS IMAGINE package to create a Surface of Digital Elevation Model Digital Elevation Model ( DEM ), but here the word elevation refers to the geophysical value in the image . This system incorporates the functions of both image processing and GIS . These functions include importing, viewing, altering, and analyzing raster and vector data sets . 6-Combining geophysical single band images into one image (multi–band image) with three bands. After creating various geophysical “single band ” images, each three of these images can be combined in one image with three layers “three bands”. Each one of these layers is represented in one color. The resulted image is RGB image. PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq To avoid low brightness and obtaining a clear representation for subsurface image of gravity and aeromagnetic data sets is produced by using the results of 6th degree polynomial residuals and displaying a resulted image as three layer image (RGB), by using the principal component ( PC ) and aeromagnetic / gravity ratio as the third input band ( Fig.7 a and b ) . Results show that the effect of the third layer is very regional in both third layer ( aeromagnetic / gravity ratio and PC ) . Thus the output geophysical image consists practically two layers only, Green-gravity and blue-aeromagnetic on red background . From the latter image ( Fig.8 ), it is clearly shown that the main anomalies have been well emphasized and one may indicate these anomalies and their structures by their DEM values in order to describe their signature in the images as shown in table1 . separate the residual field ( or the local field which is related to shallow structures ) from the trend field ( which is the regional field and related to deeper structures ) . For each original image, two images have been obtained for each degree as shown in figures 4 and 5 for gravity and aeromagnetic respectively . The application of polynomial filtering gives clear signatures for both local and general trend anomalies. It is clearly shown that the sizes of these anomalies are directly proportional to the degree of the polynomial degree. Thus the 6th degree polynomial (and 5th degree to the some extent) are the best degrees for residual separation as they reflect well the local anomalies and their related subsurface structures. So values resulted from applying the MATLAB program to the 6th degree polynomial were converted to raster images as shown in figure 6 . g p y g Thus the 6th degree polynomial (and 5th degree to the some extent) are the best degrees for residual separation as they reflect well the local anomalies and their related subsurface For gravity images, there is a great similarity between images of 5th and 6th trends with low pass filter (LPF) results and residuals with high pass filter ( HPF) results .The main structures appeared in HPF and polynomial residual images are N. Rumaila, S. PDF created with pdfFactory Pro trial version www.pdffactory.com Rumaila, N. Zubair ,S. Zubair, Nahr-Umr, Siba and Ratawi anticlinal structures besides Basrah depression. Conclusions From this work one can say that there is an ability to overcome the disadvantages of using contour maps to study the geophysical fields. The use of conventional contour plots is completely superseded by the processing techniques. Therefore many geophysical maps can be individually processed and displayed in pseudo-color imagery. Generally, the main conclusions derived from this study are: For aeromagnetic images, the general trend resulted from LPF and polynomial surface images are more complex than those in gravity images. Images of HPF and polynomial residuals reflect more anomalies referring to the same subsurface structures, but with less reflection to Nahr-Umr, Siba and Ratawi structures and better reflection for N. and S. Zubair structures. 1- The data are quickly manipulated when imagery is produced and the analyses are speedily performed and extremely powerful for the acquisition of geological information. 2- Polynomial filtering with 5th and 6th degree showed the best separation techniques among others degrees by allowing the 240 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Geophysical Features in Northern Ireland. INT. J. Remote Sensing: 14, 1, 161-17, 1993. Geophysical Features in Northern Ireland. INT. J. Remote Sensing: 14, 1, 161-17, 1993. major geophysical structures and features in southern Iraq to be delineated. 3- An integrated image of gravity and aeromagnetic data sets is produced using the results of 6th degree polynomial residuals and displaying a resulting image as RGB image is a powerful technique to analyze the geophysical data sets of gravity and magnetic data. [2] Sharma, P. V. Environmental and Engineering Geophysics. Second Edition. Cambridge University Press, 1997. [3] Sabins , F.F. Remote Sensing ; Principles and Interpretation , 2nd ed. , Remote Sensing Enterprise, Inc. , New York United Kingdom [4] Hamdullah, A. H. A Synergistic Study of Digitally Processed Geophysical and Satellite Datasets. M.Sc. Thesis, University of Technology, Baghdad,Iraq,2007. 4- Interpretation of geophysical images shows the association of negative residual gravity anomalies with N. Zubair, N and S. Rumaila, Nahr-Umr, Ratawi subsurface anticlinal structures and Basrah depression. The reversed negative sign of anomalies could be due to deep- seated light core of probably salt beds, whereas positive residuals are associated with Siba anticlinal structure. Jabal-Sanam is another positive gravity anomaly but with lower residual than the surrounding area. This contrast may be caused by salt dome. [5] Karim,H.H. Structural nature of lower Mesopotamian region from geophysical observations. Proceedings of the 3rd workshop on oceanography of Khor Al-Zubair. Marine Science Centre Publications (16),14 – 26 ,1992. [6] Karim H.H. (1998). Developmental stages and tectonic stability of southern Mesopotamia during recent geological history. Marina Mesopotamica : 13,2, 149- 174,1998. 5- The difference between gravity and aeromagnetic results is attributed to the fact that magnetic method is sensitive to magnetized rock, so that most magnetic effects observed by airborne way are essentially the same as if the sediments were not present at all. Besides the complexity nature of the vertical distribution of rocks in the studied area. [7] Al-Marsoumi, A. H. Petroleum Geology of Mesopotamia ( general review ) . The Mesopotamian Petroleum Researches,2006. http://www.geologyofmesopotamia.co m/Quaternary.htm. [8] Drury, S. A. Remote sensing of structure in temperate agriculture terrains . Geological magazine : 123, 113-121,1986. References [1] Gibson, P. J. Evaluation of Digitally Processed Geophysical Data Sets for the Analyses of 241 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Table (1) Anomolies associated with the main features Anomaly number in figure (8) Feature Name 6th degree polynomial residual anomalies DEM values Magnetic anomaly (gamma) Gravity anomaly (mgal) 1 N. Rumaila +1.5 -1.5 2 N. Rumaila +0.5 -2.0 3 S. Rumaila +1.0 -1.0 4 Nahr-Umr +0.6 -2.5 5 Ratawi -4.0 -2.0 6 N. Zubair -0.5 -2.3 7 S. Zubair +5.0 -0.5 8 Siba +1.5 +0.7 9 Basrah depression -2.0 -1.0 10 Jabal Sanam -1.9 +0.9 242 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (1) Area of study Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (1) Area of study 243 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (2) Original Bouguer gravity image Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (2) Original Bouguer gravity image Figure (2) Original Bouguer gravity image 244 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (3) Original aeromagnetic image Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Figure (3) Original aeromagnetic image 245 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq for Identification of Geological Features in Southern Iraq Figure (4) Output figures of applying 1st to 6th degrees polynomials for gravity data. Residual images ( right side); trend surface images ( left side ) (To be continued) th Figure (4) Output figures of applying 1st to 6th degrees polynomials for gravity data. Residual images ( right side); trend surface images ( left side ) (To be continued) 246 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Southern Iraq Figure (4) ( continued ) Figure (4) ( continued ) Figure (4) ( continued ) PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Southern Iraq Figure (5) Output figures of applying 1st to 6th degrees polynomials for aeromagnetic data. Residual images ( right side ); trend surface images ( left side ) , (To be continued) Figure (5) Output figures of applying 1st to 6th degrees polynomials for aeromagnetic data. Residual images ( right side ); trend surface images ( left side ) , (To be continued) 248 PDF created with pdfFactory Pro trial version www.pdffactory.com 248 PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (5) (continued) Figure (5) (continued) Figure (5) (continued) Figure (5) (continued) b a 249 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq c d Figure (6) Residual images and trend surfaces of 6th degree polynomial: a- Residual gravity data; b- Residual magnetic data; c- Trend surface gravity data; d-Trend surface magnetic data. d d c c Figure (6) Residual images and trend surfaces of 6th degree polynomial: a- Residual gravity data; b- Residual magnetic data; c- Trend surface gravity data; d-Trend surface magnetic data. 250 PDF created with pdfFactory Pro trial version www.pdffactory.com 250 PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq A b Figure (7) Three layers images (RGB mages) a- Red- principal component (PC); Green- gravity and Blue- aeromagnetic. b- Red- aeromagnetic / gravity ratio; Green- gravity and Blue- aeromagnetic. Eng. & Tech. Journal, Vol. 28, No. PDF created with pdfFactory Pro trial version www.pdffactory.com 2, 2010 A b Figure (7) Three layers images (RGB mages) Figure (7) Three layers images (RGB mages) a- Red- principal component (PC); Green- gravity and Blue- aeromagnetic. a- Red- principal component (PC); Green- gravity and Blue- aeromagnetic. b- Red- aeromagnetic / gravity ratio; Green- gravity and Blue- aeromagnetic. 251 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Eng. & Tech. Journal, Vol. 28, No. 2, 2010 Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (8) Two layer geophysical image. Green- gravity; Blue- magnetic on a red background . 1 and 2- N. Rumaila ; 3- S. Rumaila ; 4- Nahr-Umr ; 5- Ratawi ; 6- N. Zubair ; 7- S. Zubair ; Siba ; 9- Basrah depression and 10- Jabal Sanam . Digitally Processed Geophysical Data Sets for Identification of Geological Features in Southern Iraq Figure (8) Two layer geophysical image. Green- gravity; Blue- magnetic on a red background . 1 and 2- N. Rumaila ; 3- S. Rumaila ; 4- Nahr-Umr ; 5- Ratawi ; 6- N. Zubair ; 7- S. Zubair ; Siba ; 9- Basrah depression and 10- Jabal Sanam . 252 PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com
https://openalex.org/W3082359147
https://www.frontiersin.org/articles/10.3389/fnbot.2020.00060/pdf
English
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A Brain-Inspired Model of Theory of Mind
Frontiers in neurorobotics
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cc-by
15,124
Edited by: Jun Tani, Okinawa Institute of Science and Technology Graduate University, Japan Reviewed by: Jing Zhao, Yanshan University, China Xiaoqian Mao, Qingdao University of Science and Technology, China *Correspondence: Yi Zeng yi.zeng@ia.ac.cn Keywords: theory of mind, false-belief task, brain inspired model, self-experience, connection maturation, inhibitory control A Brain-Inspired Model of Theory of Mind Yi Zeng 1,2,3,4*†, Yuxuan Zhao 1†, Tielin Zhang 1, Dongcheng Zhao 1,4, Feifei Zhao 1 and Enmeng Lu 1 Yi Zeng 1,2,3,4*†, Yuxuan Zhao 1†, Tielin Zhang 1, Dongcheng Zhao 1,4, Feifei Zhao 1 and Enmeng Lu 1 1 Research Center for Brain-Inspired Intelligence, Institute of Automation, Chinese Academy of Sciences, Beijing, China, 2 Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Beijing, China, 3 National Laboratory of Pattern Recognition, Institute of Automation, Chinese Academy of Sciences, Beijing, China, 4 School of Artificial Intelligence, University of Chinese Academy of Sciences, Beijing, China Theory of mind (ToM) is the ability to attribute mental states to oneself and others, and to understand that others have beliefs that are different from one’s own. Although functional neuroimaging techniques have been widely used to establish the neural correlates implicated in ToM, the specific mechanisms are still not clear. We make our efforts to integrate and adopt existing biological findings of ToM, bridging the gap through computational modeling, to build a brain-inspired computational model for ToM. We propose a Brain-inspired Model of Theory of Mind (Brain-ToM model), and the model is applied to a humanoid robot to challenge the false belief tasks, two classical tasks designed to understand the mechanisms of ToM from Cognitive Psychology. With this model, the robot can learn to understand object permanence and visual access from self-experience, then uses these learned experience to reason about other’s belief. We computationally validated that the self-experience, maturation of correlate brain areas (e.g., calculation capability) and their connections (e.g., inhibitory control) are essential for ToM, and they have shown their influences on the performance of the participant robot in false-belief task. The theoretic modeling and experimental validations indicate that the model is biologically plausible, and computationally feasible as a foundation for robot theory of mind. ORIGINAL RESEARCH published: 28 August 2020 doi: 10.3389/fnbot.2020.00060 1. INTRODUCTION Theory of Mind (ToM) is the ability to infer and understand other people’s mental states to predict their behavior (Premack and Woodruff, 1978). It is a fundamental cognitive ability for the social brain. One of the most critical milestones in the ToM development is gaining the ability to attribute false belief: that is, to recognize that others can have beliefs about the world that are diverging (Wimmer and Perner, 1983). There is a wide variety of false-belief task (Huang and Liu, 2017; Scott and Baillargeon, 2017), but most of them can be divided into unexpected transfer task (Wimmer and Perner, 1983), unexpected contents task (Perner et al., 1987), and appearance- reality distinction (Flavell et al., 1983). Flavell et al. (1983) present a classical unexpected transfer task, Sally-Anne Test: Sally first placed a marble into her basket; then, she left the scene, and the marble was transferred by Anne and hidden in her box. Then Sally returned, and children were asked a belief question “Where will Sally look for her marble?” If the children pointed Received: 22 May 2020 Accepted: 27 July 2020 Published: 28 August 2020 Citation: Zeng Y, Zhao Y, Zhang T, Zhao D, Zhao F and Lu E (2020) A Brain-Inspired Model of Theory of Mind. Front. Neurorobot. 14:60. doi: 10.3389/fnbot.2020.00060 Received: 22 May 2020 Accepted: 27 July 2020 Published: 28 August 2020 Abbreviations: ToM, theory of mind; STS, superior temporal sulcus; TPJ, temporo-parietal junction; IPL, inferior parietal lobule; pSTS, posterior superior temporal sulcus; PCC, posterior cingulate cortex; ACC, anterior cingulate cortex; mPFC, medial prefrontal cortex; vmPFC, ventral medial prefrontal cortex; dmPFC, dorsal medial prefrontal cortex; IFG, inferior frontal gyrus; vPMC, ventral premotor cortex; M1, primary motor cortex. 2.1. Computational Models 2.1. Computational Models Berthiaume et al. (2013) presented a constructivist connectionist model to simulate the false-belief task. The model encoded the location of an object, whether an agent has observed the object’s movement, and the location where the agent came back to search. With the increased hidden units to improve computational power, the model would predict the correct search in two different false belief tasks—the approach task and the avoidance task. Their model was the first computational model to autonomously construct and transit between structures and to cover the two major false-belief task transitions. They suggested the view that the source of the transition is not developed in the understanding of beliefs, but changes in auxiliary skills such as: executive function, understanding and using representations, working memory, or language. Goodman et al. (2006) built two Bayesian models named CT model (copy theorist) and PT model (perspective theorist). Beliefs were only correlated to the location of the toy in the former model, and in the later model, the belief was not only correlated to the toy’s location but also Sally’s visual access, i.e., could Sally saw the toy moved or not. With the increase of resources and complexity in the PT model, the model could pass the false-belief task. Asakura and Inui (2016) designed a Bayesian framework that integrates theory-theory and simulation theory for false belief reasoning in the unexpected- contents task. This framework predicted other’s belief by the self model and others model which were responsible for simulation- based and theory-based reasoning, respectively. In their opinion, the multiplicative effect of the ability to understand diverse beliefs and knowledge access could predict children’s false belief ability. Their model provided good fits to a variety of ToM scale data for preschool children. Rabinowitz et al. (2018) designed a ToM neural network to learn how to model other agents by meta-learning. They constructed an observer who could collect agent’s behavioral traces, and its goal was to predict the agent’s future behavior. They applied the proposed ToMnet model in simple grid world environments, showing that the observer could model agents effectively and passed Sally Anne Test. And the observer needed not to be able to execute the behaviors itself. O’Laughlin and Thagard (2000) built a connectionist network From our point of view, self-experience in autobiographical memory and its utilization to infer other’s belief or predict other’s action is fundamental and crucial to the ToM. 2. RELATED WORKS In this section, we briefly review several related works, including the computational models, the false belief tasks, and the related brain regions of ToM. Citation: Zeng Y, Zhao Y, Zhang T, Zhao D, Zhao F and Lu E (2020) A Brain-Inspired Model of Theory of Mind. Front. Neurorobot. 14:60. doi: 10.3389/fnbot.2020.00060 August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org Brain-Inspired Theory of Mind Model Zeng et al. to the previous location of the marble, it meant that the children could understand that Sally held a false belief about the marble’s location. Most 4-year-olds could point to the correct location, but most 3-year-olds failed—they predict that Sally will find her marble in the box. neural network model and is fundamentally based on brain plasticity principles. The rest of this paper is organized as follows: Section 2 reviews the related work of computational models, the false belief tasks, and the brain regions in the ToM. In section 3, the architecture of the Brain-ToM model, the concrete neural network architecture, the Voltage-driven Plasticity centric Spiking Neural Networks (VPSNN), and the inhibitory control mechanism are introduced. The experimental settings, the experimental results and analyses are given in section 4. Some discussions and conclusions are drawn in sections 5, 6, respectively. As indicated in Asakura and Inui (2016), although ToM research has made progress on empirical findings and theoretical advances, relatively few efforts have been made from the biological plausible computational models’ perspective, especially for false belief understanding. Based on findings of neural correlates and mechanisms of the false-belief task, we propose and build a Brain-inspired model of Theory of Mind (Brain-ToM model). And we challenge the false-belief task by incorporating the proposed model to humanoid robots. In this paper, we only focus on non-verbal unexpected transfer tasks as described below, including how to learn to understand object permanence and visual access from self-experience and use them to infer other’s belief and predict their behavior. The object permanence is the ability to understand that objects continue existence even it cannot be perceived (Piaget and Cook, 1952). 2.1. Computational Models It is also mentioned as self-projection in Buckner and Carroll (2007) or using memories to understand others (Moreau et al., 2013). It enables real understanding of the self and others as well as their relationships, and utilize them to infer others’ mental states based on personal experience from the self point of view. This perspective seems somewhat missing in existing research about the computational model. In our opinion, an agent who can infer other’s belief and predict their behavior should have the capability of self-other distinction as the premise. So in Zeng et al. (2016, 2017), we proposed a brain-inspired robot bodily self-model with the neural mechanisms of bodily self-perception based on extensions to primate mirror neuron system, and apply it to the humanoid robot for self-recognition. In this paper, based on the related findings for neural correlates and mechanisms of the ToM, we propose a Brain-ToM model to make the humanoid robot learn from self-experience. With the Brain-ToM model, the robot can pass the non-verbal unexpected transfer tasks adapted from Senju et al. (2011) and Southgate et al. (2007). The efforts may also provide a possible computational model and hints on how infant infers and understands other people’s beliefs. Compared to the previous model, the characteristics of our model are with relatively more solid details from the biological brain. It explores the effect of self-experience as a core and is with considerations on the maturation of correlated brain areas (e.g., calculation capability) and their connections (e.g., inhibitory control). Besides, the model is naturally a brain-inspired spiking August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org 2 Brain-Inspired Theory of Mind Model Zeng et al. 2.3. Brain Regions in Theory of Mind Several brain regions, including the mPFC, bilateral TPJ, and precuneus, have been consistently found to be activated in various mentalizing tasks in healthy individuals (Green et al., 2015). Schurz et al. (2014) meta-analyzed 757 activation foci reported from 73 imaging studies of ToM that involved 1,241 participants, and their meta-analysis contained six different task groups—False belief vs. photo, Trait judgments, Strategic games, Social animations, Mind in the eyes, and rational actions. They found the mPFC and bilateral posterior TPJ were activated in all task groups. In false belief vs. 3. METHODS 3.1. Architecture of the Brain-ToM Model The architecture of the Brain-ToM model is shown in Figure 1. The STS is sensitive to biological motion, and in our computational model, its function is to visually encode biological motion (Grossman and Blake, 2002). The TPJ is considered as a crucial area in self-other distinction (Eddy, 2016; Bardi et al., 2017), controls representations relating to the self and other (Eddy, 2016), and involvement in self perspective-taking as well as other perspective-taking (Vogeley et al., 2001; van der Meer et al., 2011). There is no consensus on the anatomical definition of the extent and precise location of the TPJ (Igelstrom and Graziano, 2017), but in general, the TPJ contains two anatomically distinct regions including the IPL and pSTS (Abu-Akel and Shamay- Tsoory, 2011; Schurz et al., 2014; Igelstrom and Graziano, 2017). In our computational model, the TPJ is used to distinguish self and others, store self and other-relevant stimuli, and decide the output sequence of self and other-relevant stimuli. Southgate et al. (2007) used an anticipatory looking measure to test whether 2-year-olds infants have the ability of false belief understanding. In the familiarization trials, the puppet hid an object in the left or right box, then left the scene. The actor reached through the corresponding window after doors illuminated with the simultaneous chime. Note that “doors illuminated with simultaneous chime” indicated that the actor was going to reach the object. In one test trial, the puppet hid the object in the left box then move it to the right box. After the actor turned around, the puppet removed the object from the scene. In another test trial, the puppet hid the object in the left box, then the actor turned around. The puppet moved the object to the right box and hid it, then remove the object from the scene. For both test trails, the actor turned back and doors illuminated with simultaneous chime after the object was removed from the scene. Most infants could gaze toward the correct window. Their data demonstrated that 25-month-old infants had the ability of false belief understanding. The details of this experiment were illustrated in the figure of Southgate et al. (2007). The IPL is considered as a critical area in distinguishing the self from others and identifying the body ownership in our robot bodily self-model in Zeng et al. 2.2. False Belief Tasks There is a wide variety of false-belief tasks, here we introduce two non-verbal unexpected transfer tasks that will be adapted to verify the validity of the model. Senju et al. (2011) investigated whether 18-month-olds infants would use their own past experience of visual access to attribute perception and consequent beliefs to other people. Infants are divided into two groups, one group wore opaque blindfolds, and another wore trick blindfolds which looked opaque but were actually transparent. The opaque blindfold and trick blindfold looked identical. The test stage is the same as Southgate’s as described below. The puppet hid an object in the left box. After the actor wore the same blindfold, the puppet removed the object from the scene. The opaque blindfold group expected the actor to behave according to false belief, and the trick blindfolds did not. Their results show that 18-month-olds used self-experience with the blindfold to assess the actor’s visual access and predict their behavior. 2.1. Computational Models photo stories task group, they found TPJp, IPL, precuneus, posterior cingulate gyrus, mPFC connectivity clusters 3 and 4, ventral parts of the mPFC, anterior cingulate gyrus, right anterior temporal lobe, and adjacent parts of the insula be activated. Molenberghs et al. (2016) conducted a series of activation likelihood estimation (ALE) meta-analyses on 144 datasets (involving 3,150 participants) to address the brain areas that implicated in specific types of ToM tasks. In terms of commonalities, consistent activation was identified in the medial prefrontal cortex and bilateral temporoparietal junction. Schurz and Perner (2015) reviewed nine current neurocognitive theories of how the ToM was implemented in the brain and evaluate them based on the results from a recent meta-analysis by Schurz et al. (2014). From theories about cognitive processes being associated with certain brain areas, they deduced predictions about which areas should be engaged by the different types of ToM tasks. These brain areas contain the mPFC, the pSTS, the TPJ, and the IPL. whose nodes represent the relevant event in the false-belief task, and passed the false-belief task by modifying the connection weight of excitatory links and inhibitory links. Milliez et al. (2014) presented a spatio-temporal reasoning system SPARK, which included a well-designed model of object position hypotheses and generated beliefs. They enabled the robot to passed the Sally-Anne test and performed well in dialog disambiguation. Patacchiola and Cangelosi (2020) proposed a developmental cognitive architecture for trust and ToM in humanoid robots. This architecture was inspired by psychological and biological observations. And it based on an actor-critic (AC) framework, an epigenetic robotic architecture (ERA), and a Bayesian network (BN). These modules represent the functions of the corresponding brain regions in ToM, and they could uncover the detailed mechanisms of trust-based learning in children and robots. Finally, they reproduced psychological experiments with the iCub humanoid robot, and the results are coherent with the real experimental data from children. Frontiers in Neurorobotics | www.frontiersin.org 3. METHODS (2016, 2017), and some studies have indicated that it will be activated during lower- order self-perception (Schurz and Perner, 2015; Igelstrom and Graziano, 2017). So in our computational model, the IPL is used to store self-relevant stimuli. The pSTS (Frith and Frith, 1999; Schurz and Perner, 2015) is concerned with representing August 2020 | Volume 14 | Article 60 3 Zeng et al. Brain-Inspired Theory of Mind Model FIGURE 1 | The Brain-ToM model (including major functional brain areas, pathways, and their interactions). FIGURE 1 | The Brain-ToM model (including major functional brain areas, pathways, and their interactions). other-perspective are conflictive (Hartwright et al., 2012, 2015), and is suggested to inhibit self-belief to obtain correct task performance in the false-belief task (Mossad et al., 2016). Another function of IFG is encoding action goals and responding to goal-driven motions (Hamzei et al., 2016). The vPMC encodes kinematics based on motion goals from IFG, the encoded information is sent to M1. M1 encodes the strength and orientation of motion and controls the concrete motion execution (Georgopoulos et al., 1986). the actions of others and perspective taking (Frith and Frith, 2006). In our computational model, the pSTS is used to store other-relevant stimuli. The precuneus is often activated during visuo-spatial imagery, episodic memory retrieval, self- processing operations (Cavanna and Trimble, 2006), and retrieving previous experiences (Molenberghs et al., 2016). And a main function of the precuneus in ToM is mental imagery to represent the perspective of another person (Cavanna and Trimble, 2006; Schurz et al., 2013, 2014) or modeling other people’s views (Vogeley et al., 2004). In our computational model, the precuneus is the critical area for a machine to learn visual access from its own experience and uses it to infer other people’s visual access. The PCC is the caudal part of the cingulate cortex, and the precuneus lies posterior and superior to the PCC (Leech and Sharp, 2014). In our computational model, the PCC receives the information from precuneus and sends it to ACC. As indicated in Green et al. (2015) and Jiang et al. (2016), the specific roles that brain areas have in the mentalization processes is not clear. 3. METHODS Based on the neuroimaging studies as described above, we propose four pathways for robots learning from self-experience and uses it in the false-belief task, they are self-experience learning pathway, motivation understanding pathway, reasoning about one’s own belief pathway and reasoning about other people’s belief pathway. p The anterior paracingulate cortex is often considered to be a part of the ACC and is used for representing mental states “decoupled” from reality (Gallagher and Frith, 2003). In our computational model, the ACC is the critical area in acquiring the ability of object permanence and then used it for belief reasoning. The mPFC contains vmPFC and dmPFC. The vmPFC has typically been associated with self-referential processing, and the dmPFC has typically been associated with others-referential processing (Abu-Akel and Shamay-Tsoory, 2011; Denny et al., 2012; Jiang et al., 2016; Molenberghs et al., 2016). In our computational model, the mPFC is used to store the result of belief reasoning from ACC: the vmPFC stores the result of self-belief reasoning, and the dmPFC stores the other’s belief reasoning. The anterior paracingulate cortex is often considered to be a part of the ACC and is used for representing mental states “decoupled” from reality (Gallagher and Frith, 2003). In our computational model, the ACC is the critical area in acquiring the ability of object permanence and then used it for belief reasoning. The self-experience learning pathways is consist of object permanence learning pathway [Precuneus/PCC →ACC] and the visual access learning pathway [STS →TPJ(IPL) → Precuneus/PCC]. The mPFC contains vmPFC and dmPFC. The vmPFC has typically been associated with self-referential processing, and the dmPFC has typically been associated with others-referential processing (Abu-Akel and Shamay-Tsoory, 2011; Denny et al., 2012; Jiang et al., 2016; Molenberghs et al., 2016). In our computational model, the mPFC is used to store the result of belief reasoning from ACC: the vmPFC stores the result of self-belief reasoning, and the dmPFC stores the other’s belief reasoning. The test pathways are consist of motivation understanding pathway, reasoning about one’s own belief pathway, and reasoning about other people’s belief pathway. The motivation understanding pathway is STS →pSTS → TPJ(IPL) →IFG. The reasoning about one’s own belief pathway contains belief reasoning pathway [STS →TPJ(IPL) →Precuneus/PCC → ACC →MPFC(vMPFC)] and the motor response pathway [MPFC(vMPFC) →IFG →vPMC →M1]. The reasoning about other people’s belief pathway contains the true belief reasoning pathway and the false belief reasoning pathway. Frontiers in Neurorobotics | www.frontiersin.org 3. METHODS The IFG is a critical area for the inhibition process: self-perspective inhibition and self-belief inhibition. The IFG inhibits self-perspective when self perspective and August 2020 | Volume 14 | Article 60 4 Zeng et al. Brain-Inspired Theory of Mind Model The true belief reasoning pathway contains belief reasoning pathway [STS →TPJ(pSTS) →Precuneus/PCC →ACC → MPFC(dMPFC)] and the motor response pathway [MPFC(dMPFC) →IFG→vPMC →M1]. Here the turning-around state of the robot itself is detected by the degree to which its head is twisted, and the turning-around state of the other robot is detected by whether its face or back is recognized. The false belief reasoning pathway contains the belief reasoning pathway [STS →TPJ →IFG →TPJ(pSTS) → Precuneus/PCC →ACC →MPFC(dMPFC)] and the motor response pathway [MPFC →IFG →MPFC (dMPFC) →IFG →vPMC →M1]. IPL/pSTS/TPJoutput = [objectt, boxt, blindfoldt, turnt] IPL/pSTS/TPJoutput = [objectt, boxt, blindfoldt, turnt] STSt = [idt, objectt, boxt, blindfoldt, turnt] We detect this information using traditional template matching methods and represent the result by neurons with an input synaptic current I of 1.0 or 0.0. The idt uses two neurons to represent the identification of self or others. For the identification of idt, we use the Fast R-CNN to recognize others at time t. More details could be found in our previous work (Zeng et al., 2017). The objectt and boxt are both tuples consist of object or box identification information and its location information respectively. For the identification of the objectt, boxt, blindfoldt, we first collect their image templates, and then use the traditional template matching method to identify them at time t. The location of objectt or boxt is calculated by the distance between the center of the black rectangles and the center of the object or box at time t. The blindfoldt uses two neurons to represent the wearing state of the blindfold (wear or not wear) and uses another two neurons to represent whether there is a blindfold at time t. Here we define the state of self as wearing a blindfold if the blindfold covers most areas of its visual field, and define the state of others as wearing a blindfold if the blindfold covers the other’s face. The turnt uses two neurons to represent the state of turning. Frontiers in Neurorobotics | www.frontiersin.org 3.2.1. STS The ζ is the forgetting factor. In the training stage, the target output of Precuneus/PCC is the perceived location of the object (active the unseen signal if no object is detected). The output of Precuneus/PCC is encoded by the input synaptic current I of either 1.0 or 0.0 in the perception neurons, The STS encodes the processed results of visual perception and body information of the self and others at time t. STSt = [idt, objectt, boxt, blindfoldt, turnt] STSt = [idt, objectt, boxt, blindfoldt, turnt] 3.2.3. Precuneus/PCC The Precuneus/PCC is used for visual access learning. Here we use the VPSNN based on our previous work (Tielin et al., 2018) to train the robot to learn visual access. 3.2. Concrete Neural Network Architecture 3.2. Concrete Neural Network Architecture The concrete neural network architecture of the model is shown as Figure 2, and it uses the leaky integrate-and-fire model (LIF) neurons. This section describes (1) the input and output encoding information of different brain areas, (2) the Voltage- driven Plasticity-centric Spiking Neural Networks (VPSNN) used in Precuneus/PCC and ACC for visual access learning and object permanence learning respectively, and (3) the inhibitory control mechanism which was used to select correct output information of TPJ or mPFC. The input information of Precuneus/PCC contains the current information from the output of TPJ and the previous information from working memory, and it could be represented as Precuneus/PCCinputt = TPJoutputt + WMt 3.2.2. TPJ The input information of the TPJ is directly from STS, as TPJinput = STSt In the reasoning about other people’s belief pathway of false belief reasoning, the conflict between self-and-other perspective in TPJ will activate IFG, then IFG will inhibit self-relevant stimuli in IPL. So the others-relevant stimuli in pSTS will be the first output of TPJ, then it sends to Precuneus/PCC. In the motor response pathway of false belief reasoning, the conflict between self-belief and other’s belief in mPFC will activate the IFG, then the IFG inhibits self-belief in vmPFC. So the other’s belief in dmPFC will be the output of mPFC, then it will be sent to IFG for encoding action goals. and the information is divided into self-relevant stimuli and others-relevant stimuli by the idt, and then stored in IPL and pSTS, respectively. The information in IPL, pSTS, and the output of TPJ are encoded as. IPL/pSTS/TPJoutput = [objectt, boxt, blindfoldt, turnt] Precuneus/PCCoutputt = [location1, location2, unseen] Precuneus/PCCoutputt = [location1, location2, unseen] There are 160 trials in the training process. Each training trial contains two images collected from the robot as shown in Figure 4. The first image is collected when putting the various objects in one location as shown in Figure 4a. The second image is collected when the robot is asked “Where is the [object label]?” in three scenes: (1) when the blindfold is interposed (Figures 4b,c), (2) when the robot has turned around, (3) when the object is moved to another location. For example, the first image is collected at time t −1, and the second image is collected at time t. The input of the VPSNN is Precuneus/PCCinputt, and the target output of the VPSNN is Precuneus/PCCoutputt. The Precuneus/PCCinputt receives two inputs: one is the raw TPJoutputt and the other is the Precuneus/PCCinputt−1 with a forgetting factor. In each training trial, there is no previous information from working memory when collecting the first image, so the Precuneus/PCCinputt−1 is equal to the TPJoutputt−1. The Precuneus/PCCoutputt is the perceived location of the object at time t. The robot trains the self-experience of visual access to August 2020 | Volume 14 | Article 60 5 Brain-Inspired Theory of Mind Model Zeng et al. FIGURE 2 | The concrete neural network architecture of Brain-ToM model (WM denotes working memory). architecture of Brain-ToM model (WM denotes working memory). encoded by the input synaptic current I of either 1.0 or 0.0 in the related neurons, i.e., the wearing of the blindfold or the turning around of the robot in the process of visual access learning, then uses it to infer itself and other robot’s visual access in the Opaque-and-Transparent Blindfold Test and Turn Around Test. ACCoutputt+1 = [belieflocation1, belieflocation2] There are 50 trials in the training process. Each training trial contains three images collected from the visual sensor, when (1) the various objects are put in one location (Figure 5b), (2) an object is hidden in the box (Figure 5c), and (3) the box is moved away (Figure 5d). For example, the first, second, third image is collected at time t −1, t, t + 1, respectively. The input of the VPSNN is ACCinputt, and the target output of the VPSNN is ACCoutputt+1. The ACCinputt receives two inputs: one is the raw Precuneus/PCCoutputt and the other is the ACCoutputt−1 with a forgetting factor. Precuneus/PCCoutputt = [location1, location2, unseen] The ACCoutputt−1 is the perceived location of the object at time t −1. To train the ability of object permanence in the robot, we make the robot always perceive the location of the object at the end of each training trial. The ACCoutputt+1 is the perceived location of the object at time t + 1. The robot 3.2.4. ACC The ACC is used for object permanence learning. Here we use the VPSNN to train the robot to learn object permanence. The input information of ACC contains the current information from the output of Precuneus/PCC and the previous information from working memory, and it could be represented as ACCinputt = Precuneus/PCCoutputt + WMt ACCinputt = Precuneus/PCCoutputt + WMt ACCinputt = Precuneus/PCCoutputt + WMt ACCinputt = Precuneus/PCCoutputt + WMt 3.3.1. The LIF Neuron Model τm dV dt = −(V −VL) −gE gL (V −VE) (1) τE dgE dt = −gE + η X j∈NE wj,iδt (2) τm dV dt = −(V −VL) −gE gL (V −VE) (1) (1) (2) mPFCoutputt =    dmPFCt if the question is “Where is the ladybird according to the blue robot" vmPFCt if the question is “Where is the ladybird according to yourself ?" The basic neuron model in VPSNN is the LIF model, which describes the dynamics of the membrane potential of V and synaptic-weight-related gE, as shown in Equations (1) and (2). Once the pre-synaptic neurons fire, there is a non-linear increment of gE, which will then propagate into V. The gL is leaky conductance, VL is leaky potential, τm and τE are conductance decay, η is the learning rate, and VE is reversal potential. where where WMt = ζ × ACCoutputt−1 WMt = ζ × ACCoutputt−1 The ζ is the forgetting factor. In the training stage, the target output of ACC is the location of the object. The output of ACC is August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org 6 Brain-Inspired Theory of Mind Model Zeng et al. trains itself a belief that objects are still where it has last located them, even they are out of its field of visual perception, and then uses it to infer itself and other robot’s belief in the Opaque-and- Transparent Blindfold Test and Turn Around Test. trains itself a belief that objects are still where it has last located them, even they are out of its field of visual perception, and then uses it to infer itself and other robot’s belief in the Opaque-and- Transparent Blindfold Test and Turn Around Test. model (Tielin et al., 2018), which is a shallow feed-forward SNN and may well simulate input-output signals with the integration of supervised learning (with an additional teaching signal given directly to the output layer neurons) and unsupervised learning (tuned with biologically plasticity principles, e.g., STDP, and homeostatic membrane potential). We train the visual access learning in Precuneus/PCC first, then the object permanence learning in ACC. p Two three-layer SNN architectures are designed for Precuneus/PCC (with 24 input neurons, three hidden neurons, and two output neurons) and ACC (with three input neurons, three hidden neurons, and two output neurons), respectively, as shown in Figure 2. The VPSNN includes four steps, namely: feed-forward information (including both membrane potential and spikes) propagation, unsupervised homeostatic state learning, supervised last layer learning, and passively updating synaptic weights based on STDP rules. In this paper, we take advantage of these four steps for the fast network tuning and update the methodologies of giving teaching signals from single SNN to two SNNs together for the better model integration. 3.2.5. mPFC The input of mPFC is identical to the output of ACC and distinguishes between the self-belief and other-belief by the source of the information: IPL or pSTS. The vmPFC and dmPFC both use two neurons to store the self-belief and other-belief about the location of the object. mPFCinputt = ACCoutputt dmPFCt = mPFCinputt if the source is IPL vmPFCt = mPFCinputt if the source is pSTS vmPFCt = mPFCinputt if the source is pSTS The output of mPFC depends on the questions, which are set as follows: if the question is “Where is the ladybird according to the blue robot?” the other-belief stored in dmPFC tries to be the output of mPFC; if the question is “Where is the ladybird according to yourself?” the self-belief stored in vmPFC tries to be the output of mPFC. 3.3.2. The Feed Forward Propagation The information propagation in the LIF neuron is slower compared with giving input directly into V. However, this is a specially designed procedure that will make the network- tuning focusing more on the homeostatic membrane potential adjustment and STDP learning. The information (especially the membrane potential) will be propagated from pre-synaptic neurons (e.g., Vj) into the post-synaptic neurons (e.g., Vi), and the whole feed-forward procedure VFF i is shown in Equation (3), in which the Vth is the firing threshold of the neurons. IFGinputt =    Iinhibit result neuron TPJ self −perception inhibition Iinhibit result neuron mPFC self −belief inhibition mPFC encode the action goal IFGinputt =    Iinhibit result neuron TPJ self −perception inhibition Iinhibit result neuron mPFC self −belief inhibition mPFC encode the action goal IFG uses the same number of neurons as IPL and as vmPFC to inhibit self-perception information and self-belief information, respectively, and uses another two neurons to encode the action goal, which is later sent to vPMC to control the robot’s actions. The details of the inhibitory control mechanism of IFG could be found in section 3.4.    τm dVi dt = −(Vi −VL) −gE gL (Vi −VE) τE dgE dt = −gE + η PN j wj,iVj Vi = VL, Tref = T0 if (Vi > Vth) VFF i = Vi (3) With the exception in the above mentioned evaluation, the synaptic plasticity only takes place in Precuneus/PCC and ACC in the process of training, while the weights of the other connections between various areas remain unchanged in the experiment. (3) 3.2.6. IFG In the proposed model, IFG receives inputs from three sources: (1) the inhibit result neurons in TPJ that could stimulate IFG for self-perception inhibition, (2) the inhibit result neurons in mPFC that could stimulate IFG for self-belief inhibition, and (3) some other neurons in mPFC that could stimulate IFG to encode the action goal. p Potential Learning For the mathematically modeling of brain regions such as Precuneus/PCC and ACC, here we select a standard VPSNN The basic homeostatic mechanism occurs in the input-output balance of the single neuron, described as the Equation (4). August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org 7 Brain-Inspired Theory of Mind Model Zeng et al. FIGURE 3 | Inhibitory control of one single neuron in reasoning about other’s belief. At time t1, t2, and t3, the firing pattern of pSTS and IPL are identical, which means that the other-relevant stimuli and self-relevant stimuli are identical. The inhibitory neurons can inhibit self-relevant stimuli successfully. But at time t4 and t5, the other-relevant stimuli and self-relevant stimuli are in conflict with each other, and the inhibitory neurons cannot inhibit self-relevant stimuli. The inhibit result neurons will fire and stimulate IFG activation, while IFG activation will inhibit the firing of inhibit result neuron. The inhibit result neurons will combine with neurons in pSTS to generate other-relevant stimuli output firstly. Then the temporary neuron stimulates IPL, because the electrovital currents in pSTS and the inhibitory neuron is zero at this moment, self-relevant stimuli will be the second output of TPJ. Compared to the process of self-perspective inhibition, the only difference is that the inhibitory control in TPJ is used to decide the sequence of information output, and the inhibitory control in mPFC is used to decide which belief to export. Therefore, the process of self-belief inhibition does not require the involvement of temporary neuron. 1Ei = Vi −   N X j wj,iVj −Vth,i   (4) (4) The entire network homeostatic state can be represented as the addition of all of the neurons in each layer, i.e., 1E = P i∈N 1Ei. Moreover, after the calculation (the detailed methodologies are shown in paper Tielin et al., 2018), the homeostatic membrane potential VHomeo i can be updated according to Equation (5). The ηi is the learning rate. The entire network homeostatic state can be represented as the addition of all of the neurons in each layer, i.e., 1E = P i∈N 1Ei. Moreover, after the calculation (the detailed methodologies are shown in paper Tielin et al., 2018), the homeostatic membrane potential VHomeo i can be updated according to Equation (5). The ηi is the learning rate. 3.4. Inhibitory Control Mechanism The inhibitory control is used to select correct output information of TPJ or mPFC, and it can be divide into inner and outside inhibitory control. The inner inhibitory control cannot inhibit predominant information from self when the related information of self and others is conflictive, so we use the outside inhibitory control from IFG to inhibit the predominant information. Inhibitory control of one single neuron is shown in Figure 3. The IPL neurons (or vmPFC neurons) and pSTS neurons (or dmPFC neurons) receive electrovital currents of self-relevant stimuli and other-relevant stimuli, respectively. The input of inhibit neurons and temporary neurons depend on reasoning about other’s belief (contains self-perspective inhibition and self-belief inhibition) or self-belief (contains 4. EXPERIMENTS In this section, we introduce the experimental settings and the result of our proposed model. 1wj,i ∝VjV ′ i (8) 1wj,i ∝VjV ′ i (8) (8) Frontiers in Neurorobotics | www.frontiersin.org p Potential Learning 1VHomeo i = −ηi Vi − PN j wj,iVj −PN j Vth,i  −(Vi −VL) −gE gL (Vi −VE) (5) (5) (5) With the integration of Equations (3) and (5), the update rule of each neuron state Vi is shown in Equation (6). The t is the training time slot and T is the total training time in SNN learning. FIGURE 3 | Inhibitory control of one single neuron in reasoning about other’s belief. At time t1, t2, and t3, the firing pattern of pSTS and IPL are identical, which means that the other-relevant stimuli and self-relevant stimuli are identical. The inhibitory neurons can inhibit self-relevant stimuli successfully. But at time t4 and t5, the other-relevant stimuli and self-relevant stimuli are in conflict with each other, and the inhibitory neurons cannot inhibit self-relevant stimuli. The inhibit result neurons will fire and stimulate IFG activation, while IFG activation will inhibit the firing of inhibit result neuron. The inhibit result neurons will combine with neurons in pSTS to generate other-relevant stimuli output firstly. Then the temporary neuron stimulates IPL, because the electrovital currents in pSTS and the inhibitory neuron is zero at this moment, self-relevant stimuli will be the second output of TPJ. Compared to the process of self-perspective inhibition, the only difference is that the inhibitory control in TPJ is used to decide the sequence of information output, and the inhibitory control in mPFC is used to decide which belief to export. Therefore, the process of self-belief inhibition does not require the involvement of temporary neuron. 1Vi = t T 1VFF i +  1 −t T  1VHomeo i (6) (6) STDP-Based Weights Consolidation STDP-Based Weights Consolidation An additional teaching signal will be given to the network for guiding the proper network output. Here we add teaching signals into the last layer of SNN in the training procedure, as shown in Equation (7), in which VT is teacher signal state, ηc is the learning rate. dVi = −ηc (Vi −VT) (7) (7) other-perspective inhibition and other-belief inhibition), and the former is used to inhibit stimuli, the later is used to temporarily store uninhibited stimuli. In the process of self-perspective inhibition or self-belief inhibition, the input electrovital currents of inhibit neuron are equal to other-relevant stimuli, and the temporary neuron is equal to self-relevant stimuli (only in TPJ). So other-relevant stimuli will be the first output, and self-relevant stimuli will be the second output in TPJ, and other’s belief will be exported in mPFC. In the process of other-perspective inhibition and other-belief inhibition, the inhibit electrovital currents are very big that it can completely inhibit other-relevant stimuli, and the temporary neuron is equal to other-relevant stimuli (only in TPJ). Therefore, in TPJ, self-related stimulus will be output first, other-related stimulus will be output second, and then self-belief will be output in mPFC. STDP rules (Bi and Poo, 2001; Dan and Poo, 2004; Bengio et al., 2015a,b) are further used for the knowledge consolidation from the membrane potential to synaptic weights, e.g., the synaptic weights could be passively updated by the changes of the pre- and post-synaptic neuron states. The function is as shown in Equation (8), in which the V ′ i is the derivative value of Vi. (8), in which the V ′ i is the derivative value of Vi. 4.1.3. Maturation Test The ability for the ToM comes with individual development process (Grosse Wiesmann et al., 2017). Grosse Wiesmann et al. (2017) discussed the influence of white matter structure on ToM by tract-based spatial statistics analysis and probabilistic tractography. They found that “the developmental breakthrough in false belief understanding is associated with age-related changes in local white matter structure in temporoparietal regions, the precuneus, and medial prefrontal cortex, and with increased dorsal white matter connectivity between temporoparietal and inferior frontal regions.” And they thought “the emergence of mental state representation is related to the maturation of core belief processing regions and their connection to the prefrontal cortex” (Grosse Wiesmann et al., 2017). But their research focused on the 3- and 4-year-old children in the explicit false-belief tasks, and did not include younger infants who cannot pass the implicit false-belief task. They did not test whether this finding is also associated with an implicit task, because of the difficulties in performing MRI with toddlers. We suggest that the ability of learning object permanence is the prerequisite for the ToM. As indicated in Piaget and Cook (1952) and Bruce and Muhammad (2009), Piaget defined six developmental stages of object permanence. During the early stages (Stage I, Stage II, Stage III), children failed to find a hidden object. During Stage IV (8–12 months) children can retrieve an object when its concealment is observed. But they cannot find the object when it is continuously moving. During Stage V (12– 18 months), the children can retrieve an object when it is hidden several times within his or her view. In summary, when an object was hidden in location A and then hidden in location B, the children would try to find the object in location A during Stage IV and would try to find it in location B during Stage V. With similar principles, here we enable all of the robots in the experiment acquire the ability of learning object permanence from their own self experiences, and in our model, ACC acts as a central role to realize this cognitive function (Gallagher and Frith, 2003). Figure 5 shows the visual inputs of the robots in this process. 4.1.1. Opaque-and-Transparent Blindfold Test The Opaque-and-Transparent Blindfold Test is adapted from Senju et al. (2011). We enable all the robots to learn the visual access of blindfold from self experiences, as the infants’ experience in Senju’s experiment. In the proposed model, this process takes place in Precuneus (Vogeley et al., 2004; Cavanna and Trimble, 2006; Schurz et al., 2013, 2014). The robots are divided into two groups—the opaque blindfold group and the transparent blindfold group. The opaque blindfold and transparent blindfold look identical, at least the robot cannot distinguish them from appearance, but the transparent blindfold can make the robot who wears it see through, and the opaque blindfold cannot. Figure 4 presents the visual inputs of the robot in the opaque blindfold group (with Movie S1) and the transparent blindfold group (with Movie S2). In this stage, the robots could not observe other robots wearing the blindfold, as the infants in Senju’s experiment, so they have no opportunity to learn the property of the blindfold from the third-person point of view. Frontiers in Neurorobotics | www.frontiersin.org 4.1. Experimental Settings We deploy the computational model to humanoid robotics and use the Opaque-and-Transparent Blindfold Test and Turn Frontiers in Neurorobotics | www.frontiersin.org August 2020 | Volume 14 | Article 60 8 Brain-Inspired Theory of Mind Model Zeng et al. 4.1.2. Turn Around Test The Turn Around Test is adapted from Southgate et al. (2007). 4.1.2. Turn Around Test The Turn Around Test is adapted from Southgate et al. (2007). Around Test to validate the Brain-ToM model. Furthermore, we test the effect of self-experience, maturation of correlate brain areas (e.g., calculation capability) and their connections (e.g., inhibitory control) on the performance of participant robot in Opaque-and-Transparent Blindfold Test. The robot learns the visual access of turning around from self-experience. The Turn Around Test is similar to the Opaque- and-Transparent Blindfold Test. The diversity of belief is caused by different blindfolds in the Opaque-and-Transparent Blindfold Test, and in Turn Around Test, it is caused by the behavior of turn around. The visual inputs of the participant robot are shown in Figure 7. As with the Opaque-and-Transparent Blindfold Test, the participant robot also be asked two questions: “Where is the ladybird according to the blue robot?” and “Where is the ladybird according to yourself?” And we determine whether the robot can pass the task by detecting the direction of the finger. 4.1.1. Opaque-and-Transparent Blindfold Test The Opaque-and-Transparent Blindfold Test is adapted from Senju et al. (2011). 4.1.3. Maturation Test (a) An object is put on either of the black rectangles. (b) Visual inputs of the opaque blindfold group (with Movie S1). We interpose blindfold between the eyes of the robot and the object, then ask it “Where is the [object label]?” The robot will reply with the location of the object or with the fact that it did not see it. (c) Visual inputs of the transparent blindfold group (with Movie S2). The process is the same as the opaque blindfold group. FIGURE 4 | Visual access learning of blindfold from self-experience. (a) An object is put on either of the black rectangles. (b) Visual inputs of the opaque blindfold group (with Movie S1). We interpose blindfold between the eyes of the robot and the object, then ask it “Where is the [object label]?” The robot will reply with the location of the object or with the fact that it did not see it. (c) Visual inputs of the transparent blindfold group (with Movie S2). The process is the same as the opaque blindfold group. FIGURE 5 | Object permanence learning from self-experience. (a) The black rectangles are used to indicate the candidate positions of the object. (b) An object is put on either of the black rectangles, the robot can detect the location of the object—left side or right side. (c) The yellow box and the green box are used to hide the object. The robot cannot perceive the object in its visual field, hence cannot find it. It is similar to the early stages in Piaget’s Stages of Object Permanence. (d) The boxes are removed, and the robot can perceive the object’s location in its visual field. FIGURE 5 | Object permanence learning from self-experience. (a) The black rectangles are used to indicate the candidate positions of the object. (b) An object is put on either of the black rectangles, the robot can detect the location of the object—left side or right side. (c) The yellow box and the green box are used to hide the object. The robot cannot perceive the object in its visual field, hence cannot find it. It is similar to the early stages in Piaget’s Stages of Object Permanence. (d) The boxes are removed, and the robot can perceive the object’s location in its visual field. FIGURE 6 | Visual inputs of participant robot in the test stage. 4.1.3. Maturation Test Although the developmental neural basis for the implicit false-belief task is still not very clear, we hypothesize that the developmental process in implicit false belief understanding is relevant with explicit one, and will also be associated with the maturation of correlate brain areas and their connections. We aim to test this hypothesis by our computation model, and apply it to the Brain-ToM model that we developed for machine intelligence. The maturation of correlate brain areas could be regarded as calculation capability in our model, and the calculation capability increases with the maturation of brain areas. The calculation capability in this model is proportional to the number of neurons in the hidden layer. We simulate immature Precuneus/PCC by reducing the number of the neurons in its own hidden layer, then verify the effect of this condition on the performance of the participant robot. In the test stage, participant robots use the Brain-inspired Robot Bodily Self Model which we proposed in Zeng et al. (2016, 2017) to distinguish self and others. As in the experiment of Senju et al. (2011), the actor robot will try to find the hidden object in the box before the final test. By this way, the participant robot could understand that the actor has the same cognitive ability (e.g., visual ability) and the goal of the actor robot (Movie S3). In the final test, the opaque blindfold group and the transparent blindfold group are tested with the same process as shown in Figure 6. Then the participant robot be asked two questions: “Where is the ladybird according to the blue robot?” and “Where is the ladybird according to yourself?” We determine whether the robot can pass the task by detecting the direction of the finger which makes the results more intuitive. The maturation of the connection between brain areas is critical for information transmission and information integration, especially inhibitory connection and control. The inhibitory control is generally considered as a key mechanism in false-belief task (Leslie and Polizzi, 1998; Scott and Baillargeon, 2017), and we think that the maturation of connections between IFG and TPJ, IFG and vmPFC are the neural basis of self- perspective inhibition and self-belief inhibition, respectively. We August 2020 | Volume 14 | Article 60 9 Brain-Inspired Theory of Mind Model Zeng et al. FIGURE 4 | Visual access learning of blindfold from self-experience. 4.1.3. Maturation Test (a) The blue robot in the left is the actor, and the red robot in right is the participant who should infer the actor’s belief. The middle screen in the (a), and the remaining figures are the visual inputs of the participant robot. (b) An object (ladybird) is put on the left black rectangle. (c) The ladybird is hidden in the yellow box. (d) The blindfold is interposed between the actor (the blue robot) and the object (ladybird), and the object is moved to the right side. (e) The ladybird is hidden in the green box. (f) Finally, the blindfold is removed. FIGURE 6 | Visual inputs of participant robot in the test stage. (a) The blue robot in the left is the actor, and the red robot in right is the participant who should infer the actor’s belief. The middle screen in the (a), and the remaining figures are the visual inputs of the participant robot. (b) An object (ladybird) is put on the left black rectangle. (c) The ladybird is hidden in the yellow box. (d) The blindfold is interposed between the actor (the blue robot) and the object (ladybird), and the object is moved to the right side. (e) The ladybird is hidden in the green box. (f) Finally, the blindfold is removed. FIGURE 6 | Visual inputs of participant robot in the test stage. (a) The blue robot in the left is the actor, and the red robot in right is the participant who should infer the actor’s belief. The middle screen in the (a), and the remaining figures are the visual inputs of the participant robot. (b) An object (ladybird) is put on the left black rectangle. (c) The ladybird is hidden in the yellow box. (d) The blindfold is interposed between the actor (the blue robot) and the object (ladybird), and the object is moved to the right side. (e) The ladybird is hidden in the green box. (f) Finally, the blindfold is removed. August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org 10 Zeng et al. Brain-Inspired Theory of Mind Model FIGURE 7 | Turn Around Test. An object (ladybird) is put on the left black rectangle and hidden it in the yellow box firstly. In the false belief condition, the object was moved to the other box when the actor robot turned around (Movie S6). 4.1.3. Maturation Test And in true belief task, the actor robot did not turn around when the object was moved to the other box (Movie S7). FIGURE 7 | Turn Around Test. An object (ladybird) is put on the left black rectangle and hidden it in the yellow box firstly. In the false belief condition, the object was moved to the other box when the actor robot turned around (Movie S6). And in true belief task, the actor robot did not turn around when the object was moved to the other box (Movie S7). n object (ladybird) is put on the left black rectangle and hidden it in the yellow box firstly. In the false belief condition, the object was e actor robot turned around (Movie S6). And in true belief task, the actor robot did not turn around when the object was moved to th simulate immature connections between IFG and TPJ, IFG and vmPFC by set the synaptic weights as 0, then verify the effect of this condition on the performance of the participant robot. (15.2 ms). In the process of reasoning about self-belief, the time consumption of false belief condition (15.4 ms) and true belief condition (15 ms) are similar. In traditional true belief task, the time consumption will be shorter. The perception in TPJ is identical, hence the IFG will not be activated in this task and the time consumption is reduced. Frontiers in Neurorobotics | www.frontiersin.org 4.2. Experimental Results and Analyses In this section, we present the results of our model. Besides, we analyze the temporal and spatial activation of different brain areas during different tasks, the effect of self-experience, maturation of correlate brain areas (e.g., calculation capability) and their connections (e.g., inhibitory control) on the performance of participant robot in Opaque-and-Transparent Blindfold Test. p Here we provide the time consumption in different tasks. Based on the belief about the object location, the task can be divided into reasoning about actor robot’s false-belief task (other-incongruent condition) and true belief task (other- congruent condition), reasoning about participant robot’s own belief task which contains self-incongruent condition (self-belief is divergent from other’s) and self-congruent condition (self- belief is corresponding with other’s). Figure 8 shows the temporal and spatial activation of different brain areas during different tasks. This process only contains the perception conflict stage (as shown in Figure 6d) and motion response stage which have critical differences in different tasks. The time consumptions in STS and vPMC of different tasks are similar, about 448 and 500 ms, respectively. So we select 400-900 ms to show the process and the difference in different tasks. Reasoning about other’s belief in the transparent group can be regarded as true belief task, but it must be noted that, the information from self-perspective and other-perspective are identical in tradition true belief task, while they conflict with each other in this task which is originally from Senju et al. (2011) based on human studies of 18-month-olds. For example, in the transparent blindfold task, the participant robot perceived from self-perspective that “I (participant robot) saw the object moved to the green box without blindfold” and the participant robot perceived from other-perspective that “the actor robot saw the object moved to the green box with blindfold,” so the information is conflictive in this task. And in traditional true belief task, the information is identified as “I (participant robot) saw the object was moved to the green box” and “the actor saw the object was moved to the green box” without the difference caused by the transparent blindfold. 4.2.1. Opaque-and-Transparent Blindfold Test To show the function of IFG easily, we make the arrow mark in the figure. FIGURE 8 | The temporal and spatial activation of different brain areas during different tasks. The time consumption in STS (visually encoding biological motion), Precuneus/PCC (inferring visual access), ACC (belief reasoning), IFG (encoding action goals), and vPMC/M1 (encoding kinematics/motion execution) are similar in different tasks. In the process of TPJ activation (deciding the output sequence of self and other-relevant stimuli) in other-incongruent condition (red line) and other-congruent condition (green line), IFG is activated to inhibit self-perspective. In the process of mPFC activation (deciding belief for motor response) in other-incongruent condition, IFG is activated to inhibit self-belief. In the tasks of self-incongruent condition (black line) and self-congruent condition (blue line), the IFG is only activated in the process of encoding action goals. The value of total time consumption is self-congruent condition < self-incongruent condition < other-congruent condition < other-incongruent condition. To show the function of IFG easily, we make the arrow mark in the figure. Our focus is the activation sequence of brain areas and reaction time in the various task, such as the other-incongruent condition spends more time than self-congruent condition rather than the numerical value of time consumption, these result is consistent with the functional neuroimaging studies in Mossad et al. (2016) and Dohnel et al. (2012). question “Where is the [object label]?” In the test stage, we put the blindfold on the desk or interpose it at other positions where the actor’s visual inputs are not blocked. Both of the groups can infer the visual access of actor robot correctly, and conclude that the actor robot could see the object move to the right side. And self-belief is corresponding to other’s belief in both groups. This additional test could prove that the actor robot does not use low- level features such as whether the blindfold exists when inferring other’s mental state, and it also proves the effect of the bodily model in this task. In the process of inferring visual access which corresponds to Figure 6d, even though the visual inputs in both groups are identical, the output is different when the participant robot infer other’s visual access with different self-experience. 4.2.1. Opaque-and-Transparent Blindfold Test The opaque blindfold group can be regarded as the false belief condition, as the actor robot’s belief is inconsistent with the representations of reality. When asking the participant robot “Where is the ladybird according to the blue robot?” the participant robot will point to the yellow box on the left side. And when asking the participant robot “Where is the ladybird according to yourself?” the participant robot will point to the green box on the right side (Movie S4). The transparent blindfold group can be regarded as the true belief condition, as the actor robot’s belief is consistent with the representations of reality. When asking the participant robot the upper two questions, for both of them, the participant robot will point to the green box on the right side (Movie S5). We repeat this experiment 20 times, the robot could pass the task every time, and we calculate the mean value and standard deviation of time consumption in different brain areas. To make the results more visible and clear, the data from STS, Precuneus/PCC, ACC, vPMC/M1 are excluded, because their time consumption is similar in different tasks. We select the time consumption in TPJ, IFG, and mPFC as examples. The time consumption of false belief reasoning (87.7 ms) is longer than true belief reasoning (69.8 ms). And the time consumption of reasoning about other’s belief (78.8 ms) is longer than self-belief August 2020 | Volume 14 | Article 60 11 Zeng et al. Brain-Inspired Theory of Mind Model FIGURE 8 | The temporal and spatial activation of different brain areas during different tasks. The time consumption in STS (visually encoding biological motion), Precuneus/PCC (inferring visual access), ACC (belief reasoning), IFG (encoding action goals), and vPMC/M1 (encoding kinematics/motion execution) are similar in different tasks. In the process of TPJ activation (deciding the output sequence of self and other-relevant stimuli) in other-incongruent condition (red line) and other-congruent condition (green line), IFG is activated to inhibit self-perspective. In the process of mPFC activation (deciding belief for motor response) in other-incongruent condition, IFG is activated to inhibit self-belief. In the tasks of self-incongruent condition (black line) and self-congruent condition (blue line), the IFG is only activated in the process of encoding action goals. The value of total time consumption is self-congruent condition < self-incongruent condition < other-congruent condition < other-incongruent condition. Frontiers in Neurorobotics | www.frontiersin.org 4.2.1. Opaque-and-Transparent Blindfold Test In other words, when inferring visual access of another person by self- experience, the opaque blindfold group will know the actor cannot see the moving object, and the transparent blindfold group will know the actor can. When inferring self visual access, the perception of visual inputs and the result of precuneus are identical in both groups. August 2020 | Volume 14 | Article 60 4.2.3. Maturation Test The maturation of correlate brain areas will be regarded as calculation capability in our model, and the calculation capability increases with the maturation of brain areas. The calculation capability in this model is proportional to the number of neurons in the hidden layer. For example, If the neuron number involved in the calculation of the hidden layer in Precuneus/PCC is two or fewer, even though participant robot can learn visual access of blindfold, it failed in inferring other’s visual access. As indicated in Myowa-Yamakoshi et al. (2011), 8-month- old infants and 12-month-old infants had experienced being blindfolded, when they saw a blindfolded actor did a successful goal-directed action which normally could not succeed with a blindfold, 12-month-old infants will look longer, but 8- month-old infants will not. We think that with the maturation of correlate brain areas as well as their connections, more neurons and synaptic connections will be included in the task processing. The maturation of the connection between brain areas is critical for information transmission and information integration, especially inhibitory connection and control. The inhibitory control is generally considered as a key mechanism in false-belief task (Leslie and Polizzi, 1998; Scott and Baillargeon, 2017), and we think that the maturation of connections between IFG and TPJ, IFG and vmPFC are the neural basis of self- perspective inhibition and self-belief inhibition respectively. As shown in Figure 9, the inhibitory control uses inhibitory neurons and temporary neurons which store information temporarily for the selection of correct output information of TPJ or mPFC. In the process of reasoning about other’s belief, the inhibitory neurons in TPJ and mPFC receive other-relevant information from STS or ACC, respectively. The information of inhibitory neurons in TPJ is identical with the information of other-relevant stimuli in pSTS, and the information of inhibitory neurons in mPFC is identical with the information of other’s belief in dmPFC. The temporary neurons in TPJ receive self-relevant information from STS, and the temporary neurons’ information is identical with the information of self-relevant stimuli in IPL. The self-perspective inhibition takes place in TPJ and the self-belief inhibition takes place in mPFC. Then we test the effect of these connections in the false-belief task, and observe that the different maturation of connections leads to different permanence in the task. 4.2.2. Turn Around Test In the false belief condition, the actor robot’s belief is inconsistent with the representations of reality. When asking the participant robot “Where is the ladybird according to the blue robot?” the participant robot will point to the yellow box on the left side. And when asking the participant robot “Where is the ladybird according to yourself?” the participant robot will point to the green box on the right side (Movie S6). In the process of inferring other’s visual access, the IFG will not be activated when self-perspective and other-perspective is identical, as shown in Figures 6b,c,f. If the self-perspective and other-perspective are in conflict with each other in the process of reasoning about other’s belief, the IFG will be activated to inhibit the information of self-perspective, as shown in Figures 6d,e. In the true belief condition, the actor robot’s belief is consistent with the representations of reality. When asking the participant robot the upper two questions, for both of them, the participant robot will point to the green box on the right side (Movie S7). In the process of motor response in reasoning about other’s belief, the IFG will inhibit self-belief if the beliefs are conflictive. So the other’s belief in dmPFC will be the output of mPFC. Then IFG receives input from mPFC and encodes action goals to control vPMC to action. We repeat this experiment 20 times, the robot could pass the task every time. The mechanism of Turn Around Test is similar to the Opaque-and-Transparent Blindfold Test, the only difference is that the self-perspective and other-perspective are identical in true belief task of Turn Around Test. So IFG was not activated in this stage. We select the time consumption in TPJ, IFG, and mPFC as examples. The time consumption of false belief reasoning (87.3 ms) is longer than true belief reasoning (16.1 ms). And the time consumption of reasoning about other’s In addition, we also test the effect of blindfold position in this task. In the visual access of blindfold learning stage, we add a new phase: we put the blindfold on the desk or interpose it at others position to make that both the blindfold and object can be perceived by the participant robot, and we also ask the August 2020 | Volume 14 | Article 60 12 Brain-Inspired Theory of Mind Model Zeng et al. 5. DISCUSSION belief (51.7 ms) is longer than self-belief (15.3 ms). In the process of reasoning about self-belief, the time consumption of false belief condition (15.4 ms) and true belief condition (15.2 ms) are similar. In this section, we will discuss the characteristics of the model, the reasons why robot experiments and cognitive experiments are not completely consistent, and the possible mechanisms of why toddlers fail in high inhibition tasks. g Compared to the previous models which we introduce in the related works, our model explores and is fundamentally based on the role of self-experience. In our model, robots learn to understand object permanence and visual access of blindfold or turn around from self-experience, then use it to infer other’s belief and predict their actions. All of the participant robots learn the ability of understanding object permanence from the same experience. In the Opaque-and-Transparent Blindfold Test, they are divided into opaque blindfold group and transparent blindfold group. Even though the visual inputs of both groups in the test stage are identical, the different experience with an opaque blindfold or transparent blindfold leads to different performances. In Turn Around Test, the different behaviors of the actor robot in the test stage result in different performances. Compared with the recently published work from Patacchiola and Cangelosi (2020): (1) Our model is based on spiking neural networks, and just uses the STDP to successfully reproduce the complex cognitive function of ToM, hence more biological plausible. And their model is based on an actor-critic (AC) framework, an epigenetic robotic architecture (ERA) and a Bayesian network (BN). (2) Our model considered more brain regions that have been consistently found in many experimental paradigms of ToM, such as the TPJ that used for self-other distinction, the IFG that used for self- perspective inhibition and self-belief inhibition, etc. (3) Our model is used for the false belief task, which is one of the most classical and widely used experimental paradigms of ToM, and their model challenges a different task. The two studies have complementary contributions to the ToM models through bio-inspired mechanisms. Frontiers in Neurorobotics | www.frontiersin.org FIGURE 9 | The effect of IFG connection in the false-belief task. The solid circles indicate that the neurons have been activated. “No IFG” means the connection between IFG and TPJ or IFG and vmPFC is immature, and ‘With IFG’ means the connection between IFG and TPJ or IFG and vmPFC is mature. (A) Both of the connections between IFG and TPJ, IFG and vmPFC are immature. The first output of TPJ (TPJ_1) is different from IPL or pSTS because the self-perspective inhibition failed which caused by the immature connection between IFG and TPJ, and the second output (TPJ_2) is identical with IPL. The inferring visual access result of PC_1 and PC_2 in Precuneus/PCC are identical, and the belief reasoning result of ACC_1 and ACC_2 in ACC are identical. In the process of motor response, the InR neurons have not been activated because the belief in dmPFC and vmPFC are identical, so the output of mPFC is identical with dmPFC. The behavior of the participant robot to predict the actor robot’s action is the same as the action caused by self-belief, so it failed in the false-belief task. (B) The connection between IFG and TPJ is immature, and the connection between IFG and vmPFC is mature. The participant robot also failed in this task because the result of inferring other’s visual access is wrong which causes by the immature connection between IFG and TPJ. (C) The connection between IFG and TPJ is mature, and the connection between IFG and vmPFC is immature. The activated InR neurons activate IFG to inhibit self-perspective. The TPJ_1 and TPJ_2 could be output correctly. The participant robot could infer other’s belief correctly, but it cannot inhibit the effect of self-belief without IFG. Both of the two candidate responses are activated when asked the question “Where is the ladybird according to the blue robot.” The participant robot will tend to select action directed by self-belief and failed in the false-belief task. (D) Both of the connections between IFG and TPJ, IFG, and vmPFC are mature. With the connection to IFG, the participant robot will inhibit self-perspective in TPJ and inhibit self-belief in mPFC, then succeed in this task. on whether they can pass the task. And in the test trial, they removed the object from the scene to make infants pass the task easier. 4.2.3. Maturation Test In this figure, the inhibitory neurons (In) are used to inhibit information in IPL or vmPFC, and the inhibitory result (InR) is the result of their interaction. If the connections between IFG and TPJ, IFG and vmPFC are mature, the activated neurons of InR will activate IFG to inhibit the information in InR neurons, and then make the correct information as the output of TPJ and mPFC. These connections will not influence the process of reasoning about self-belief. Through the integration of biological inspirations and computational modeling, we suggest that the self-experience, maturation of correlated brain areas (e.g., calculation capability) and connection between brain areas (e.g., inhibitory control) will have great influence on the participant’s performance in the false-belief task. As indicated in Scott and Baillargeon (2017), the false belief tasks contain spontaneous-response and elicited-response tasks that belong to the implicit task and explicit task, respectively. The difference in spontaneous-response and elicited-response tasks is that the former investigates the capacity of false belief understanding by spontaneous behavior such as anticipatory- looking, preferential-looking, etc with a non-verbal task, and the latter investigates this capacity by answering direct questions that predict agent’s behavior who has a false belief with the verbal task. Children can pass the spontaneous-response task before 2 years old, but they can not pass the elicited-response tasks until about 4 years old. Our tasks on robots are not completely consisted with Senju et al. (2011) on 18-month- olds infants and Southgate et al. (2007) on 2-year-olds infants. Both of them used spontaneous-response to test the infants August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org 13 Zeng et al. Brain-Inspired Theory of Mind Model Frontiers in Neurorobotics | www.frontiersin.org SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnbot. 2020.00060/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnbot. 2020.00060/full#supplementary-material In summary, we propose a Brain-ToM model to enable machines to acquire the ability of ToM through learning and inferring based on self-experience. We validate the model by deploying it on humanoid robots. Our model successfully enabled the robot to pass the false-belief task, which is a classical task designed to understand the nature and mechanisms of ToM from Cognitive Psychology. The model and its application on robots show that current understanding on the mechanisms of the ToM can be computationally unified into a consistent framework and enable the robots to be equipped with the initial cognitive ability of ToM. Movie S1 | Visual access learning of blindfold from self-experience (opaque blindfold group). This process enables the participant robot understand that other robots wearing a blindfold cannot see. Movie S1 | Visual access learning of blindfold from self-experience (opaque blindfold group). This process enables the participant robot understand that other robots wearing a blindfold cannot see. Movie S2 | Visual access learning of blindfold from self-experience (transparent blindfold group). This process enables the participant robot understand that other robots wearing a blindfold can see. Movie S3 | Familiarization phase. This process enables the participant robot understand the goal of the actor robot. Movie S4 | Opaque-and-transparent blindfold test (opaque blindfold group). The details in opaque blindfold group of blindfold test. Movie S5 | Opaque-and-transparent blindfold test (transparent blindfold group). The details in transparent blindfold group of blindfold test. DATA AVAILABILITY STATEMENT Movie S6 | Turn around test (false-belief task). The details in false-belief task of turn around test. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. Movie S7 | Turn around test (true belief task). The details in true belief task of turn around test. FUNDING This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences [Grant No. XDB32070100], the Beijing Municipality of Science and Technology [Grant No. Z181100001518006], the CETC Joint Fund [Grant No. 6141B08010103], the Major Research Program of Shandong Province [2018CXGC1503] and the Key Research Program of Frontier Sciences of Chinese Academy of Sciences [Grant No. ZDBS-LY-JSC013]. This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences [Grant No. XDB32070100], the Beijing Municipality of Science and Technology [Grant No. Z181100001518006], the CETC Joint Fund [Grant No. 6141B08010103], the Major Research Program of Shandong Province [2018CXGC1503] and the Key Research Program of Frontier Sciences of Chinese Academy of Sciences [Grant No. ZDBS-LY-JSC013]. This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences [Grant No. XDB32070100], the Beijing Municipality of Science and Technology [Grant No. Z181100001518006], the CETC Joint Fund [Grant No. 6141B08010103], the Major Research Program of Shandong Province [2018CXGC1503] and the Key Research Program of Frontier Sciences of Chinese Academy of Sciences [Grant No. ZDBS-LY-JSC013]. In the low inhibition task, the participant has no idea about the object’s location and one of the two candidate motor responses are activated, so the participant robot can succeed August 2020 | Volume 14 | Article 60 14 Brain-Inspired Theory of Mind Model Zeng et al. 6. CONCLUSION The computational model for the robotic ToM is regarded as one of the Grand Challenges for Artificial Intelligence and Robotics (Yang et al., 2018). Here we proposed a Brain-ToM model based on existing biological findings of ToM, and this model shows its relevance to ToM of human from the mechanism and behavior perspectives. In our task, we determine whether the robot can pass the task by detecting the direction of the finger which makes the results more intuitive. And in the test trial, we move the object to the other box. Setoh et al. (2016) found that 2.5-year-olds toddlers could succeeded in a traditional false-belief task with reduced processing demands. Toddlers could pass the elicited- intervention and low inhibition task (removing object from the scene) which is described by language and picture, but would fail in high inhibition task (moving the object to another box). They thought the reason why toddlers failed in high inhibition task is that toddlers cannot inhibit the response of the actual location of the object. We suppose that the core mechanism of belief reasoning is identical in both tasks, and the only difference should be in the process of motor response. In the motor response process of the elicited-intervention task, it may use the brain areas which control the hand movement. And in spontaneous- response task, it may use the brain areas which control the eye movement. The main reason why we use high inhibition task to replace low inhibition task is that the behavior of the robots in the true belief task is more intuitive to be understood (for the high inhibition task, the robot can point to the position of the object, while for the low inhibition task, the objects are removed outside the scene, and the robot cannot point to their positions). And we suppose that the reason why toddlers failed in high inhibition task should be related to the lack of motor response ability rather than ToM (e.g., understanding that others have beliefs that are different from one’s own). As shown in Figure 9, the connection between IFG and TPJ are matured but the connection between IFG and vmPFC are not, both of the two candidate responses are activated when the participant robot is asked “Where is the ladybird according to the blue robot.” The participant robot will tend to select action directed by self-belief rather than randomly, as the result of behavior data shown in Setoh et al. (2016) and Samson et al. (2005). AUTHOR CONTRIBUTIONS in this task. And in Samson et al. (2005), the participant who has a lesion of the right inferior and middle frontal gyri performed well in low-inhibition false-belief task but failed in the high-inhibition task. YZe conceived the initial idea. YZh and YZe designed the model, carried out, and analyzed the experiments. TZ, YZe, and DZ designed and implemented the SNN algorithm. FZ contributed to the inhibitory control mechanism. YZe, YZh, TZ, and EL wrote the manuscript. 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Rev. 12, 833–839. doi: 10.1016/j.arr.2013.06.005 Dohnel, K., Schuwerk, T., Meinhardt, J., Sodian, B., Hajak, G., and Sommer, M. (2012). Functional activity of the right temporo-parietal junction and of the medial prefrontal cortex associated with true and false belief reasoning. NeuroImage 60, 1652–1661. doi: 10.1016/j.neuroimage.2012.01.073 j Mossad, S. I., AuCoin-Power, M., Urbain, C., Smith, M. L., Pang, E. W., and Taylor, M. J. (2016). Thinking about the thoughts of others; temporal and spatial neural activation during false belief reasoning. NeuroImage 134, 320–327. doi: 10.1016/j.neuroimage.2016.03.053 Eddy, C. M. (2016). The junction between self and other? Temporo- parietal dysfunction in neuropsychiatry. Neuropsychologia 89, 465–477. doi: 10.1016/j.neuropsychologia.2016.07.030 Flavell, J. H., Flavell, E. R., and Green, F. L. (1983). Development of the appearance-reality distinction. Cogn. Psychol. 15, 95–120. doi: 10.1016/0010-0285(83)90005-1 Myowa-Yamakoshi, M., Kawakita, Y., Okanda, M., and Takeshita, H. (2011). Visual experience influences 12-month-old infants’ perception of goal-directed actions of others. Dev. Psychol. 47, 1042–1049. doi: 10.1037/a0023765 Frith, C. D., and Frith, U. (1999). Interacting minds-a biological basis. Science 286, 1692–1695. doi: 10.1126/science.286.5445.1692 O’Laughlin, C., and Thagard, P. (2000). Autism and coherence: a computational model. Mind Lang. 15, 375–392. doi: 10.1111/1468-0017.00140 Frith, C. D., and Frith, U. (2006). The neural basis of mentalizing. Neuron 50, 531–534. doi: 10.1016/j.neuron.2006.05.001 Patacchiola, M., and Cangelosi, A. (2020). A developmental cognitive architecture for trust and theory of mind in humanoid robots. IEEE Trans. Cybern. 1–13. doi: 10.1109/TCYB.2020.3002892 j Gallagher, H. L., and Frith, C. D. (2003). Functional imaging of “theory of mind”. Gallagher, H. L., and Frith, C. D. (2003). 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(2016). “Towards robot self-consciousness (i): Brain-inspired robot mirror neuron system model and its application in mirror self-recognition,” in International Conference on Brain Inspired Cognitive Systems (Beijing), 11–21. doi: 10.1007/978-3-319-49 685-6_2 Yang, G.-Z., Bellingham, J., Dupont, P. E., Fischer, P., Floridi, L., Full, R., et al. (2018). The grand challenges of science robotics. Sci. Robot. 3, 1–14. doi: 10.1126/scirobotics.aar7650 Frontiers in Neurorobotics | www.frontiersin.org REFERENCES doi: 10.1006/nimg.2001.0789 p yg Leech, R., and Sharp, D. J. (2014). The role of the posterior cingulate cortex in cognition and disease. Brain 137(Pt 1), 12–32. doi: 10.1093/brain/awt162 Leslie, A. M., and Polizzi, P. (1998). Inhibitory processing in the false belief task: two conjectures. Dev. Sci. 1, 247–253. doi: 10.1111/1467-7687.00038 Vogeley, K., May, M., Ritzl, A., Falkai, P., Zilles, K., and Fink, G. R. (2004). Neural correlates of first-person perspective as one constituent of human self-consciousness. J. Cogn. Neurosci. 16, 817–827. doi: 10.1162/089892904 970799 Milliez, G., Warnier, M., Clodic, A., and Alami, R. (2014). “A framework for endowing an interactive robot with reasoning capabilities about perspective- taking and belief management,” in Ro-Man: the IEEE International Symposium on Robot and Human Interactive Communication (Edinburgh), 1103–1109. doi: 10.1109/ROMAN.2014.6926399 Wimmer, H., and Perner, J. (1983). Beliefs about beliefs: representation and constraining function of wrong beliefs in young children’s understanding of deception. Cognition 13, 103–128. doi: 10.1016/0010-0277(83)90004-5 August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org Frontiers in Neurorobotics | www.frontiersin.org 16 Zeng et al. Brain-Inspired Theory of Mind Model Yang, G.-Z., Bellingham, J., Dupont, P. E., Fischer, P., Floridi, L., Full, R., et al. (2018). The grand challenges of science robotics. Sci. Robot. 3, 1–14. doi: 10.1126/scirobotics.aar7650 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Zeng, Y., Zhao, Y., and Bai, J. (2016). “Towards robot self-consciousness (i): Brain-inspired robot mirror neuron system model and its application in mirror self-recognition,” in International Conference on Brain Inspired Cognitive Systems (Beijing), 11–21. doi: 10.1007/978-3-319-49 685-6_2 Copyright © 2020 Zeng, Zhao, Zhang, Zhao, Zhao and Lu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. August 2020 | Volume 14 | Article 60 Frontiers in Neurorobotics | www.frontiersin.org 17
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https://repozytorium.uwb.edu.pl/jspui/bitstream/11320/4972/1/CNISK_1%281%29_2016_G_Kedzierska_Opinia_bieglego_w_slynnych_procesach_przeciwko_kobietom.pdf
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Opinia biegłego w słynnych procesach przeciwko kobietom
Czasopismo Naukowe Instytutu Studiów Kobiecych
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DR GRAŻYNA KĘDZIERSKA Niepaństwowa Wyższa Szkoła Pedagogiczna w Białymstoku DOI 10.15290/cnisk.2016.01.01.05 DOI 10.15290/cnisk.2016.01.01.05 DR GRAŻYNA KĘDZIERSKA 1 A. Murzynowski, Istota i zasady procesu karnego, Warszawa 1994, s. 110. Ce- chy zasady: zasada musi posiadać węzłowe znaczenie dla określenia modelu procesu; zasada powinna zawierać określoną treść ideologiczną i społeczną, wskazując cele i wartości, jakie należy osiągnąć w danej społeczności; zasada powinna dotyczyć bezpośrednio procesu, niezależnie od obowiązywania w in- nych gałęziach prawa (zasadami procesowymi nie są: zasada humanizmu, de- mokratyzmu jako uniwersalne); zasada powinna być sformułowana w sposób dyrektywny, który wyraża pewną prawidłowość, rozwiązanie organizacyjne lub zachowanie się. Podział zasad: − naczelna zasada: zasada prawdy, Streszczenie Sformułowanie „wskazań wiedzy”, zawarte w treści procesowej za- sady swobodnej oceny dowodów, odnosi się do wykorzystania przez or- gan procesowy osiągnięć nauki w postępowaniu karnym. Kluczowa jest tutaj opinia biegłego, oparta na badaniach, zgodnych z aktualnym sta- nem nauki. Niezależnie jednak od wyniku badań prezentowanego w opi- nii, nigdy nie jest ona wiążąca dla sądu. Jakie więc jest znaczenie opinii biegłego dla rozstrzygnięcia istotnych wątpliwości występujących w da- nej sprawie? Opinia biegłego zawsze miała znaczenie adekwatne do sta- nu nauki w danym okresie historycznym. Wraz z rozwojem nauki rosły możliwości badawcze biegłego, ustalanie okoliczności zdarzenia pozwa- lało rozstrzygać więcej wątpliwości. Z perspektywy przestępczości kobiet w słynnych procesach, opinie biegłych czasem nie potrafiły rozstrzygnąć wątpliwości i dawały uniewinnienie oskarżonym, bywały mylne i dopiero zbieg okoliczności decydował o ustaleniu sprawcy, prowadziły do obar- czenia winą jednej osoby i uniewinnienia drugiej, zdarzało się, że nie by- ły brane pod uwagę, co skutkowało surowym wyrokiem, najczęściej jed- nak dawały możliwość wymierzenia kary. Słowa kluczowe: ekspertyza • historia kryminalistyki • opinia bie- głego • przestępczość kobiet • zasada swobodnej oceny dowodów C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h G . K ę d z i e r s k a , O p i n i a b i e g ł e g o w s ł y n n y c h p r o c e s a c h . . . 85 THE EXPERT WITNESS’S OPINION IN WELL-KNOWN TRIALS AGAINST WOMEN Abstract − naczelna zasada: zasada prawdy, Abstract The term “indications of knowledge” used in the content of the pro- cedural principle of free appraisal of evidence refers to the judicial body using scientific developments in the area of criminal proceedings. The expert’s opinion, based on the current state of knowledge, is in such cases of utmost importance. However, whatever the expert’s opinion ev- idence, it will not be bound by the courts. So how important are the opinions of experts in settling the doubts in a criminal case? As was shown by the analysis of several well-known trials against women, the research capacity of the expert witness always corresponded to the state of science in a relevant historical period. Unfortunately, sometimes the expert witness’s opinion was not able to clear up doubts and resulted in finding a defendant not guilty. Occasionally, it was erroneous and the perpetrator was identified only as a result of coincidence. In some cases the opinion led to acquitting one person and finding another one guilty, or the opinion was not taken into account by the court at all. Neverthe- less, in most cases scientific findings of the expert witness presented in an opinion dispelled the doubts of the court which significantly influ- enced sentencing. Keywords: history of criminology • expert witness’s opinion • crime of women • the principle of free appraisal of evidence • scientific devel- opments Społecznie ważne, ogólne dyrektywy dotyczące najbardziej istotnych kwestii z zakresu procesu karnego zostały uregulowane w zasadach procesowych1. Za niezwykle istotną wśród tych kil- 1 ( 1 ) 2 0 1 6 1 ( 1 ) 2 0 1 6 1 ( 1 ) 2 0 1 6 S t u d i a i m a t e r i a ł y 86 kunastu dyrektyw uznaje się zasadę swobodnej oceny dowodów, która stanowi, że organ procesowy2 w ocenie dowodów kieruje się swoim przekonaniem nieskrępowanym ustawowymi regułami oceny, ukształtowanym pod wpływem wskazań wiedzy, doświad- czenia życiowego i zasad logicznego rozumowania. Kluczowe, dla poniższych rozważań w treści tej zasady jest sformułowanie wskazań wiedzy, które odnosi się do wykorzystania przez organ procesowy osiągnięć nauki w przeprowadzaniu dowodów. Zasada swobodnej oceny dowodów jest w prawie procesowym bezwarun- kowo statuowana i wskazuje na brak możliwości wartościowania jakichkolwiek dowodów. − zasady materialno-procesowe: zasada wychowawczego oddziaływania wy- miaru sprawiedliwości, zasada legalizmu, zasada ścigania z urzędu, − zasady dotyczące formy procesu: zasada skargowości, zasada kontradykto- ryjności, zasada publiczności, zasada instancyjności, − zasady dotyczące stanowiska sądu: zasada niezawisłości sędziowskiej, za- sada udziału czynnika społecznego, zasada uczciwego procesu, − zasady dotyczące sytuacji prawnej oskarżonego: zasada domniemania nie- winności, zasada prawa do obrony, − zasady dotyczące postępowania dowodowego: zasada swobodnej oceny do- wodów, zasada bezpośredniości. 2 Organ procesowy jest uczestnikiem postępowania karnego. W zależności od etapu postępowania karnego organem jest np. prokurator, Policja, Straż Gra- niczna lub sąd. 3 Ekspertyza jest to zespół czynności badawczych wymagających wiadomości spe- cjalnych i dlatego wykonywanych na zlecenie organu procesowego, zakończonych opinią mogącą mieć charakter dowodu w procesie. Definicja ta została sformuło- wana przez Profesora Tadeusza Hanauska, kryminalistyka i kryminologa Uniwer- sytetu Jagiellońskiego. Szerzej na temat badań w ramach ekspertyzy, biegłego i opinii: W. Kędzierski, Ekspertyza. Biegli [w:] Kryminalistyka. Wybrane zagadnie- nia techniki, red. G. Kędzierska i W. Kędzierski, Szczytno 2011, s. 450 i n. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h − zasady materialno-procesowe: zasada wychowawczego oddziaływania wy- miaru sprawiedliwości, zasada legalizmu, zasada ścigania z urzędu, 3 Ekspertyza jest to zespół czynności badawczych wymagających wiadomości spe- cjalnych i dlatego wykonywanych na zlecenie organu procesowego, zakończonych opinią mogącą mieć charakter dowodu w procesie. Definicja ta została sformuło- wana przez Profesora Tadeusza Hanauska, kryminalistyka i kryminologa Uniwer- sytetu Jagiellońskiego. Szerzej na temat badań w ramach ekspertyzy, biegłego i opinii: W. Kędzierski, Ekspertyza. Biegli [w:] Kryminalistyka. Wybrane zagadnie- nia techniki, red. G. Kędzierska i W. Kędzierski, Szczytno 2011, s. 450 i n. − zasady materialno-procesowe: zasada wychowawczego oddziaływania wy- miaru sprawiedliwości, zasada legalizmu, zasada ścigania z urzędu, − zasady dotyczące formy procesu: zasada skargowości, zasada kontradykto- ryjności, zasada publiczności, zasada instancyjności, − zasady dotyczące stanowiska sądu: zasada niezawisłości sędziowskiej, za- sada udziału czynnika społecznego, zasada uczciwego procesu, − zasady dotyczące sytuacji prawnej oskarżonego: zasada domniemania nie- winności, zasada prawa do obrony, − zasady dotyczące postępowania dowodowego: zasada swobodnej oceny do- wodów, zasada bezpośredniości. O j iki i k l i d 2 Organ procesowy jest uczestnikiem postępowania karnego. W zależności od etapu postępowania karnego organem jest np. prokurator, Policja, Straż Gra- niczna lub sąd. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h Abstract Opinia biegłego oparta na badaniach zgodnych z aktualnym stanem nauki nigdy nie jest jednak wiążą- ca dla sądu, jest tylko środkiem dowodowym i podlega swobodnej ocenie, tak jak inne środki dowodowe. Opinia jest końcowym wynikiem badań podejmowanych przez biegłego w ramach czynności procesowej, jaką jest ekspertyza3. Biegły to osoba posiadająca wiadomości specjalne, wezwana do zbadania i wyjaśnienia okoliczności istotnych dla rozstrzygnięcia sprawy. Wiadomości specjalne, które stanowią atrybut biegłego, są wiedzą, która w określonym czasie przekracza zasób informacji przeciętnie wykształconego, dorosłego człowieka w dziedzinie na- 2 Organ procesowy jest uczestnikiem postępowania karnego. W zależności od etapu postępowania karnego organem jest np. prokurator, Policja, Straż Gra- niczna lub sąd. 3 Ekspertyza jest to zespół czynności badawczych wymagających wiadomości spe- cjalnych i dlatego wykonywanych na zlecenie organu procesowego, zakończonych opinią mogącą mieć charakter dowodu w procesie. Definicja ta została sformuło- wana przez Profesora Tadeusza Hanauska, kryminalistyka i kryminologa Uniwer- sytetu Jagiellońskiego. Szerzej na temat badań w ramach ekspertyzy, biegłego i opinii: W. Kędzierski, Ekspertyza. Biegli [w:] Kryminalistyka. Wybrane zagadnie- nia techniki, red. G. Kędzierska i W. Kędzierski, Szczytno 2011, s. 450 i n. G . K ę d z i e r s k a , O p i n i a b i e g ł e g o w s ł y n n y c h p r o c e s a c h . . . 87 uki, techniki, sztuki lub rzemiosła, przy czym wiedza ta obejmuje tak teorię, jak i umiejętności praktyczne. Istotą każdej ekspertyzy są czynności badawcze opierające się na wiedzy z zakresu okre- ślonej dyscypliny naukowej. Według kryterium, które stanowi ro- dzaj wiedzy wykorzystywanej podczas czynności badawczych, klasycznie ekspertyzy dzieli się na kryminalistyczne (a wśród nich są daktyloskopijne, mechanoskopijne, pisma, traseologiczne, broni) oraz inne, takie jak: biologiczne, chemiczne, toksykologicz- ne, medyczne, informatyczne. Opinia jest twierdzeniem formułowanym przez biegłego na podstawie wniosków osiągniętych z wyników badań. Sąd nigdy nie jest związany twierdzeniem biegłego i zawsze dokonuje swo- bodnej oceny każdej opinii. Swobodnej – nie oznacza jednak do- wolnej, co podkreśla się tak w doktrynie, jak i w orzecznictwie. Zdarza się, że nawet opinia z badań daktyloskopijnych4 nie zosta- je uznana przez sąd za dowód w sprawie. Nie umniejsza to w żadnym razie roli badań i opinii biegłego w ustalaniu okolicz- ności istotnych dla rozstrzygnięcia sprawy. Autorzy literatury faktu5 oraz badacze dosyć chętnie zajmują się przestępczością kobiet, wyraźnie faworyzując przestępstwo zabójstwa dokonywane przez kobiety. 4 Daktyloskopia jest metodą identyfikacji człowieka na podstawie śladów linii papilarnych palców rąk. Jest to metoda najlepiej zbadana a jej ustalenia nie budzą wątpliwości. Identyfikacja daktyloskopijna opiera się na trzech cechach linii papilarnych: niezmienności, nieusuwalności i indywidualności. 5 Do tej grupy należy chyba zaliczyć książki pisane na podstawie wywiadów, charakteryzujące się dużą społeczną uwagą i poczytnością, jak K. Bonda, B. Lach, Zbrodnia niedoskonała, Chorzów 2009, ss. 247 lub K. Bonda, Polskie morderczynie, Warszawa 2008, ss. 304. linii papilarnych: niezmienności, nieusuwalności i indywidualności. 5 Do tej grupy należy chyba zaliczyć książki pisane na podstawie wywiadów, charakteryzujące się dużą społeczną uwagą i poczytnością, jak K. Bonda, B. Lach, Zbrodnia niedoskonała, Chorzów 2009, ss. 247 lub K. Bonda, Polskie morderczynie, Warszawa 2008, ss. 304. 4 Daktyloskopia jest metodą identyfikacji człowieka na podstawie śladów linii papilarnych palców rąk. Jest to metoda najlepiej zbadana a jej ustalenia nie budzą wątpliwości. Identyfikacja daktyloskopijna opiera się na trzech cechach linii papilarnych: niezmienności, nieusuwalności i indywidualności. Abstract Zdarzenia tego typu mają znaczny stopień sensacyjności właśnie z powodu płci sprawcy. Od wieków zabójstwo, w którym oskarżano kobietę budziło żywe zainteresowanie tak specjalistów, jak i szerokich rzesz społeczeń- stwa. W wielu takich przypadkach konieczność ustalenia zgodne- go z prawdą przebiegu zdarzenia była motorem rozwoju wiedzy kryminalistycznej i nie tylko. Zważywszy poczynione na wstępie uwagi, w artykule podjęto próbę odpowiedzi na pytanie: Jakie znaczenie ma opinia biegłego w ustalaniu okoliczności istotnych 1 ( 1 ) 2 0 1 6 88 S t u d i a i m a t e r i a ł y dla rozstrzygnięcia sprawy w słynnych procesach przeciwko ko- bietom? dla rozstrzygnięcia sprawy w słynnych procesach przeciwko ko- bietom? Materiał do analizy stanowiły opisy przypadków uzyskane z literatury oraz ogólnie dostępnego źródła informacji – Internetu. Wybrano siedem przypadków, w których o popełnienie zabójstwa oskarżano kobietę. Rozstrzygnięcie wątpliwości pojawiających się w trakcie postępowania karnego wymagało, aby organy proceso- we skorzystały z wiedzy biegłych różnych specjalności. Przypadki obejmują dosyć duży zakres czasowy, a niektóre pokazują rozwój badań kryminalistycznych, z zakresu medycyny sądowej oraz toksykologii, stymulowanych potrzebami praktyki. Przestępczość kobiet była i jest silnie związana z rolą, jaką kobiety pełniły i pełnią w społeczeństwie. Większość kobiecych przestępstw to zdarzenia o małej szkodliwości społecznej, ale ko- biety dopuszczały się w przeszłości i dzisiaj także zabójstw, a właśnie te przypadki zapadają w pamięć i zapisują się na kar- tach niechlubnej historii kobiecych zbrodni. Są to również spra- wy, w których najczęściej organy ścigania i wymiaru sprawiedli- wości zwracają się o pomoc do biegłych. W przeszłości odbyło się niemało widowiskowych procesów, w których na ławie oskarżo- nych zasiadały kobiety. Orzeczenia sądów w tych procesach w dużym stopniu były uzależnione od opinii biegłych. Dzisiaj opinia biegłego potrafi wyjaśnić wiele wątpliwości i zro- zumieć okoliczności przestępstwa, jednak w całkiem nieodległych czasach zdarzało się, że nie była ona w stanie pokonać zastrzeżeń organu wymiaru sprawiedliwości. Potrzebę obiektywnej pomocy prokuratorom i sędziom w ustalaniu okoliczności istotnych dla rozstrzygnięcia sprawy widziano już bardzo dawno, ale stan na- uki był poważną blokadą jakichkolwiek racjonalnych działań w tej dziedzinie. Ilustracją takiej sytuacji, trwającej kilka stuleci, są procesy czarownic. W XV wieku, mocą swojego autorytetu, Kościół Kato- licki potwierdził istnienie czarownic, a w 1487 r. ukazało się dzie- ło dwóch dominikanów Hainricha Kraemera i Jacoba Sprengera pt. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h 8 Na przykład latem 1775 r. w Doruchowie, wsi położonej pod Kępnem w powiecie ostrzeszowskim oskarżono o czary czternaście wiejskich kobiet, za- sądzono je na spalenie po pławieniu i torturach a następnie spalono. Dramat rozpętała żona dziedzica Doruchowa, która żaliła się na prześladowania czarow- nic, twierdząc, że wszystkie jej dolegliwości pochodzą od uroków. S. Szenic, Pi- taval wielkopolski, Poznań 1977, s. 9 i n. 9 Arszenik nazywano trucizną wszech czasów. Jako pierwszy wyodrębnił go w VIII wieku arabski alchemik Geber. Był nazywany „trucizną królewską”, „proszkiem dziedziczenia”, bo jako biały, bezsmakowy proszek łatwo było go domieszać do potraw i napoi. Zaczyna działać po kilku godzinach od spożycia, wywołując objawy jak przy zatruciu pokarmowym. Toksykologia u zarania swo- ich dziejów za podstawowy problem uznała opracowanie metod wykrywania tej trucizny w tkankach zwłok. Znaczny udział w tych poszukiwaniach mają polscy badacze. J. Widacki, Stulecie polskich detektywów, Kraków 1992, s. 104, 105. 7 Dwa inne warunki uznania za czarownicę polegały na ustaleniu, czy po kłótni z kobietą zdarzały się nieszczęśliwe wypadki i czy widziano, jak postać kobiety czyni szkodę. Ostatni z wymienionych warunków nazywano dowodem widmo- wym. 6 S. Wrzesiński, Potępieńcy średniowiecznej Europy, Kraków 2007, s. 65; D. Alexander, Czarownice, wróżbici, szamani. Podróż przez świat mitów i magii, Warszawa 2008, s. 59. y j y D. Alexander, Czarownice, wróżbici, szamani. Podróż przez świat mitów i magii, Warszawa 2008, s. 59. 7 Dwa inne warunki uznania za czarownicę polegały na ustaleniu, czy po kłótni z kobietą zdarzały się nieszczęśliwe wypadki i czy widziano, jak postać kobiety czyni szkodę. Ostatni z wymienionych warunków nazywano dowodem widmo- wym. 6 S. Wrzesiński, Potępieńcy średniowiecznej Europy, Kraków 2007, s. 65; D. Alexander, Czarownice, wróżbici, szamani. Podróż przez świat mitów i magii, Warszawa 2008, s. 59. 7 Dwa inne warunki uznania za czarownicę polegały na ustaleniu, czy po kłótni z kobietą zdarzały się nieszczęśliwe wypadki i czy widziano, jak postać kobiety czyni szkodę. Ostatni z wymienionych warunków nazywano dowodem widmo- wym. 8 Na przykład latem 1775 r. w Doruchowie, wsi położonej pod Kępnem w powiecie ostrzeszowskim oskarżono o czary czternaście wiejskich kobiet, za- sądzono je na spalenie po pławieniu i torturach a następnie spalono. Dramat rozpętała żona dziedzica Doruchowa, która żaliła się na prześladowania czarow- nic, twierdząc, że wszystkie jej dolegliwości pochodzą od uroków. S. Szenic, Pi- taval wielkopolski, Poznań 1977, s. 9 i n. 9 Arszenik nazywano trucizną wszech czasów. Jako pierwszy wyodrębnił go w VIII wieku arabski alchemik Geber. Był nazywany „trucizną królewską”, „proszkiem dziedziczenia”, bo jako biały, bezsmakowy proszek łatwo było go domieszać do potraw i napoi. Zaczyna działać po kilku godzinach od spożycia, wywołując objawy jak przy zatruciu pokarmowym. Toksykologia u zarania swo- ich dziejów za podstawowy problem uznała opracowanie metod wykrywania tej trucizny w tkankach zwłok. Znaczny udział w tych poszukiwaniach mają polscy badacze. J. Widacki, Stulecie polskich detektywów, Kraków 1992, s. 104, 105. 6 S. Wrzesiński, Potępieńcy średniowiecznej Europy, Kraków 2007, s. 65; D. Alexander, Czarownice, wróżbici, szamani. Podróż przez świat mitów i magii, Warszawa 2008, s. 59. 7 Dwa inne warunki uznania za czarownicę polegały na ustaleniu, czy po kłótni z kobietą zdarzały się nieszczęśliwe wypadki i czy widziano, jak postać kobiety Abstract Młot na czarownice, w którym opisano cechy czarownicy, ro- C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h G . K ę d z i e r s k a , O p i n i a b i e g ł e g o w s ł y n n y c h p r o c e s a c h . . . 89 dzaje uprawianych czarów i sposoby zwalczania czarodziejstwa6. Żeby jednak doszło do skazania kobiety za uprawianie czarów, konieczna była jej identyfikacja jako czarownicy. Dokonywano te- go przez szczególny rodzaj badania. Otóż jeżeli na ciele kobiety ujawniono znamię, wbijano w nie szpilkę. Płynąca po takim za- biegu krew miała wskazywać, że znamię jest punktem karmienia diabła, co oczywiście było równoznaczne ze stwierdzeniem, że ko- bieta była czarownicą7. Tak przedstawiał się ówczesny rodzaj ba- dań identyfikacyjnych. Procesy o czary – chociaż z punktu widze- nia obowiązującego wówczas prawa nie można im nic zarzucić – są przykładem niesłusznego sądzenia kobiet i skazywania ich za czyny, których w rzeczywistości nie popełniły. Kobiety w tych procesach były ofiarami systemów politycznych, wierzeń i zabo- bonów8, a prymitywne badanie nazywane dowodem reprezento- wało ówczesną naukę. Przez wiele wieków trucizny były niewykrywalne, nie było me- tod ich oznaczania w płynach ustrojowych i tkankach ludzkich. Niewątpliwie trucizną może być każda substancja. Wszystko zale- ży od ilości, sposobu podania i wrażliwości organizmu, który sub- stancję przyjmuje. Popularne były trucizny roślinne, ale to arsze- nik9 był uważany za substancję powszechnie stosowaną właśnie 1 ( 1 ) 2 0 1 6 1 ( 1 ) 2 0 1 6 1 ( 1 ) 2 0 1 6 S t u d i a i m a t e r i a ł y 90 przez kobiety do pozbawiania życia niewygodnych osób. W Rzy- mie tylko w roku 184 p.n.e. za trucicielstwo skazano prawie dwa tysiące kobiet10. Najsłynniejszą trucicielką była wtedy Lukusta, która działała w sferach rzymskiej arystokracji11. W roku 1850 w Belgii popełniono zbrodnię, której wyjaśnienie wymagało zaangażowania biegłego chemika. Dwoje sprawców, hrabia Hippolit Visart de Bocarmé i jego żona Lidia12, dokonało zabójstwa Gustawa Fougnies. Zbrodnię popełniono w zamku Bi- tremont. Gustaw był bratem Lidii, a motywem były pieniądze. , , , 12 J. Thorwald, Stulecie detektywów, Kraków 1992, s. 314. , y , , 13 Aleksander Lacassangge (1843–1924), francuski lekarz i kryminolog. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h 10 Z. Majchrzyk, Kiedy kobieta zabija, Warszawa 2009, s. 34. j y y j 11 A. Krawczuk, Neron, Warszawa 1988, s. 90. 10 Z. Majchrzyk, Kiedy kobieta zabija, Warszawa 2009, s. 34. 11 A. Krawczuk, Neron, Warszawa 1988, s. 90. 12 J. Thorwald, Stulecie detektywów, Kraków 1992, s. 314. 13 Aleksander Lacassangge (1843–1924), francuski lekarz i kryminolog. j y y j A. Krawczuk, Neron, Warszawa 1988, s. 90. 14 G. Faix, Sûreté. Wielkie ucho Paryża, Katowice 1988. 15 [Dostęp: 12.07. 2016]. Dostępny w World Wide Web: <www.kryminalistyka. fr.pl./crime_polska_gorgonowa.php>. 16 S. K. Kolińska, Słynne procesy. Dramaty, tajemnice, zbrodnie, Warszawa 2008, s. 224. Abstract Do zabójstwa użyto nikotynę – alkaloid roślinny uważany wtedy za niewykrywalny. Aby zatrzeć ślady trucizny, zabójcy wlewali w usta Gustawa i polewali jego ciało octem winnym. Sekcja zwłok wykazała śmierć gwałtowną, wskutek wypicia żrącej substancji – jednak nie było wiadomo jakiej. Belgijski chemik Jean Servais Stas (1813–1891) przeprowadzał badanie zwłok i zastanawiał go unoszący się nad ciałem zapach tytoniu, przebijający się przez woń octu. Inspirowany swoim spostrzeżeniem, opracował metodę wykrywania nikotyny w tkankach zwłok, która pozwoliła ustalić, że Gustaw Fougnies został otruty właśnie tym alkaloidem. Sędzia śledczy, który prowadził sprawę odkrył równocześnie, że Hipolit de Bocarmé sam wytwarzał wodę kolońską z liści tytoniu. Efek- tem opinii, jaką wydał Jean Servais Stas, było skazanie Hippolita de Bocarmé na karę śmierci, a metoda wykrywania nikotyny zna- lazła zastosowanie w rozwiązywaniu spraw kryminalnych. Pod koniec XIX wieku na brzegu Rodanu niedaleko Lyonu znaleziono kufer z rozkawałkowanymi zwłokami. Zwłoki nazwano zmarłym z Millery. Ekspertyzę anatomopatologiczną przeprowa- dził Aleksander Lacassagne13, ostatecznie doprowadzając do iden- tyfikacji zwłok, co miało istotne znaczenie z punktu widzenia po- szukiwań zaginionego komornika o nazwisku Gouffé. Po zbada- niu kości zwłok obliczono wzrost zmarłego z Millery na 1,785 cm, podczas gdy komornik miał 1,78 cm. Szczegółowe oględziny kości nóg zmarłego wykazały ich zmiany chorobowe. Świadkowie ze- G . K ę d z i e r s k a , O p i n i a b i e g ł e g o w s ł y n n y c h p r o c e s a c h . . . 91 znawali, że zaginiony Gouffé utykał. Analiza uzębienia zwłok do- starczyła dowodów, że zmarły był w wieku około 50 lat, podczas gdy Gouffé w chwili zaginięcia miał lat 49. Okazało się także, że włosy zwłok i włosy ze szczotki z mieszkania komornika miały ta- kie same cechy. W ten sposób Aleksander Lacassagne udowodnił, że zmarły z Millery to ciało komornika Gouffé. Zidentyfikowanie zwłok pozwoliło oskarżyć o zabójstwo parę: Michaela Eyrauda – paryskiego sutenera i oszusta oraz Gabrielę Bombard – prosty- tutkę. Mężczyzna został skazany na karę śmierci, a jego wspól- niczka otrzymała wyrok 20 lat ciężkich robót14. 23 kwietnia 1932 r. przed sądem we Lwowie stanęła Rita Gorgonowa15. Rita z pochodzenia Dalmatynka, pracowała jako opiekunka do dzieci u architekta inż. Henryka Zaremby, którego żona przebywała w zakładzie dla psychicznie chorych. Zaremba miał córkę Elżbietę i syna Stasia. Abstract Rita związała się z Zarembą i w 1928 roku urodziła mu córkę. Dzieci Zaremby źle odbierały ten związek. 30 grudnia 1931 r. Elżbietę znaleziono martwą w willi Zaremby w Brzuchowicach. W noc jej śmierci Stasia obu- dził skowyt psa. Wstał z łóżka, poszedł do holu i tam między cho- inką a pianinem zobaczył postać kobiecą. Postać przesunęła się bardzo cicho przez odchylone drzwi na werandę i znikła. Staś pomyślał, że była to Gorgonowa16. Wpadł do pokoju Lusi, zoba- czył krew i zorientował się, że siostra nie żyje. Oględziny pokoju nie wykazały żadnych śladów wskazujących na włamanie ani na podłodze, ani na częściach okna. W holu przy wejściu do pokoju Lusi znaleziono grudki kału, a nad nim na ścianie trzy plamy krwawe. Częściowo przyprószone ślady na śniegu stóp obutych w pantofle biegły od frontu do małej weran- dy, komunikującej z sypialnią Gorgonowej, następnie rozwidlały się w kierunku piwniczki i basenu. W drzwiach małej werandy szyba była wybita na wysokości klamki. Ujawniono ślady krwi na murze niedaleko werandy oraz na drzwiach prowadzących do ma- 1 ( 1 ) 2 0 1 6 S t u d i a i m a t e r i a ł y 92 łej piwniczki. W piwniczce znaleziono chusteczkę z kilkoma różo- wymi plamkami. W basenie odkryto żelazny dżagan. Nie było na nim śladów krwi, a ogrodnik powiedział, że używał dżagana do robienia przerębli. Gorgonową zastano w futrze i w pantoflach. Na tylnej podszewce futra były ślady kału. Pantofle były mokre z du- żymi rdzawymi plamami. Prawa ręka Gorgonowej była zraniona. y y g j y W pierwszym procesie R. Gorgonową uznano za winną zabój- stwa Elżbiety Zaremby i skazano na karę śmierci. Podstawowym dowodem obciążającym było rozpoznanie Rity przez Stasia Za- rembę, który podobno zauważył jej postać w holu, za choinką. Po rewizji, we wrześniu 1932 r., Sąd Najwyższy wyrok uchylił i sprawa została rozpatrzona przez Sąd Okręgowy w Krakowie. Wtedy zakwestionowano wiarygodność Stasia jako świadka. 6 marca 1933 r. rozpoczął się następny proces. Zdaniem adwoka- ta Rity ekspertyzy przedstawione w pierwszym procesie zostały wykonane niefachowo. Teraz biegły, Jan Olbrycht, stwierdził, że kał to odchody psa. Cały czas rozpatrywano problem domniema- nego narzędzia zbrodni – dżagana, znalezionego na dnie basenu i braku na nim śladów krwi. Niestety, nie umiano jeszcze porów- nywać kształtu obrażeń, jakie powstały na czaszce z konkretnym narzędziem. Kontrowersyjny był także dowód z opinii po badaniu serologicznym śladów krwi na chusteczce Gorgonowej17. 18 J. Thorwald, op. cit., s. 362. 17 J. Widacki, op. cit., s. 202. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h Abstract Sąd jed- nak ponownie uznał Ritę za winną, orzekając tym razem tylko 8 lat więzienia, przyjmując, że zbrodnię popełniła pod wpływem silnego wzruszenia. Ritę Gorgonową również w drugim procesie skazano przede wszystkim na podstawie tak zwanego dowodu widmowego, stosowanego w czasach procesów czarownic, czyli zeznań dwunastoletniego chłopca, który widział postać Rity. Gor- gonowa wyszła na wolność po 6 latach na skutek amnestii, ogło- szonej w związku z wybuchem wojny. Annały kryminalistyki opisują także długotrwały proces fran- cuskiej wieśniaczki Marii Besnard (1896–1980)18. W roku 1949 została ona oskarżona o dwunastokrotne morderstwo na osobach swoich krewnych i znajomych. W celu rozstrzygnięcia wątpliwości G . K ę d z i e r s k a , O p i n i a b i e g ł e g o w s ł y n n y c h p r o c e s a c h . . . 93 ekshumowano 12 zwłok i wykonano wiele analiz toksykologicz- nych. W 11 ciałach badania wykonane przez dra Bernarda wyka- zały zabójczą ilość arsenu w przeliczeniu na kilogram tkanek. Śmierć każdej z tych osób przynosiła Marii Besnard dziedziczenie pokaźnego spadku. W 1961 r. w trzecim procesie oskarżoną uniewinniono, mimo że już w drugim procesie profesor P.L. Truf- fert zwracał uwagę, że bakterie glebowe są w stanie przenieść ar- sen do zwłok. Wątpliwość, skąd się wzięła tak duża ilość arsenu w zwłokach, sąd uwzględnił w trzecim procesie, uniewinniając Marię Besnard. Ówczesny stan nauki nie potrafił rozstrzygnąć istotnej niepewności w tej sprawie. Niewiele lat po zakończeniu wojny, w roku 1954 w Wormacji, pod zarzutem otrucia męża, teścia i przyjaciółki sądzono Christę Lehmann19. Zlecono ekspertyzę chemiczno-toksykologiczną i wte- dy okazało się, że w treści żołądka wszystkich ofiar znajdują się składniki substancji określanej symbolem E 605, stosowanej w ochronie roślin. Christa Lehmann została uznana winną zarzu- canych jej zabójstw i skazana na dożywotnie pozbawienie wolno- ści. Interesujące były reperkusje tej sprawy, ponieważ do 1954 r. nigdy nie dokonano zabójstwa za pomocą E 605. Natomiast pra- wie natychmiast po ogłoszeniu, że za pomocą E 605 uśmiercono trzy osoby, w Niemczech i Austrii rozpoczęła się fala zabójstw oraz samobójstw z użyciem tego środka. Kilku niemieckich che- mików rozpoczęło walkę o szybkie opracowanie metody oznacza- nia zawartości E 605 w płynach ustrojowych i tkankach osób przypadkowo zatrutych. Połowiczny sukces, który wtedy osiągnię- to pozwalał ustalać zawartości E 605 we krwi. 14 stycznia 2000 r. 19 Idem, Stulecie detektywów, wyd. 2, Kraków 2009, s. 571 i n. 20 Badania wariograficzne mają w Polsce także inną nazwę: badania poligraficz- ne. Niezależnie jednak od nazwy jest to instrumentalna metoda ustalania wiedzy osoby o realiach zdarzenia i modus operandi sprawcy. Definicję tę przedstawił prof. dr hab. Mariusz Kulicki dla badań wariograficznych wykonywanych na po- trzeby procesu karnego. Więcej na ten temat M. Kulicki, V. Kwiatkowska- Wójcikiewicz, L. Stępka, Kryminalistyka. Wybrane zagadnienia teorii i praktyki śledczo-sądowej, Toruń 2009, s. 641 i n. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h Abstract trzy kobiety – córka, konkubina i przyja- ciółka rodziny – zabiły 70-letniego Antoniego K., radcę prawnego z Chrzanowa. Najpierw próbowały go otruć pierogami z nadzie- niem z muchomorów. Gdy to nie poskutkowało, podały mu środki usypiające w kawie. Gdy zasnął, 41-letnia Anna G., konkubina, uderzyła go łomem w głowę, a córka radcy, 43-letnia Katarzyna K., dobiła ojca, zakładając na jego głowę torebkę foliową i przydu- szając go poduszką. 41-letnia Aleksandra C. M. była ich znajomą 1 ( 1 ) 2 0 1 6 S t u d i a i m a t e r i a ł y 94 i w szczegółach zaplanowała zbrodnię. Anna G., i Katarzyna K. poćwiartowały ciało, a kawałki w reklamówkach wkładały do plecaków, autobusem zawoziły do Krakowa i tam w rejonie Sal- watora wrzucały je do Wisły. Jednego dnia przewiozły głowę, ręce i nogi, następnego korpus. Po dwóch tygodniach córka zgłosiła zaginięcie ojca, twierdząc, że pojechał służbowo do Warszawy. Wystąpiła nawet w programie telewizyjnym, apelując o informacje na temat miejsca jego pobytu. Zabójstwo Antoniego K. kobiety określały między sobą kryptonimem puzzle, ponieważ łączyło się ono z rozkawałkowaniem ciała ofiary. Syn radcy do poszukiwań zaangażował jasnowidza. Ten powiedział mu, że widział las, a w nim zwłoki. Brat Katarzyny naciskał, by policja przeszukała zagajnik. Niczego jednak nie znaleziono. Trzy miesiące po zabój- stwie z Wisły wyłowiono korpus mężczyzny. Ale nikt nie powiązał tego ze zniknięciem radcy, ponieważ biegli stwierdzili, że chodzi o mężczyznę w wieku 40–55 lat, podczas gdy Antoni K. miał pięt- naście lat więcej. Sprawę umorzono. Cztery lata później córka – Katarzyna K. zgłosiła się do jed- nostki policji z anonimowym listem, w którym była informacja o zwłokach wrzuconych do Wisły. Do listu był dołączony portfel i zdjęcia zaginionego radcy. Odnalezienie ciała oznaczało stwier- dzenie zgonu radcy Antoniego K., realizację testamentu i przejęcie majątku. Ale policjantom z krakowskiego Archiwum X wszystko wydało się podejrzane. Szybko ustalono, na jakiej maszynie list został napisany. Odkryto także, że tę maszynę kupiły występują- ce w sprawie kobiety oraz szybko się jej pozbyły. Szczegółowe oględziny mieszkania Antoniego K. wykazały, że w jednym z po- mieszczeń zrywano parkiet. Kobiety nie potrafiły wyjaśnić powodu takiego remontu, który był jednak daremny, ponieważ w kącie pokoju ujawniono ślady krwi, które poddano badaniom DNA. W sprawie obie kobiety badano wariograficznie20. Pytanie o ukry- G . 21 [Dostęp: 12.07.2016]. Dostępny w World Wide Web: <http://bit.ly/2gICvfP>. 22 Weronika K., Zabiłam, oprac. Piotr Litka, Kraków 2015, ss. 320. Abstract K ę d z i e r s k a , O p i n i a b i e g ł e g o w s ł y n n y c h p r o c e s a c h . . . 95 cie zwłok było znaczące dla Katarzyny K.21 W kolejnym przesłu- chaniu córka i konkubina radcy przyznały się do zbrodni, wska- zując Aleksandrę C. M. jako kierującą zbrodnią. W lutym 2006 r. Katarzyna K. i Anna G. zostały skazane, każda na 12 lat więzie- nia, Aleksandra C. M., mimo że nie przyznała się do winy, ma do- żywocie. Anna G. po siedmiu latach wyszła na wolność za dobre sprawowanie. Po opuszczeniu więziennych murów, korzystając z pomocy dziennikarza TVN Piotra Litki, napisała książkę opisu- jącą przebieg zbrodni. Choć akcja książki została przeniesiona do fikcyjnej miejscowości Brudnowo22, a bohaterowie mają zmienio- ne imiona, łatwo doszukać się analogii do morderstwa radcy z Chrzanowa. 24 stycznia 2012 r. wieczorem w mediach podano wiadomość o zaginięciu półrocznej dziewczynki z Sosnowca. Według relacji matki – Katarzyny Waśniewskiej (ur. 10 sierpnia 1990 r.) – pod- czas spaceru tajemniczy mężczyzna uderzył ją w głowę i gdy była nieprzytomna, zabrał Madzię z wózka. W całej Polsce rozpoczęły się poszukiwania dziecka, które nie dały rezultatu. Matka, pła- cząc publicznie, prosiła porywacza, by oddał córeczkę. Ale 3 lute- go 2012 r. K. Waśniewska przyznała się prywatnemu detektywo- wi, że porwania nie było, lecz dziecko zmarło po tym, jak w domu wypadło jej z rąk. Policjanci znaleźli ciało niemowlęcia w zrujno- wanym budynku w Sosnowcu. Powołano biegłych, którzy mieli odpowiedzieć na pytania: kiedy i w jaki sposób straciła życie Ma- dzia Waśniewska? W celu rozstrzygnięcia tej wątpliwości przeprowadzono bada- nie toksykologiczne i histopatologiczne wycinków narządów we- wnętrznych i tkanek miękkich zmarłego dziecka. Takie badanie pozwala znaleźć zmiany na poziomie tkankowym i określić, czy śmierć osoby była przypadkowa, czy też nastąpiła w wyniku ce- lowego działania. Analiza toksykologiczna z kolei jest skierowana na poszukiwanie toksycznych związków chemicznych. Z eksper- tyz jasno wynikało, że przyczyną śmierci dziewczynki było udu- szenie. Krwiak w krtani i krew w płucach mogły powstać, jak 1 ( 1 ) 2 0 1 6 S t u d i a i m a t e r i a ł y 96 twierdzą biegli, tylko w wyniku uduszenia. 23 [Dostęp: 30.07.2016]. Dostępny w World Wide Web: <http://bit.ly/2fAz8pn>. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h 23 [Dostęp: 30.07.2016]. Dostępny w World Wide Web: <http://bit.ly/2fAz8pn>. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h Abstract Specjaliści nie potrafią tylko określić, czy dziecku założono worek na główkę, czy duszo- ne było poduszką, ręką lub w inny sposób. Ciało leżało kilka dni na ostrym mrozie – po rozmrożeniu tkanki uległy uszkodzeniu. Uraz głowy, traktowany na początku jako możliwa przyczyna śmierci, powstał już po zgonie, od uderzenia główką o podłogę, ścianę, ziemię. Pobierano też próbki zabrudzeń z ciała i ubranka dziecka oraz różnych miejsc, w których zwłoki Madzi mogły być ukrywane zanim umieszczono je w zrujnowanym budynku23. W wyniku badania zawartości komputera Katarzyny Waśniew- skiej ustalono, że kilka dni przed rzekomym porwaniem na stro- nach internetowych szukano informacji o organizacji pogrzebu dziecka, cenników maleńkich trumien oraz wysokości zasiłków wypłacanych rodzicom po śmierci dziecka. Sprawdzano także, ja- kie są objawy zatrucia tlenkiem węgla. We krwi Madzi biegli od- kryli hemoglobinę tlenkową, która występuje podczas zatrucia czadem. W mieszkaniu przeprowadzono oględziny pieców i prze- wodów kominowych, a opinia kominiarska miała odpowiedzieć na pytanie: czy były zatykane? Badania podejmowano z powodu po- dejrzenia, że Katarzyna Waśniewska próbowała zabić córkę co najmniej od 19 stycznia 2012 r., usiłując zatruć ją czadem, ude- rzając dzieckiem o podłogę, a nawet podduszając niemowlę przez kilka minut. W tej sytuacji 31 grudnia 2012 r. Prokuratura Okrę- gowa w Katowicach skierowała do sądu akt oskarżenia przeciwko K. Waśniewskiej z zarzutem zabójstwa Madzi. 3 września 2013 r. Sąd Okręgowy w Katowicach wymierzył Katarzynie Waśniewskiej (Rycina 8) karę 25 lat więzienia za zabicie córki oraz za zawiado- mienie o niepopełnionym przestępstwie, odrzucając wersję obro- ny, że Magda zmarła na skutek wypadku. Obrońca złożył apelację, żądając wykonania uzupełniających badań, m.in. przez biegłego laryngologa dziecięcego. Według obrony śmierć Madzi była spowodowana laryngospazmem, czyli nagłym skurczem krtani. Biegli analizowali dokumentację z sekcji zwłok dziecka i badań lekarskich przeprowadzonych za jego życia. Zgodnie stwierdzono, że co prawda historia medycyny zna przy- G . K ę d z i e r s k a , O p i n i a b i e g ł e g o w s ł y n n y c h p r o c e s a c h . . . 97 padki, w których laryngospazm był śmiertelny, jednak są one niezmiernie rzadkie i dotyczą powikłań po znieczuleniu, które w tym przypadku nie wystąpiło. Opinia biegłych była jednoznacz- na: półroczna Magda zmarła wskutek gwałtownego uduszenia. Przyczyną śmierci nie była choroba ani uraz. 24 Dowodów przeciwko Marii Capelle Lafarge dostarczyły badania pioniera toksy- kologii Mateusza Bonawentury Josepha Orfili. B. Innes, Niezbity dowód. Metody wykrywania zbrodni, Warszawa 2001, s. 48. 25 F. McLynn, Słynne procesy. Sprawy, które tworzyły historię, Warszawa 1996, s. 122 i n. 26 J Wid ki it 154 1 ( 1 ) 2 0 1 6 24 Dowodów przeciwko Marii Capelle Lafarge dostarczyły badania pioniera toksy- kologii Mateusza Bonawentury Josepha Orfili. B. Innes, Niezbity dowód. Metody wykrywania zbrodni, Warszawa 2001, s. 48. 25 F. McLynn, Słynne procesy. Sprawy, które tworzyły historię, Warszawa 1996, s. 122 i n. 26 J. Widacki, op. cit., s. 154. 27 [Dostęp: 12.07.2016]. Dostępny w World Wide Web: <http://bit.ly/2gIDFYq>. y y , , 25 F. McLynn, Słynne procesy. Sprawy, które tworzyły historię, Warszawa 1996, s. 122 i n. 27 [Dostęp: 12.07.2016]. Dostępny w World Wide Web: <http://bit.ly/2gIDFYq>. 26 J. Widacki, op. cit., s. 154. 24 Dowodów przeciwko Marii Capelle Lafarge dostarczyły badania pioniera toksy- kologii Mateusza Bonawentury Josepha Orfili. B. Innes, Niezbity dowód. Metody wykrywania zbrodni, Warszawa 2001, s. 48. Abstract Kobieta została ska- zana prawomocnym wyrokiem na 25 lat pozbawienia wolności za zabicie swojej sześciomiesięcznej córki Magdy. Wiele kobiet, nie tylko te w opisanych wyżej przypadkach są- dzonych za zabójstwo, zapisało swoją historię na kartach krymi- nalistyki. Opinie biegłego miały w ich procesach różne znaczenie. W przypadku procesu oskarżonej o zabójstwo męża Marii Capelle Lafarge w roku 1840, po raz pierwszy w naukowy sposób dowie- dziono użycia arszeniku do pozbawienia życia24, co skutkowało skazaniem Marii. Z kolei sądzona w 1856 r. Madeleine Smith25 najprawdopodobniej zabiła kochanka, który jej przeszkadzał, ale została uniewinniona, bo nie umiano usunąć wątpliwości doty- czących sposobu i czasu podania trucizny. Janinę Borowską w 1910 r. uwolniono od zarzutu zabójstwa adwokata Włodzimie- rza Lewickiego, chociaż do ustalenia przebiegu zdarzenia zasto- sowano niezwyczajny ówcześnie eksperyment. Powołano w tym celu dwóch znawców broni – oficera armii i rusznikarza, którzy strzelali do manekina ustawianego w różnych pozycjach i różnych miejscach pokoju. Ostatecznie jednak wątpliwości pozostały i bie- gli uznali, że mogło to być bardzo typowe samobójstwo, lub bar- dzo nietypowe morderstwo26. Eva Rablen27, skazana w 1929 r. za zabójstwo męża, nie przewidziała, jakie konsekwencje może przynieść kawa rozlana przypadkiem na sukienkę koleżanki. Przenikliwy śledczy zlecił ekspertyzę plamy i wtedy wykryto, że płyn zawierał także strych- ninę. Z kolei Lindy Chamberlain, oskarżona o to, że w 1980 r. za- biła córeczkę, musiała latami dowodzić swojej niewinności, po- 1 ( 1 ) 2 0 1 6 98 S t u d i a i m a t e r i a ł y nieważ australijski wymiar sprawiedliwości, mimo licznych opinii biegłych nie mógł uwierzyć, że dziecko mógł porwać i zabić dziki pies dingo. Opinia biegłego ma istotne znaczenie w ustalaniu okoliczno- ści zdarzenia. Pozwala wskazać rodzaj zdarzenia (przestępstwo, samobójstwo, nieszczęśliwy wypadek), biegły może też określić miejsce zdarzenia oraz jego czas. Badania dają możliwość pozna- nia sposobu działania sprawcy oraz rodzaju narzędzia, które za- stosowano do realizacji przestępczego działania. Zaznaczyć jed- nak należy, że opinia biegłego zawsze miała znaczenie adekwatne do stanu nauki w danym okresie historycznym. Wraz z rozwojem nauki rosły możliwości badawcze biegłego, ustalanie okoliczności zdarzenia pozwalało rozstrzygać więcej wątpliwości. Abstract W słynnych procesach przeciwko kobietom opinie biegłych: − czasem, nie potrafiły rozstrzygnąć wątpliwości i dawały uniewinnienie oskarżonym, jak w postępowaniach prze- ciwko Janinie Borowskiej i Marii Besnard, − czasem były mylne i dopiero zbieg okoliczności decydował o ustaleniu sprawcy, jak wtedy, gdy córka z koleżankami zabiła ojca, − czasem prowadziły do obarczenia winą jednej osoby i unie- winnienia drugiej, jak w przypadku Lidii de Bocarmé, − czasem nie były brane pod uwagę, co skutkowało surowym wyrokiem, jak w sprawie Rity Gorgonowej, − najczęściej jednak dawały możliwość wymierzenia kary, co przedstawiają procesy Gabrieli Bombard, Christy Lehmann i Katarzyny Waśniewskiej. Bibliografia Autobiografia Weronika K., Zabiłam, oprac. Piotr Litka, Kraków: Wydawnictwo WAM, 2015, ISBN 978-83-277-1061-1. Weronika K., Zabiłam, oprac. Piotr Litka, Kraków: Wydawnictwo WAM, 2015, ISBN 978-83-277-1061-1. 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Murzynowski A., Istota i zasady procesu karnego, Wyd. 3, Warszawa: Wydawnictwo Naukowe PWN, 1994, ISBN 83-01-11585-8. Szenic S., Pitaval wielkopolski, Wyd. 2, Poznań: Wydaw. Poznańskie, 1977. Thorwald J., Stulecie detektywów: drogi i przygody kryminalistyki, przeł. Wanda Kragen i Karol Bunsch, Kraków: Wydawnictwo Literackie, 1992, ISBN 83-08-02175-1. Thorwald J., Stulecie detektywów: drogi i przygody kryminalistyki, przeł. Wanda Kragen i Karol Bunsch, Kraków: Wydawnictwo Znak, 2009, ISBN 978-83-240-1118-6. Widacki J., Stulecie polskich detektywów, Kraków: Wydawnictwo Wa- welskie, 1992, ISBN 83-85347-04-6. Wrzesiński S., Potępieńcy średniowiecznej Europy, Kraków: Wydawnic- two EGIS, 2007, ISBN 978-83-7396-627-7. C z a s o p i s m o N a u k o w e I n s t y t u t u S t u d i ó w K o b i e c y c h
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Lack of IL-17 Receptor A signaling aggravates lymphoproliferation in C57BL/6 lpr mice
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Lack of IL-17 Receptor A signaling aggravates lymphoproliferation in C57BL/6 lpr mice Received: 12 April 2016 Accepted: 15 November 2018 Published: xx xx xxxx Received: 12 April 2016 Accepted: 15 November 2018 Published: xx xx xxxx Odilia B. J. Corneth1,2, Fleur Schaper3, Franka Luk1,5, Patrick S. Asmawidjaja1, Adriana M. C. Mus1, Gerda Horst3, Peter Heeringa   4, Rudi W. Hendriks2, Johanna Westra3 & Erik Lubberts1 Defects in Fas function correlate with susceptibility to systemic autoimmune diseases like autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE). C57BL/6 lpr (B6/ lpr) mice are used as an animal model of ALPS and develop a mild SLE phenotype. Involvement of interleukin-17A (IL-17A) has been suggested in both phenotypes. Since IL-17 receptor A is part of the signaling pathway of many IL-17 family members we investigated the role of IL-17 receptor signaling in disease development in mice with a B6/lpr background. B6/lpr mice were crossed with IL-17 receptor A deficient (IL-17RA KO) mice and followed over time for disease development. IL- 17RA KO/lpr mice presented with significantly enhanced lymphoproliferation compared with B6/ lpr mice, which was characterized by dramatic lymphadenomegaly/splenomegaly and increased lymphocyte numbers, expansion of double-negative (DN) T-cells and enhanced plasma cell formation. However, the SLE phenotype was not enhanced, as anti-nuclear antibody (ANA) titers and induction of glomerulonephritis were not different. In contrast, levels of High Mobility Group Box 1 (HMGB1) and anti-HMGB1 autoantibodies were significantly increased in IL-17RA KO/lpr mice compared to B6/lpr mice. These data show that lack of IL-17RA signaling aggravates the lymphoproliferative phenotype in B6/lpr mice but does not affect the SLE phenotype. Defects in Fas function correlate with susceptibility to several systemic autoimmune diseases such as autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE)1. Defects in Fas function correlate with susceptibility to several systemic autoimmune diseases such as autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE)1. ALPS is characterized by immune dysregulation due to an inability to regulate lymphocyte homeostasis through abnormalities in lymphocyte apoptosis or programmed cell death. This defect leads to lymphoprolifera- tive disease with clinical manifestations that can include lymphadenopathy, splenomegaly, increased risk of lym- phoma and autoimmune disease. Another hallmark for ALPS is expansion of T cells that express the alpha/beta T cell receptor but lack CD4 and CD8 (double negative (DN) T cells) in peripheral blood and tissue2. Moreover, lupus prone mice, often present with an ALPS like phenotype. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 12 April 2016 Accepted: 15 November 2018 Published: xx xx xxxx Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w Results I Increased cell numbers in lymphoid organs of IL-17RA KO/lpr mice compared to B6/lpr mice. B6/lpr and IL-17RA KO/lpr mice were followed over time and sacrificed at 10 weeks (no disease development in B6/lpr), 20 weeks (sub-clinical development of disease in B6/lpr mice) and 30 weeks of age (established disease in B6/lpr mice). At 30 weeks of age, the spleens and lymph nodes from IL-17RA KO/lpr mice were greatly enlarged compared to spleens and lymph nodes of B6/lpr mice (Fig. 1A). The total number of splenocytes was comparable between C57BL/6 (B6) wild-type and IL-17RA KO mice without the lpr mutation at 10, 20 and 30 weeks of age, and increased mildly over time in B6/lpr mice. However, the number of splenocytes in IL-17RA KO/lpr mice increased significantly over time to about twofold compared with B6/lpr mice at 30 weeks of age (Fig. 1B). Increased numbers of DN T and CD4+ T cells in IL-17RA KO/lpr mice. Both DN T and CD4+ T cells have been shown to play a role in the pathogenesis of SLE in patients31. To determine whether these subpopula- tions were increased in IL-17RA KO/lpr mice, we analyzed the splenic T cell compartment of these mice by flow cytometry. At weeks 20 and 30, an increase in the number of T cells was found in IL-17RA KO/lpr mice compared to B6/lpr mice (Fig. 2A). This was due to an increase in the total numbers of DN T cells, and to a lesser extent of CD4+ T cells (Fig. 2B,C). To further characterize the DN T cells, DN TCRβ+ analysis was performed. At week 20, higher numbers of DN TCRβ+ cells in B6.lpr were noted compared to C57BL/6 and IL-17RA KO mice which decline at week 30. In addition, the numbers of DN TCRβ+ are higher in the IL-17RA KO/lpr compared to B6.lpr mice at week 20 but not at week 30 (Fig. 2D). There was also an increase in numbers of IL-17A-producing DN T cells and CD4+ T cells in IL-17RA KO/lpr mice (Fig. 2B,C). At week 20, IL-17A+ DN TCRβ+ cells were elevated in b6.lpr and IL-17RA KO/lpr mice that decline to background levels at week 30 (Fig. 2D). These data indicate that lack of IL-17RA influenced the proliferation of CD4+ and especially DN T cells. www.nature.com/scientificreports/ www.nature.com/scientificreports/ and tissue damage8. Tissue damage, or disturbed clearance of apoptotic cells, can lead to the release of damage associated molecular pattern (DAMP) molecules, such as High Mobility Group Box-1 (HMGB1). HMGB1 can be released from activated, apoptotic and necrotic cells. Recently it was demonstrated that HMGB1 levels are increased in serum and urine of SLE patients and are related to disease activity9,10. p y Both ALPS and SLE phenotypes can spontaneously develop in lpr mouse models, carrying mutations in the Tnfrsf6 gene, encoding the Tumor Necrosis factor (TNF)-family receptor protein FAS or CD95 that has the capacity to induce apoptosis. These lpr mice are often referred to as lupus mice and display lymphoproliferation, expansion of DN T cells and autoimmune nephritis, including anti-dsDNA autoantibodies11. It is known that MRL/lpr mice are severely affected and develop autoimmune manifestations that both serologically and patho- logically show similarities to human SLE but also demonstrate the lymphoproliferative phenotype that charac- terizes ALPS12. C57BL/6-lpr (B6/lpr) mice are also used as an animal model of ALPS and present a milder SLE phenotype13. p yp Interleukin 17A (IL-17A) is a pro-inflammatory cytokine implicated in different autoimmune disorders14–16 and can be produced by several immune cells, including several T cell subsets such as T helper 17 (Th17) cells, CD8+ T cells, natural killer (NK) cells, and DN T cells17,18. IL-17A is a member of the IL-17 cytokine family and signals through a heterodimeric receptor complex composed of the IL-17 receptor A (IL-17RA) and IL-17RC subunits19. The IL-17RA subunit appears to be the common receptor subunit for most if not all IL-17 cytokine family members although the signalling pathways for IL17B and IL-17D are not fully elucidated19. Maintenance of Th17 cells, the predominant producers of IL-17 in autoimmunity, depends on IL-23 signalling20. h p p y p g g Several lines of evidence indicate that IL-17A is involved in ALPS and SLE pathology. In addition, IL-17F and IL-17C promoted Th17 cell-driven glomerular inflammation and tissue injury21,22. IL-17A inhibits Fas-induced cell death and its neutralization enhances lymphocyte apoptosis in patients with ALPS. In MRL/lpr mice anti-IL- 17A antibody treatment ameliorates the autoimmune manifestations and to a lesser extent the lymphoprolifer- ative phenotype, prolonging survival of MRL/lpr mice12. In SLE patients, IL-17A plasma levels and numbers of IL-17A producing peripheral blood mononuclear cells (PBMCs) are reported to be increased and correlate with disease severity23–25. www.nature.com/scientificreports/ Furthermore, IL-17A producing DN T cells have been found in the kidneys of lupus nephri- tis patients17. However, there are also studies in SLE patients that do not observe a relation between IL17A and disease activity26,27. y In murine models of SLE increased levels of IL-17A, and IL-23 receptor-positive cells have been observed28,29. It was demonstrated that B6/lpr mice lacking IL-23 receptor (IL-23R) signalling were completely protected against SLE development30. These mice had decreased numbers of IL-17A producing cells in the lymph nodes and decreased anti-DNA antibody production, suggesting a crucial role for the IL-23/IL-17A axis in SLE patho- genesis in this model30. g Since the IL-17RA subunit is a common receptor unit for most if not all IL-17 family members19, we inves- tigated the phenotype in aging B6/lpr mice lacking IL-17RA. Our data show that B6/lpr mice lacking IL-17RA develop a severely lymphoproliferative phenotype without effecting ANA levels. Furthermore, an increase in serum HMGB1 was noted, indicating that activated immune cells might secrete HMGB1. Lack of IL-17 Receptor A signaling aggravates lymphoproliferation in C57BL/6 lpr mice t SLE is a systemic autoimmune disease that is characterized by accumulation of large numbers of DN T cells and production of multiple autoantibodies, mostly directed against nuclear antigens. Lupus nephritis is one of the most serious manifestations3. The cause of the disease is unknown, but both genetic and environmental fac- tors play a role. Anti-nuclear antibodies (ANAs) and immune complexes can promote T cell activation and the production of interferon (IFN) type I by plasmacytoid dendritic cells (pDCs)4,5. IFN type I expression (signature) is increased in approximately half of SLE patients6. Monocyte derived DCs (mDCs) are activated by IFN type I and can induce T helper cell differentiation, enhanced B cell activation and survival through B cell activat- ing factor (BAFF) production7. This leads to the production of auto-reactive antibodies, mostly ANAs, which form immune complexes and deposits in small capillaries in for example the kidneys, inducing inflammation 1Department of Rheumatology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. 2Department of Pulmonary Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. 3Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 4Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 5Present address: Department of Internal Medicine, division of Nephrology and Transplantation, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. Odilia B. J. Corneth and Fleur Schaper contributed equally. Correspondence and requests for materials should be addressed to E.L. (email: E.Lubberts@erasmusmc.nl) Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 1 Results I mice at week 30, despite the difference in disease phenotype at that age. No difference was observed for IL-4 (data not shown), and IL-10 (Fig. 3). Plasma cell formation is enhanced in IL-17RA KO/lpr mice. As we found increased IL-6 levels in serum of IL-17RA KO/lpr mice at early disease onset and increased B cell numbers in these mice (Fig. 4A), we further analyzed activation of the splenic B cell compartment. Analysis of CD21 and CD23 expression by flow cytometry32 showed that the increase in number of B cells could be attributed primarily to an increase in the number of follicular B cells (CD19+ B220+ CD21− CD23+) and marginal zone B cells (CD19+ B220+ CD21+ CD23−) (Fig. 4B), although at week 30 also the size of the transitional B cell fraction (CD19+ B220+ CD21− CD23−) was increased in IL-17RA KO/lpr mice (Fig. 4B). Analysis of germinal centre formation showed that after an initial rise at 20 weeks of age the numbers of germinal centre B cells (PNA+ CD95+ CD19+) were decreased in B6/lpr mice at 30 weeks of age, but not significantly lower than in IL-17RA KO/lpr mice (Fig. 4B). However, flow cytometry analysis for Ig subclasses showed that the numbers of IgM, IgG1 and IgG2 plasma cells present in the spleen at 30 weeks of age was significantly higher in IL-17RA KO/lpr mice compared with B6/lpr mice (Fig. 4C). Together, these data show increased B cell activation in the absence of IL-17RA signalling in B6/ lpr mice. Autoimmune pathology is not enhanced in B6/lpr mice in absence of IL-17RA signalling. To investigate whether the increase in cytokine producing T cells and plasma cells in the spleen was associated with enhanced kidney pathology in IL-17RA KO/lpr mice, kidney slides were specifically stained and evaluated. By light microscopic analysis no thickening of the glomerular basal membrane in IL-17RA KO/lpr mice could be observed, indicating absence of kidney damage (Fig. 5A). Moreover, no increased influx of CD3-positive cells was seen in kidneys from IL17RA KO/lpr mice compared to B6/lpr mice (Fig. 5A). In addition, we found no enhanced C3 complement deposition and no enhanced IgG antibody deposition in kidneys sections from 26-week-old IL-17RA KO/lpr mice compared to B6/lpr (Fig. 5B,C). To further investigate renal inflammation, mRNA levels of the pro-inflammatory cytokine MCP-1 and the renal injury-related biomarkers NGAL and KIM-1 were assessed in kidneys. Results I The numbers of IFN-γ and IL-4 producing DN T cells and CD4+ T cells were similar in IL-17RA KO/lpr and B6/lpr mice (data not shown). Serum cytokine levels are increased in IL-17RA KO/lpr mice. To further asses the cytokine profile in B6.lpr and IL-17RA KO/lpr mice, serum cytokine levels were measured by multiplex assay. As expected, levels of IL-17A were significantly increased in IL-17RA KO/lpr mice at 10 weeks of age (Fig. 3). In addition, in young IL-17RA KO/lpr mice, IFN-γ, TNF-α and IL-22 were increased (Fig. 3). IL-6 was significantly increased at week 20, but surprisingly none of the other cytokines analysed were significantly different between the two groups of Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 2 www.nature.com/scientificreports/ Figure 1. Increased spleen and lymph node size in IL-17RA KO/lpr mice. (A) Spleens and cervical lymph nodes from 30 week old B6/lpr (left and top) and IL-17RA KO/lpr (right and bottom). (B) Total number of splenocytes (all live cells in spleen) at different ages. Bars depict wild type B6 (grey stripe), wild type IL-17RA KO (open stripe), B6/lpr (black), and IL-17RA.KO/lpr (open) mice at 10, 20 and 30 weeks of age. Mean and SEM are shown for n = 3–21 mice per group; *p < 0.05; **p < 0.01. Figure 1. Increased spleen and lymph node size in IL-17RA KO/lpr mice. (A) Spleens and cervical lymph nodes from 30 week old B6/lpr (left and top) and IL-17RA KO/lpr (right and bottom). (B) Total number of splenocytes (all live cells in spleen) at different ages. Bars depict wild type B6 (grey stripe), wild type IL-17RA KO (open stripe), B6/lpr (black), and IL-17RA.KO/lpr (open) mice at 10, 20 and 30 weeks of age. Mean and SEM are shown for n = 3–21 mice per group; *p < 0.05; **p < 0.01. Figure 1. Increased spleen and lymph node size in IL-17RA KO/lpr mice. (A) Spleens and cervical lymph nodes from 30 week old B6/lpr (left and top) and IL-17RA KO/lpr (right and bottom). (B) Total number of splenocytes (all live cells in spleen) at different ages. Bars depict wild type B6 (grey stripe), wild type IL-17RA KO (open stripe), B6/lpr (black), and IL-17RA.KO/lpr (open) mice at 10, 20 and 30 weeks of age. Mean and SEM are shown for n = 3–21 mice per group; *p < 0.05; **p < 0.01. Results I Moreover, mRNA levels of CD68 were measured to determine influx of macrophages. There was no significant difference in expression of NGAL and KIM-1 in kidneys of B6/lpr compared to IL17-RA KO/lpr com- pared, indicating no overt kidney damage in both groups (Table 1), confirming renal histology. HMGB1 levels in serum are increased in B6/lpr mice lacking IL-17RA signalling. SLE is char- acterized by circulating ANA and consumption of complement C3. Complement C3 levels did not decrease over time in B6/lpr or IL-17RA KO/lpr mice (data not shown). Serum ANA titers increased with age in both Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 3 www.nature.com/scientificreports/ Figure 2. Expansion of T cell populations in IL-17RA KO/lpr mice. (A) Total numbers of splenic T cells (CD3+) in wildtype and lpr mice. Bars depict wild type B6 (grey stripe), wild type IL-17RA KO (open stripe), B6/lpr (black), and IL-17RA.KO/lpr (open) mice at 10, 20 and 30 weeks of age. (B) Total numbers of splenic CD4+ T cells (CD3+ CD4+ CD8−) (left) and IL-17A producing CD4+ cells(right) in B6/lpr (black bars) and IL-17RA KO/lpr (open bars). (C) Double negative (DN) splenic T cells (CD3+ CD4− CD8−) (left), and IL-17A producing DN T cells (right) in B6/lpr (black bars) and IL-17RA KO/lpr (open bars) mice at different ages. (D) Double negative (DN) splenic TCRβ+ (DN TCRb+) T cells (CD3+ CD4− CD8− TCRβ+) (left), and IL-17A producing DN TCRβ+ (IL-17A in DN TCRb+) T cells (right) for wild type and lpr mice at week 20 and 30. Mean is shown for n = 9–21 animals per group; *p < 0.05; **p < 0.01. Figure 2. Expansion of T cell populations in IL-17RA KO/lpr mice. (A) Total numbers of splenic T cells (CD3+) in wildtype and lpr mice. Bars depict wild type B6 (grey stripe), wild type IL-17RA KO (open stripe), B6/lpr (black), and IL-17RA.KO/lpr (open) mice at 10, 20 and 30 weeks of age. (B) Total numbers of splenic CD4+ T cells (CD3+ CD4+ CD8−) (left) and IL-17A producing CD4+ cells(right) in B6/lpr (black bars) and IL-17RA KO/lpr (open bars). (C) Double negative (DN) splenic T cells (CD3+ CD4− CD8−) (left), and IL-17A producing DN T cells (right) in B6/lpr (black bars) and IL-17RA KO/lpr (open bars) mice at different ages. Results I (D) Double negative (DN) splenic TCRβ+ (DN TCRb+) T cells (CD3+ CD4− CD8− TCRβ+) (left), and IL-17A producing DN TCRβ+ (IL-17A in DN TCRb+) T cells (right) for wild type and lpr mice at week 20 and 30. Mean is shown for n = 9–21 animals per group; *p < 0.05; **p < 0.01. groups (Fig. 6A), but ANA titers were not significantly higher in IL-17RA KO/lpr mice than in B6/lpr mice at any age. HMGB1 levels were determined in serum by Western blot (see Fig. 6C for a representable example of the Western blot). HMGB1 was low in control C57BL/6 wild type or IL-17RA KO mice (Fig. 6B). Remarkably, at 10 weeks of age HMGB1 levels were already increased in IL-17RA KO/lpr mice compared to B6/lpr mice (Fig. 6B). Furthermore, anti-HMGB1 antibody levels, which were low in C57BL6 and IL-17RA KO mice (Fig. 6B) increased in IL-17RA KO/lpr mice with age and reached a significantly higher level than in B6/lpr mice (Fig. 6B). To inves- tigate the source of HMGB1, both kidney and spleen sections of (the same) mice were stained for HMGB1 (Fig. 6D). Similar nuclear HMGB1 expression, no cytoplasmic HMGB1 and no apparent release of extracellular HMGB1 due to activation were observed in kidneys of both IL-17RA KO/lpr and B6/lpr mice (Fig. 6D). In spleen sections however, we did observe extracellular HMGB1. Moreover, HMGB1 negative nuclei (blue staining) were present, which suggests active release of HMGB1 from these cells, but there was no difference in HMGB1 expres- sion between IL-17RA KO/lpr and B6/lpr mice (Fig. 6D). Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w Discussionh This study demonstrates that deletion of IL-17RA signaling markedly aggravates the lymphoproliferation in B6/ lpr mice, but does not affect SLE pathology. The greatly increased spleen and lymph node size in IL-17RA KO/lpr mice, characterized by expansion of CD4+ T cells, CD8+ T cells, DN T cells and B cells indicates an important role for IL-17RA signaling in limiting the lymphoproliferative phenotype in B6/lpr mice. Remarkably however, neither serum ANA titers, antibody deposition in the kidney nor kidney damage were different between IL-17RA KO/lpr and B6/lpr mice, showing that these hallmarks of SLE are independent of IL-17RA signaling in this model. Furthermore, increased serum HMGB1 levels followed by elevated anti-HMGB1 antibody levels were seen in IL-17RA KO/lpr mice compared to B6/lpr mice. A strong lymphoproliferative phenotype was found in IL-17RA KO/lpr mice leading to visible lymphadeno- megaly. The phenotype was accompanied by increased lymphocyte numbers of which enhanced DN T cell pro- liferation was most prominent. Total numbers of IL-17A-cytokine producing DN T cells and CD4+ T cells were increased at 20 and 30 weeks of age in IL-17RA KO/lpr mice compared with B6/lpr mice. This increase is most likely a consequence of loss of a negative feedback loop limiting IL-17A production through IL-17RA signalling. Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 4 www.nature.com/scientificreports/ Figure 3. IL-17A, IL-6, IL-10, TNF-α, IFN-γ, and IL-22 levels in B6/lpr and IL-17RA KO/lpr mice. Serum cytokine levels as measured by multiplex at 10, 20 and 30 weeks of age in B6.lpr (black circles) and IL17RA KO/ lpr (open circles); *p < 0.05; **p < 0.01. Figure 3. IL-17A, IL-6, IL-10, TNF-α, IFN-γ, and IL-22 levels in B6/lpr and IL-17RA KO/lpr mice. Serum cytokine levels as measured by multiplex at 10, 20 and 30 weeks of age in B6.lpr (black circles) and IL17RA KO/ lpr (open circles); *p < 0.05; **p < 0.01. Figure 4. Expansion of B cell populations in IL-17RA KO/lpr mice. (A) Total numbers of splenic B cells (CB19+) in wildtype and lpr mice. Bars depict wild type B6 (grey stripe), IL-17RA KO (open stripe), B6/lpr (black), and IL-17RA.KO/lpr (open) mice at 10, 20, and 30 weeks of age. Discussionh (C) Quantitative analysis of C3 and IgG deposition in kidney sections of 30 week old B6.lpr and IL-17R KO/lpr. Box and Whiskers plot, median and interquartile range are shown for n = 5–9 mice per group. Figure 5. No induction of kidney damage in both IL 17RA KO/lpr and B6/lpr mice. (A) Representative PAS staining, HE staining, CD3 staining, in kidneys of 30 week old B6.lpr and IL-17R KO/lpr (10×). Inserts show glomerulus in detail (40×). Representative pictures are shown for n = 6 animals per group. (B) Representativ staining of C3 and IgG deposition in kidneys of 30 week old B6/lpr and IL-17R KO/lpr (10×). (C) Quantitat analysis of C3 and IgG deposition in kidney sections of 30 week old B6.lpr and IL-17R KO/lpr. Box and Whiskers plot, median and interquartile range are shown for n = 5–9 mice per group. B6/lpr IL-17RA KO/lpr CD68 0.0009 (0.0003–0.027) 0.007 (0–0.024) MCP-1 0.0002 (0–0.003) 0.0005 (0–0.009) NGAL 0.0001 (0–0.018) 0.001 (0–0.018) KIM-1 0.0001 (0–0.0221) 0 (0–0.0221) Table 1. IL-17RA KO does not affect renal mRNA levels of CD68, MCP-1, NGAL or KIM-1. mRNA analysis in kidney tissue of b6/lpr and IL-17RA ko/lpr for CD68, MCP-1 (monocyte chemotactic protein-1), KIM-1 (kidney injury molecule-1), NGAL (neutrophil gelatinase-associated lipocalin). Data are presented as median (range) of relative expression compared to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). B6/lpr IL-17RA KO/lpr CD68 0.0009 (0.0003–0.027) 0.007 (0–0.024) MCP-1 0.0002 (0–0.003) 0.0005 (0–0.009) NGAL 0.0001 (0–0.018) 0.001 (0–0.018) KIM-1 0.0001 (0–0.0221) 0 (0–0.0221) Table 1. IL-17RA KO does not affect renal mRNA levels of CD68, MCP-1, NGAL or KIM-1. mRNA analysis in kidney tissue of b6/lpr and IL-17RA ko/lpr for CD68, MCP-1 (monocyte chemotactic protein-1), KIM-1 (kidney injury molecule-1), NGAL (neutrophil gelatinase-associated lipocalin). Data are presented as median (range) of relative expression compared to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This indicates that IL-17RA signaling plays a more significant role in generating negative feedback to regulate the IL-17 inflammatory response rather than functioning in effector signaling to aggravate or initiate disease in these mice. It might be that IL-17RA signaling can limit cell proliferation or survival of CD4+ and DN T cells in particular. In normal transgenic IL-17RA KO mice, loss of this regulatory function can apparently be rescued by other factors, however, additional loss of FAS-FASL signaling induces expansion of immune cells. A role for IL-17RA signaling has been shown in disease progression in lupus prone BXD2 mice33,34. Discussionh [B] Total numbers of splenic follicular B cells (CD19+ B220+ CD21− CD23+),marginal zone B cells (CD19+ B220+ CD21+ CD23−), transitional B cells (CD19+ B220+ CD21− CD23−), and germinal centre B cells (CD19+ B220+ PNA+ CD95+) in B6/ lpr and IL-17RA KO/lpr mice at different ages. (C) Total numbers of IgM, IgG1 and IgG2 producing CD138+ plasma cells at different ages. Mean and SEM are shown for n = 9–21 animals per group; *p < 0.05; **p < 0.01; ***p < 0.001. Figure 4. Expansion of B cell populations in IL-17RA KO/lpr mice. (A) Total numbers of splenic B cells (CB19+) in wildtype and lpr mice. Bars depict wild type B6 (grey stripe), IL-17RA KO (open stripe), B6/lpr (black), and IL-17RA.KO/lpr (open) mice at 10, 20, and 30 weeks of age. [B] Total numbers of splenic follicular B cells (CD19+ B220+ CD21− CD23+),marginal zone B cells (CD19+ B220+ CD21+ CD23−), transitional B cells (CD19+ B220+ CD21− CD23−), and germinal centre B cells (CD19+ B220+ PNA+ CD95+) in B6/ lpr and IL-17RA KO/lpr mice at different ages. (C) Total numbers of IgM, IgG1 and IgG2 producing CD138+ plasma cells at different ages. Mean and SEM are shown for n = 9–21 animals per group; *p < 0.05; **p < 0.01; ***p < 0.001. Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 5 www.nature.com/scientificreports/ Figure 5. No induction of kidney damage in both IL-17RA KO/lpr and B6/lpr mice. (A) Representative PAS staining, HE staining, CD3 staining, in kidneys of 30 week old B6.lpr and IL-17R KO/lpr (10×). Inserts show a glomerulus in detail (40×). Representative pictures are shown for n = 6 animals per group. (B) Representative staining of C3 and IgG deposition in kidneys of 30 week old B6/lpr and IL-17R KO/lpr (10×). (C) Quantitative analysis of C3 and IgG deposition in kidney sections of 30 week old B6.lpr and IL-17R KO/lpr. Box and Whiskers plot, median and interquartile range are shown for n = 5–9 mice per group. Figure 5. No induction of kidney damage in both IL-17RA KO/lpr and B6/lpr mice. (A) Representative PAS staining, HE staining, CD3 staining, in kidneys of 30 week old B6.lpr and IL-17R KO/lpr (10×). Inserts show a glomerulus in detail (40×). Representative pictures are shown for n = 6 animals per group. (B) Representative staining of C3 and IgG deposition in kidneys of 30 week old B6/lpr and IL-17R KO/lpr (10×). Discussionh As BXD2 mice have a mixed C57BL/6 and DBA genetic background, it is possible that strain specific genes can explain these discrepancies, as normal germinal center formation in IL-17RA KO mice on a C57BL/6 back- ground in collagen induced arthritis and after influenza infection were found35. tl A recent study demonstrated that IL-17 signaling drives type I interferon induced proliferative crescentic glomerulonephritis (cGN) in lupus prone mice36. Impaired infiltration of alternatively activated macrophages into the kidney was observed. However, no signs of enhanced lymphoproliferative phenotype was reported in the IL-17RA deficient B6.lpr mice which may be due to the poly I:C induced TLR3 stimulation with enhanced type I interferon induction in this cGN lupus model before significant lymphoproliferation starts to develop spontane- ously as we have shown in this study.if y y Schmidt et al. reported that IL-17A deficiency did not affect the morphologic or functional parameters in MRL/lpr mice with lupus nephritis, nor did IL-17A neutralization affect the clinical course of nephritis in NZB/ NZW mice37, which is in line with our results. In MRL/lpr mice, HMGB1 levels correlate with disease progression38. In the present study HMGB1 and anti-HMBG1 levels were significantly increased in lpr mice lacking IL-17RA while no difference in ANA levels were observed between IL-17R KO/lpr and B6/lpr mice. A morphological hallmark of human and experimental lupus nephritis is the trafficking of inflammatory cells into the kidneys which was not observed in both B6/lpr and IL-17RA KO/lpr mice during the time span of our study. Thus, in both B6/lpr and IL-17RA KOL/lpr mice there was no induction of lupus nephritis which suggests that there is no direct involvement of HMGB1 and potential immune complex formation with anti-HMGB1 in driving nephritis in these models. HMGB1 can be released from the spleen as was observed in IL-17RA KO/lpr mice. Subsequently, HMGB1 can have an effect on apoptosis of cells or interfere in phagocytosis of apoptotic cells39–41 which may partly explain the enhanced lymphoprolifera- tive phenotype in IL-17RA KO/lpr mice. Importantly, in a sepsis induced mouse model it was shown that admin- istration of recombinant HMGB1 induced splenomegaly, lymphocytosis and splenocyte priming42. Previously, a role for IL-23 and IL-23R signaling in the SLE phenotype of B6/lpr and MRL/lpr mice was reported29,30,43. Discussionh When crossed with IL-17RA KO mice, BXD2 mice are partially protected against disease development. This can be attributed to impaired germinal center reactions in these mice. In the present study, no decrease in the number of PNA+ germinal center B cells in the spleens of IL-17RA KO/lpr mice was found at any age. Instead, the number of germinal center B cells appeared to be slightly increased, albeit not significantly. Importantly, ANA titers are Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 6 www.nature.com/scientificreports/ Figure 6. HMGB1 levels are increased in serum of IL-17RA KO/lpr mice. (A) Serum ANA titers by immunofluorescence for B6/lpr mice (black bars) and IL-17RA KO/lpr (open bars). Mean and SEM are shown for n = 5–9 animals per group. (B) Serum HMGB1 levels measured by Western Blot and serum anti-HMGB1 levels measured by ELISA in wildtype B6 mice, B6/lpr, and IL-17RA KO/lpr mice at different ages. Line indicates median. (C) Representative Western blot of HMGB1 in sera of lpr mice, 1 = positive HMGB1 control, 2 = Biorad molecular weight marker, 3–18 = mouse lpr sera. (D) Kidney and spleen HMGB1 staining 30 week old B6/lpr and IL-17R KO/lpr (10×). Representative pictures are shown for n = 6 animals per group. *p < 0.05; **p < 0.01; ***p < 0.001. Figure 6. HMGB1 levels are increased in serum of IL-17RA KO/lpr mice. (A) Serum ANA titers by immunofluorescence for B6/lpr mice (black bars) and IL-17RA KO/lpr (open bars). Mean and SEM are shown for n = 5–9 animals per group. (B) Serum HMGB1 levels measured by Western Blot and serum anti-HMGB1 levels measured by ELISA in wildtype B6 mice, B6/lpr, and IL-17RA KO/lpr mice at different ages. Line indicates median. (C) Representative Western blot of HMGB1 in sera of lpr mice, 1 = positive HMGB1 control, 2 = Biorad molecular weight marker, 3–18 = mouse lpr sera. (D) Kidney and spleen HMGB1 staining 30 week old B6/lpr and IL-17R KO/lpr (10×). Representative pictures are shown for n = 6 animals per group. *p < 0.05; **p < 0.01; ***p < 0.001. not decreased in IL-17RA KO/lpr mice, suggesting that in B6/lpr mice ANA development is IL-17RA independ- ent. Materials and Methods i Mice. C57BL/6-lpr/lpr (B6/lpr) mice were purchased from The Jackson Laboratory, USA, and IL-17 receptor A knock-out (IL-17RA KO) mice on a C57BL/6 background were kindly provided by dr. J. Tocker, Amgen, Seattle, USA. For genotyping of the IL-17RA construct, 5′- CTTGTGTAGCGCCAAGTG, 5′-AGCTGCTGTTAGCACTTTGC and 5′- CGTACGCACACACTCTCGA primers were used. For genotyp- ing of the lpr construct, 5′- GTAAATAATTGTGCTTCGTCAG, 5′-TAGAAAGGTGCACGGGTGTG and 5′- CAAATCTAGGCATTAACAGTG primers were used. The mouse lines were crossed to generate IL-17RA KO B6/lpr mice. Mice were housed under SPF conditions in the Erasmus Medical Center Animal Facility (EDC) and provided with food and water ad libitum. All experiments were approved by the Erasmus MC Animal Ethical Committee (DEC) and were performed according to strict governmental and international guidelines on animal experimentation. Mice were sacrificed at the age of 10, 20 or 30 weeks. Blood was drawn for collection of serum and spleens, kidneys and cervical lymph nodes were harvested. Flow cytometry for B- and T cells. Spleens were harvested and single cell suspensions prepared using 100 µm filters. Flow cytometry for B and T cells was performed as previously described35. Anti-CD19, anti-B220, anti-CD21, anti-CD23, anti-IgM, anti-CD3, anti-CD4, anti-CD8, anti-IL-17A, anti-IL-4 and anti-IFN-γ anti- bodies were obtained from eBioscience (San Diego, CA, USA), anti-IgD, anti- CD95, anti-CD138, anti-IgG1 and anti-IgG2ab antibodies from BD BioSciences, anti-IL-10 antibody was purchased from Biolegend (San Diego, CA, USA) and biotinylated peanut agglutinin (PNA) from Sigma-Aldrich (St Louis, USA).l ) y p gg ( ) g ( ) Samples were measured on a FACS Canto II HTS or a LSR II flow cytometer (BD BioSciences) and analysis was performed using FlowJo v7.6 research software (Tree Star Inc. Ashland, OR). Histology. Kidney tissue samples from 30 week-old female IL-17RA KO lpr and B6/lpr mice were fro- zen in Tissue-Tec O.C.T. Compound (Sakura Finetek Europe B.V) and stored at −80 °C or embedded in for- malin. Two µm sections of formalin-fixed, paraffin-embedded kidney tissues were cut and were routinely stained with haematoxylin or eosin (H&E) and periodic acid- Schiff (PAS) for evaluation of kidney pathology. Complement C3 and IgG staining was performed on 5 µm frozen kidney sections with 1 µg/ml rabbit anti-C3 antibody (Thermoscientific) followed by goat-anti-rabbit IgG-HRP (Dako). For IgG staining rabbit anti-mouse IgG-HRP (Dako) was used. Peroxidase activity was detected with DAB and sections were counterstained with Mayer’s hematoxylin. Discussionh In IL-23R KO/lpr mice, which do not develop lupus, the total number of DN T cells as well as the total number of IL-17A producing DN T cells and CD4+ T cells was decreased. This was accompanied by lower serum IgG and ANA levels, decreased IgG and complement deposition in the kidneys and absence of kidney damage30. These Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 7 www.nature.com/scientificreports/ data suggest an important role for IL-17A in the IL-23 targeting approach of SLE although direct effects of IL-23 in disease development cannot be excluded. data suggest an important role for IL-17A in the IL-23 targeting approach of SLE although direct effects of IL-23 in disease development cannot be excluded. p By deletion of IL-17RA potentially several IL-17 family members are not functional as this subunit is involved in signaling of many if not all IL-17 family members. Therefore, this study goes beyond the role of IL-17A alone. Interestingly, IL-23 signaling is not deficient in the IL-17RA KO/lpr mice. It has been shown that CISK (Act1) knockout mice on a FcγRIIB background showed greatly improved survival and were largely protected from the development of glomerulonephritis44. CISK (Act1) is part of the IL-17 receptor signaling pathway and loss of CISK blocks signaling by all IL-17 cytokines and to a lesser extent loss of IL-17A44. However, BAFF can acti- vate B cells and myeloid cells via the BAFF receptor to activate CISK (Act1), indicating that the CISK/Act1 KO may have a broader impact in different signaling pathways in the development of SLE than the IL-17RA KO/ lpr in our study14. A proposed mechanism is that IL-17RA deficiency induces enhanced lymphoproliferation and that this leads to release of HMGB1 from activated or dying cells in spleens and lymph nodes, inducing a pro-inflammatory loop. However, evidence for this mechanism is not investigated in the present study. It is also possible that other IL-17 family member(s) are involved in controlling the lymphoproliferative phenotype in B6/ lpr mice. Further studies are needed to identify these mechanisms. However, our data clearly demonstrate that IL-17RA signaling is involved in the regulation of the lymphoproliferative phenotype in B6/lpr mice indicating that caution should be taken to modulate IL-17RA signaling in lymphoproliferative prone individuals. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Serum cytokine levels were quantified with a Multiplex panel (ProcartaPlex Mouse Simplex; Affymetrix eBioscience, Vienna, Austria) according to the manufacturer’s instructions. Samples were measured on a Luminex 100 System (Luminex, Austin, TX, USA) and data was analyzed with StarStation software, version 2.3 (AppliedCytometry, Birmingham, UK). The following cytokines were assessed: IL-4, IL-6, IL-10, IL-17A, IL-22, IFN-γ and TNF-α. γ HMGB1 levels were measured by Western Blotting as described previously for human serum9. Detection of HMGB1 on blots was performed with polyclonal anti-HMGB1-biotin (Thermoscientific, Etten-Leur, the Netherlands), and streptavidin-IRDye800 (LI-COR Biotechnology, Lincoln, NE, USA). Blots were scanned with an Odyssey infrared Imaging System (LI-COR Biotechnology) and analyzed with Odyssey software. In each blot a cell lysate made of Jurkat cells was run as a standard. HMGB1 levels were presented as the fluorescence intensity against the standard. g Levels of anti-HMGB1 were measured by in-house ELISA. Costar polystyrene plates were coated with 1 µg/ml recombinant HMGB1 (Sigma, St. Louis, MO, USA) and mouse sera were added in dilutions of 20 and 80 times. Detection of antibodies was done with rabbit anti- mouse IgG-HRP (Dako, Glostrup, Denmark) and TMB color reaction. Levels of anti-HMGB1 were calculated against a standard curve of a monoclonal anti-HMGB1 (R&D systems, Abingdon, United Kingdom). Statistical analysis. Data was analyzed using Prism software v5.04 (GraphPad Software Inc. La Jolla, CA) or comparisons, a non-parametric Mann-Whitney U test was used. P-values < 0.05 were considered significant Statistical analysis. Data was analyzed using Prism software v5.04 (GraphPad Software Inc. La Jolla, CA). For comparisons, a non-parametric Mann-Whitney U test was used. P-values < 0.05 were considered significant. References Yung, S. & Chan, T. M. Anti-DNA antibodies in the pathogenesis of lupus nephritis–the emerging mechanisms. Autoimmun Rev 7, 317–21 (2008). 9. Abdulahad, D. A. et al. High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus. Arthritis Res Ther 13, R71 (2011). y p yh 0. Abdulahad, D. A. et al. Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations Arthritis Res Ther 14, R184 (2012).hi h 11. Theofilopoulos, A. N. & Dixon, F. J. 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Decreased plasma IL22 levels, but not increased IL17 and IL23 levels, correlate disease activity in patients with systemic lupus erythematosus. Ann. Rheum. Dis. 68, 604–606 (2009). 27. Zhao, X. F. et al. Increased serum interleukin 17 in patients with systemic lupus erythematosus. Mol. Biol. Rep. 37, 81–85 (2010). 28. Wen, Z. et al. Materials and Methods i All sections were scored digitally after examination using a Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01, Hamamatsu Photonics K.K., Japan) and quantified ((number of positive pixels* 0.5) + number of strong positive pixels/total pixels) with software of ImageScope Viewer (V11.2.0.780 Aperio, e-Pathology Solution, CA, USA).fi HMGB1 and CD3 staining was performed on 2 µm paraffin sections using polyclonal anti-HMGB1 (Abcam, Cambridge, UK) and polyclonal anti-CD3 (Dakocytomation). RNA isolation. Total RNA was extracted from 20 10 µm thin kidney cryo-sections using RNeasy Mini plus Kit (Qiagen, Westburg, Leusden, The Netherlands) according to the manufacturer’s instructions. Integrity of RNA was determined by agarose gel electrophoresis. RNA quantity (OD-260) and quality (OD-260/OD-280) were determined using an ND-1,000 UV-Vis spectrophotometer (NanoDrop Technologies, Rockland, DE). cDNA was synthesized from 1 µg RNA using M-MLV Reverse Transcriptase and oligo(dT) 24 (Life Technologies, USA). mRNA expression of IFN-γ, TNF-α, IL-6, HMGB1, MCP-1 (monocyte chemotactic protein-1), KIM-1 (kidney injury molecule-1), NGAL (neutrophil gelatinase-associated lipocalin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was measured by the real-time quantitative PCR system (ABI Prism 7900HT Sequence Detection System, Applied Biosystems, USA) with specific Taqman probes. The amount of target was normalized to an endogenous reference (GAPDH) and expressed as relative expression (2−ΔCT). Serum analysis. ANA titers were measured by immunofluorescence on HEp-2000 coated glass slides (Biomedical Diagnostics, Eindhoven, the Netherlands), using serial dilutions of mouse serum in PBS, and rabbit anti-mouse IgG-FITC (Dako, Glostrup, Denmark) for detection. Complement levels were measured using a com- mercial ELISA, according to the manufactures instructions (GenWay Biotech, San Diego, USA). 8 Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ 35. Corneth, O. B. et al. Absence of interleukin-17 receptor a signaling prevents autoimmune inflammation of the joint and leads to a Th2-like phenotype in collagen-induced arthritis. Arthritis Rheumatol. 66, 340–349 (2014). h 36. Ramani, K. & Biswas, P. S. Interleukin 17 signaling drives Type I Interferon induced proliferative crescentic glomerulonephri lupus-prone mice. Clin. Immunol. 162, 31–36 (2016).h g g y lupus-prone mice. Clin. Immunol. 162, 31–36 (2016).h p p C , ( ) 37. Schmidt, T. et al. Function of the Th17/interleukin-17A immune response in murine lupus nephritis. Arthritis Rheumatol. 67, 475–487 (2015). ( ) 8. Jiang, W. & Pisetsky, D. S. Expression of high mobility group protein 1 in the sera of patients and mice with systemic lupu erythematosus. Ann Rheum Dis 67, 727–8 (2008).l y ( ) 39. Zhu, X. et al. Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation. J. Clin. Invest. 125, 1098–1110 (2015). ( ) 0. Liu, G. et al. High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine J. Immunol. 181, 4240–4246 (2008).f 1. Friggeri, A. et al. HMGB1 inhibits macrophage activity in efferocytosis through binding to the alphavbeta3-integrin. Am. J. Physiol Cell. Physiol. 299, C1267–76 (2010).l y 42. Valdes-Ferrer, S. I. et al. HMGB1 mediates splenomegaly and expansion of splenic CD11b+ Ly-6C(high) inflammatory monocytes in murine sepsis survivors. J. Intern. Med. 274, 381–390 (2013). p ( ) 43. Kyttaris, V. C., Kampagianni, O. & Tsokos, G. C. Treatment with anti-interleukin 23 antibody ameliorates disease in lupus-prone mice. Biomed. Res. Int. 2013, 861028 (2013). ( ) 4. Pisitkun, P. et al. Interleukin-17 cytokines are critical in development of fatal lupus glomerulonephritis. Immunity 37, 1104–1115 (2012). Acknowledgements g We thank H. Moorlag, J. Bijzet, and M.D. Brem for excellent technical support. This project was funded by the Dutch Arthritis Foundation (Reumafonds, grant no DAA 0801043). Author Contributions O.B.J.C. and F.S. participated in the acquisition, analysis and interpretation of data and drafting of the manuscript. F.L., P.S.A., A.M.C.M. and G.H. participated in the acquisition of data. P.H., R.W.H., J.W. and E.L. participated in the study conception and design, analysis and interpretation of data and critically revised the manuscript. All authors read and approved the manuscript. References Interleukin-17 expression positively correlates with disease severity of lupus nephritis by increasing anti-do stranded DNA antibody production in a lupus model induced by activated lymphocyte derived DNA. PLoS One 8, e58161 (20 29 Zh Z K tt i V C & T k G C Th l f IL 23/IL 17 i i l h iti J I l 183 3160 3169 (2009) y p p y y p y , ( ) 29. Zhang, Z., Kyttaris, V. C. & Tsokos, G. C. The role of IL-23/IL-17 axis in lupus nephritis. J. Immunol. 183, 3160-3169 (2009). 29. Zhang, Z., Kyttaris, V. C. & Tsokos, G. C. The role of IL-23/IL-1 g yh p p ( ) 30. Kyttaris, V. C., Zhang, Z., Kuchroo, V. K., Oukka, M. & Tsokos, G. C. Cutting edge: IL- 23 receptor deficiency prevent development of lupus nephritis in C57BL/6-lpr/lpr mice. J Immunol 184, 4605-9 (2010). 31. Shivakumar, S., Tsokos, G. C. & Datta, S. K. T cell receptor alpha/beta expressing double-negative (CD4−/CD8−) and CD4+ T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis. J Immunol 143, 103-12 (1989). 32. Oliver, A. M., Martin, F., Gartland, G. L., Carter, R. H. & Kearney, J. F. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur J Immunol 27, 2366–74 (1997). 33. Hsu, H. C. et al. Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice. Nat Immunol 9, 166–75 (2008).h autoimmune BXD2 mice. Nat Immunol 9, 166–75 (2008). ( ) 4. Ding, Y. et al. IL-17RA Is Essential for Optimal Localization of Follicular Th Cells in the Germinal Center Light Zone To Promote Autoantibody-Producing B Cells. J Immunol (2013). Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 9 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-39483-w. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports | (2019) 9:4032 | https://doi.org/10.1038/s41598-019-39483-w 10
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Relationship Between Fruit and Vegetables Intake and Common Mental Disorders in Youth: A Systematic Review
Public health reviews
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Relationship Between Fruit and Vegetables Intake and Common Mental Disorders in Youth: A Systematic Review Julia Dabravolskaj 1*, Shelby Marozoff 1, Katerina Maximova 2,3, Sandra Campbell 4 and Paul J. Veugelers 1 1School of Public Health, University of Alberta, Edmonton, AB, Canada, 2MAP Centre for Urban Health Solutions, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada, 3Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada, 4John W. Scott Health Sciences Library, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada Objective: Recent evidence suggests that adequate fruit and vegetables intake (FVI) might be associated with lower risk of common mental disorders (CMDs) in adults, but studies in youth are also beginning to emerge and are synthesized in this systematic review. Methods: Online databases were searched from inception to 30 October 2020 to locate cross-sectional, cohort, and case-control studies focusing on the FVI and CMDs in youth (i.e., 10–18 years old). The risk of bias of studies was assessed using Joanna Briggs Institute Critical Appraisal Tool and the Newcastle-Ottawa quality assessment scale. Edited by: Diogo Costa, Bielefeld University, Germany Edited by: Diogo Costa, Bielefeld University, Germany Reviewed by: Laura Orlando, University of Toronto, Canada June Kloubec, Bastyr University, United States Conclusion: The lack of associations between FVI and CMDs in youth, along with consistent associations in adults, might be explained by the accumulation of risk theoretical model and methodological challenges. *Correspondence: Julia Dabravolskaj dabravol@ualberta.ca Keywords: adolescents, youth, mental health and wellbeing, common mental disorders, healthy diet, vegetables and fruit, depression, anxiety Received: 12 December 2021 Accepted: 31 August 2022 Published: 20 September 2022 Received: 12 December 2021 Accepted: 31 August 2022 Published: 20 September 2022 Results: Among 3,944 records identified, 12 studies (8 cross-sectional, 1 case-control, and 3 prospective cohort studies) were included in the final synthesis. None of the prospective cohort studies identified a statistically significant association between FVI and CMDs in youth, although inconsistent associations were reported in cross-sectional and case-control studies. Public Health Reviews SYSTEMATIC REVIEW published: 20 September 2022 doi: 10.3389/phrs.2022.1604686 Public Health Reviews SYSTEMATIC REVIEW Inclusion Criteria Two reviewers [JD and SM] independently reviewed the titles and abstracts on the Covidence platform [19]. [JD and SM] documented and compared reasons for exclusion. Bibliographies of included papers were reviewed for relevant papers independently by [JD and SM]. Disagreements during the screening process were resolved by consensus. We included observational (i.e., cohort, case-control, and cross-sectional) studies that focused on FVI, measured combined or separately, and CMDs in community-dwelling adolescents (see detailed inclusion and exclusion criteria in Table 1). Inclusion of primary studies was not limited by sex, ethnicity, or any socioeconomic determinants of health. Studies that included adolescents but also extended outside the specified age range (i.e., 10–18 years old) were assessed on a case-by-case basis as to whether they could meaningfully contribute to the systematic review. If data were duplicated in more than one study, only the latest study or the study with the largest sample size was considered. Studies that did not report any estimates of the association of interest and did not provide any data that could be used to calculate any measures of association were excluded. With one in five adolescents living with mental disorders worldwide [12], there is a recognized need for the development and implementation of effective primary prevention strategies [13]. Recent systematic reviews [14, 15] of observational studies point to the association between adherence to high quality diet (i.e., rich in fruit, vegetables, legumes, nuts and whole grains [16]) and lower incidence of CMDs in a general population of youth. However, diet quality is often conceptualized and measured differently, making it difficult to compare results across different studies. To circumvent this challenge, fruit and vegetables intake (FVI) is often used as a simple indicator of overall diet quality [17]. A systematic review [18] of 16 cross-sectional, 9 cohort, and 2 case-control on the association between FVI and mental disorders in adults showed that the highest category of FVI was associated with up to 17% lower risk of depression in cohort studies, with higher magnitudes of the associations (i.e., up to 25% lower risk of depression) observed in cross- sectional studies. Moreover, every 100-g increase in fruit and vegetable intake was associated with a 3% reduced risk of depression in cohort studies. However, to our knowledge, evidence on this association in adolescents has not yet been synthesized. Data Extraction and Management Data Extraction and Management Upon finalizing the list of included studies, [JD and SM] independently extracted the following data: study details (first author, title, publication year, journal, objectives of the study, study design, follow-up years, study duration, recruitment procedures utilized, description of the exposure(s) and exposure assessment tools, comparator, description of the outcome(s) and outcome assessment tools, sample size, mean age or age range at baseline, sex of participants included in the study); analysis and results (i.e., statistical methods used to produce the measure and magnitude of association, standard error, standard deviation for the exposure and control groups, 95% CI, p-value, confounders adjusted for in the analysis); and author conclusions. If data in a selected study was missing or lacked sufficient details, [JD] contacted corresponding authors for additional information. Where the results of several models where presented, data were extracted for all models. Study-specific methods and results (e.g., statistical methods used, comparators, effect measures) are presented in Supplementary Table S1 (Supplementary File S3). Citation: Mental disorders constitute a significant global public health burden [1–3] and result from an intricate interplay of multiple factors. This interplay is particularly potent during adolescence (i.e., 10–19 years old) when more than three-quarters of all life-time mental disorders, especially common mental disorders (CMDs) such as depression and anxiety, manifest for the first time [4, 5]. In fact, the cumulative probability of CMDs rises steeply from around 5% in early adolescence to as high as 20% by the end of adolescence [6]. Mental disorders in adolescence are associated with many long-term negative psychosocial outcomes, including low educational attainment, poor work Dabravolskaj J, Marozoff S, Maximova K, Campbell S and Veugelers PJ (2022) Relationship Between Fruit and Vegetables Intake and Common Mental Disorders in Youth: A Systematic Review. Public Health Rev 43:1604686. doi: 10.3389/phrs.2022.1604686 Dabravolskaj J, Marozoff S, Maximova K, Campbell S and Veugelers PJ (2022) Relationship Between Fruit and Vegetables Intake and Common Mental Disorders in Youth: A Systematic Review. Public Health Rev 43:1604686. doi: 10.3389/phrs.2022.1604686 September 2022 | Volume 43 | Article 1604686 Public Health Reviews | Owned by SSPH+ | Published by Frontiers Diet and Adolescent Mental Disorders Dabravolskaj et al. systematic review was registered on PROSPERO (CRD42020148625, 1 August 2020). systematic review was registered on PROSPERO (CRD42020148625, 1 August 2020). performance [7], difficulty developing stable relationships and social networks, unemployment [8], as well as future mental disorders [9], substance abuse, and suicide [6]. Often underdiagnosed [4], they can be difficult to manage due to limited effectiveness of available treatment options [10] and high rates of recurrence [6] and comorbidity [11]. Inclusion Criteria This paper fills in this gap, with particular attention given to the methodological aspects of available studies to inform future research in the field of nutritional psychiatry in youth. Exclusion criteria Non-human subjects; institutionalized adolescents; studies focusing solely on children (<10 years old) or adults (≥19 years old) Non-human subjects; institutionalized adolescents; studies focusing solely on children (<10 years old) or adults (≥19 years old) Other diet constructs (e.g., eating behaviors) considered alone, rather than in combination with FVI. Other diet constructs (e.g., eating behaviors) considered alone, rather than in combination with FVI. Other mental disorders (including those with anxiety and/or depressive components, eating disorders, psychological distress, attention deficit hyperactivity disorder). For studies where outcomes were measured with a single question (as opposed to a validated scale), reviewers assessed if the question explicitly stated or implied mental disorders other than the outcome of interest TABLE 2 | Quality assessment of cross-sectional studies included in the systematic review (systematic review, all countries, up to 2020). Cross-sectional studies Were the criteria for inclusion in the sample clearly defined? Were the study subjects and the setting described in detail? Was the exposure measured in a valid and reliable way? Were confounding factors identified? Were strategies to deal with confounding factors stated? Were the outcomes measured in a valid and reliable way? Was appropriate statistical analysis used? Arat, 2017 [23] No No Yes Yes Yes No Yes Arat, 2015 [24] No No Yes No No No No Hoare et al., 2019 [25] No Yes Yes Yes Yes Yes Yes Hoare et al., 2014 [26] No Yes Yes Yes Yes Yes Yes Hoare et al., 2018 [27] No Yes Yes Yes Yes Yes Yes Park et al., 2018 [31] No Yes Yes Yes Yes Yes Yes Hong and Peltzer, 2017 [28] No Yes Yes Yes Yes No Yes Liu et al., 2020 [29] Yes Yes Yes Yes Yes No Yes Park et al., 2018 [30] Yes Yes Yes Yes Yes No Yes Winpenny et al., 2018 [32] No Yes Yes Yes Yes Yes Yes 2 | Quality assessment of cross-sectional studies included in the systematic review (systematic review, all countries, up to 2020). Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines (Supplementary File S4). Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines (Supplementary File S4). As part of the quality assessment process, we assessed whether important confounding factors [particularly, socioeconomic status (SES) which can affect both person’s diet and mental health outcomes] were identified and adjusted for in the included studies. Search Strategy gy A medical librarian [SC (Sandra Campbell)] searched the following databases: Prospero, Wiley Cochrane Library, Ovid Embase, Ovid Medline, Ovid PsycInfo, EBSCO CINAHL, ProQuest Dissertations and Theses Global, Food Science and Technology Abstracts (WOS) and CAB Abstracts (WOS). Each of the databases was searched from inception to 30 October 2020. The search strategy included both text words and controlled vocabulary (e.g., MeSH, EMTREE, etc.) for the terms “fruits or vegetables” and “anxiety or depression” (see Supplementary File S1). Studies limited to adults and very young children were excluded. In addition, bibliographies of relevant studies and researcher- identified databases were hand searched. All identified records were exported to Covidence systematic review software [19], and duplicates were automatically removed (see PRISMA flowchart, Supplementary File S2). Language restriction was not applied; when necessary, a native language speaker was identified in the research community at the University of Alberta and asked to translate the paper, assess eligibility of the study and extract data. This Risk of Bias Assessment The Newcastle-Ottawa quality assessment scale [20] was used to assess the quality of cohort and case-control studies. The Joanna Briggs Institute (JBI) Critical Appraisal Tool for cross-sectional studies [21] was used to assess the quality of cross-sectional studies. One question was excluded from this tool (i.e., “Were objective, standard criteria used for measurement of the condition?”), since the focus was on the general population rather than specific diagnostic methods or clinical populations. September 2022 | Volume 43 | Article 1604686 Public Health Reviews | Owned by SSPH+ | Published by Frontiers 2 Diet and Adolescent Mental Disorders Dabravolskaj et al. TABLE 1 | Inclusion and exclusion criteria (systematic review, all countries, up to 2020). Inclusion criteria Population Community-dwelling 10–18 years old adolescents; general population if the association was studied in adolescents as part of subgroups analyses Exposure of interest FVI measured in terms of frequency of consumption or servings or grams per day (80 g was considered one serving, World Health Organization, 2004 [22]) Outcome of interest Common mental disorders (i.e., depression, anxiety, or co-morbid depression and anxiety), as diagnosed by physicians, using validated tools, or self-reporting Exclusion criteria Discrepancies resulting from the independent application of quality assessment tools by [JD and SM] were resolved by consensus. Since the systematic review aimed to map the existing literature and highlight methodological challenges and areas for further research, studies were not excluded based on risk of bias assessment. The utility of introducing a qualitative score has long been discredited [33]; instead, quality assessments of the selected cross-sectional, cohort and case-control studies are summarized in Tables 2–4, respectively. RESULTS The search yielded 3,944 records to assess for eligibility, 903 duplicates were removed. Among 117 records that were examined in full, 12 studies were included in the final analysis. The articles were published between 2012 and 2020 and included analysis of data collected in United States, United Kingdom, Greece, Australia, Canada, South Korea, Saint Lucia, Egypt, Saint Vincent and the Grenadines, Djibouti, Morocco, Myanmar, Zambia, Tanzania, Venezuela, Grenada, Lebanon, China, Indonesia, Thailand, Uganda, Tunisia, Botswana, Sri Lanka, India, Seychelles, Guyana, Ecuador, Jordan, Argentina, and Kenya. Table 5 and Supplementary Table S1 (see Supplementary File S4) provide detailed information on the study characteristics and results of the included studies, respectively. Eight studies [23–27, 28–30] used cross-sectional design, three studies [31, 32, 34] were prospective cohort studies (two [31, 32] included analyses of both cross-sectional and Data Analysis and Reporting Considering substantial heterogeneity between studies in terms of study design, exposure and outcome definition and assessment methods, included covariates, measures of association, and results of the risk of bias assessment, the authors refrained from pooling data in a meta-analysis. Moreover, the number of studies available was not sufficient to pool in sub-group analyses. Therefore, a narrative synthesis is provided. The review follows September 2022 | Volume 43 | Article 1604686 Public Health Reviews | Owned by SSPH+ | Published by Frontiers 3 Diet and Adolescent Mental Disorders Dabravolskaj et al. longitudinal data), and one [35] was a case-control study. Sample i d f 603 [32] t 65 528 [28] d l t i servings or grams per day. Food frequency questionnaire [34], f d di t di [32] d 24 h di t ll [25] d i TABLE 3 | Quality assessment of cohort studies included in the systematic review (systematic review, all countries, up to 2020). Cohort studies Representativeness of the exposed cohort: A: truly representative of the average (describe) in the community; B: somewhat representative of the average in the community; C: selected group of users; D: no description of the derivation of the cohort Selection of the non exposed cohort: A: drawn from the same community as the exposed cohort; B: drawn from a different source; C: no description of the derivation of the non- exposed cohort Ascertainment of exposure: A: secure record; B: structured interview; C: written self report; D: no description Demonstration that outcome of interest was not present at start of study: A: yes; B: no. Public Health Reviews | Owned by SSPH+ | Published by Frontiers Data Analysis and Reporting Comparability of cohorts on the basis of the design or analysis: A: study controls for socioeconomic status; B: study controls for any additional factor Assessment of outcome: A: independent blind assessment; B: record linkage; C: self report; D: no description Was follow- up long enough for outcomes to occur: A: yes; B: no Adequacy of follow up of cohorts: A: complete follow up - all subjects accounted for; B: subjects lost to follow up unlikely to introduce bias (≥70% follow-up) or description provided of those lost; C: follow up rate <70% and no description of those lost; D: no statement Hoare et al., 2016 [31] B A C A B C A (2 years) B McMartin et al., 2012 [34] A A C A A and B B A (3 years) A Winpenny et al., 2018 [32] B A C A A and B C A (3 years) C TABLE 3 | Quality assessment of cohort studies included in the systematic review (systematic review, all countries, up to 2020). Cohort studies Representativeness of the exposed cohort: A: truly representative of the average (describe) in the community; B: somewhat representative of the average in the community; C: selected group of users; D: no description of the derivation of the cohort Selection of the non exposed cohort: A: drawn from the same community as the exposed cohort; B: drawn from a different source; C: no description of the derivation of the non- exposed cohort Ascertainment of exposure: A: secure record; B: structured interview; C: written self report; D: no description Demonstration that outcome of interest was not present at start of study: A: yes; B: no. Comparability of cohorts on the basis of the design or analysis: A: study controls for socioeconomic status; B: study controls for any additional factor Assessment of outcome: A: independent blind assessment; B: record linkage; C: self report; D: no description Was follow- up long enough for outcomes to occur: A: yes; B: no Adequacy of follow up of cohorts: A: complete follow up - all subjects accounted for; B: subjects lost to follow up unlikely to introduce bias (≥70% follow-up) or description provided of those lost; C: follow up rate <70% and no TABLE 3 | Quality assessment of cohort studies included in the systematic review (systematic review, all countries, up to 2020). Data Analysis and Reporting TABLE 4 | Quality assessment of case-control studies included in the systematic review (systematic review, all countries, up to 2020). Case- control studies Is the case definition adequate: A: yes, with independent validation; B: yes (e.g., record linkage, self reports); C: no description Representativeness of the cases: A: consecutive or obviously representative series of cases; B: potential for selection biases or not stated Selection of controls: A: community controls; B: hospital controls; C: no description Definition of controls: A: no history of disease (endpoint); B: no description of source Comparability of cases and controls on the basis of the design or analysis: A: study control for socioeconomic status; B: study controls for additional factors Ascertainment of exposure (A: secure record (e.g., surgical records); B: structured interview blinded to case/control status; C: interview not blinded to case/control status; D: written self report or medical record only; E: no description Same method of ascertainment for cases and control: A: yes; B: no Non- response rate: A: same rate for both groups; B: non respondents described; C: rate different and no designation Kim et al., 2015 [35] B B A A B D A C TABLE 4 | Quality assessment of case-control studies included in the systematic review (systematic review, all countries, up to 2020). diagnoses of internalizing disorders, and the rest of the studies [23, 24, 28–30] used single-item self-reported questions to assess the outcome(s) of interest. Subgroup analysis based on gender was conducted in five studies [25–27, 31, 32]. familial history of depression and physical activity, were not measured. It is unclear whether assessors were blinded to the research question or outcome assessment. Among three prospective cohort studies, samples were representative of 10–11 years old children in corresponding communities in one study [34]. In all cohort studies, non- exposed cohorts were drawn from the same community as exposed cohorts and samples appeared free from mental disorders at the beginning of the studies. All studies ascertained exposure using self-reported dietary measures, and in all but one [34] mental disorders were self-reported. One study [31] included a two-year follow-up, while the other two studies included three-year [32, 34] follow-ups. One study [34] had complete follow up. Another study [31] had 74.5% participation rate, but participants lost to follow-up (3.2% refusal, 7.2% unavailable, 15.1% relocated) were unlikely to introduce bias. Data Analysis and Reporting Hoare et al., 2016 [31] B A C A B C A (2 years) B McMartin et al., 2012 [34] A A C A A and B B A (3 years) A Winpenny et al., 2018 [32] B A C A A and B C A (3 years) C longitudinal data), and one [35] was a case-control study. Sample sizes ranged from 603 [32] to 65,528 [28] adolescents in cross- sectional studies, from 472 [31] to 3,757 [34] in cohort studies, and 849 [35] in the case-control study. Except for one study that focused exclusively on female youth [35], all other studies included approximately equal numbers of male and female youth. Age distribution was within the prespecified age limits (i.e., 10–18 years old) in all but one study [25] that included a range of 9–13 years old children attending Grade 5 and 6, with 58% of the sample being older children (11–13 years old). servings or grams per day. Food frequency questionnaire [34], four-day diet diary [32], and 24-h dietary recall [25] were used in one study each. Single-item dietary assessment questions were part of larger questionnaires on various lifestyle behaviours in nine studies [23, 24, 26–31, 35]. The questions referred to fruit, vegetable, or combined intake in the past 12 months [34, 35], 30 days [23, 29], 7 days [24, 28, 30], and the day before [26, 27, 31]. All studies assessed depression, two assessed anxiety [23, 29], and one study [34] included common symptoms of depression and anxiety (combined) as the outcome of interest. Six studies used the following validated questionnaires to assess the outcomes of interest: COOPS/WONCA questionnaire [25], SMFQ [26, 31], CES-D [27], Korean version of the Beck Depression Inventory [35], and Moods and Feelings Questionnaire [32]. One study [34] included physician Only self-reported dietary assessment instruments were used. Eight studies [23–25, 27–30, 35] assessed intakes of fruit and vegetables separately, while five studies [26, 29, 31, 32, 34] assessed combined FVI. Seven studies [23, 24, 27–30, 35] measured FVI in terms of frequency of FV consumption, while five studies [25, 26, 31, 32, 34] assessed FVI in terms of Public Health Reviews | Owned by SSPH+ | Published by Frontiers September 2022 | Volume 43 | Article 1604686 4 Diet and Adolescent Mental Disorders Dabravolskaj et al. Data Analysis and Reporting One study [32] reported a follow-up rate of less than 70%; the analysis of differences between participants and non-participants was not available, making it difficult to assess the possibility of selection bias. All studies adjusted for sex/ gender. Other confounding factors adjusted for included: parental education, and the school participants attended [31]; age, SES, other lifestyle behaviours (smoking, physical activity, alcohol consumption, sleep), friendship quality, self-esteem, family functioning, %body fat, medication use, total energy Public Health Reviews | Owned by SSPH+ | Published by Frontiers Quality Assessment Of 10 cross-sectional studies, eight [23–28, 31, 32] omitted the criteria for inclusion in the sample, and five [23, 24, 28–30] did not include validated scales to assess CMDs. Causal claims in respect to the focal association were made in two [23, 24] cross- sectional studies. All (except one [24]) studies identified and adjusted for at least some important confounding factors: four studies [25, 27, 28, 30] included indicators of SES and one [23] adjusted for food insecurity, two studies stratified by ethnicity [24] and gender [31]. There was one [35] case-control study. Community controls with no history of disease were selected from the same source population as the cases (i.e., adolescent youth attending the University Health Center for annual routine health examinations). FFQ with 63 food items and the Korean version of the Beck Depression Inventory (K-BDI) were included in the same questionnaire. Importantly, participants with pre-existing psychological conditions or those taking medication for depression were excluded from the study. Some of the most important confounding factors, such as SES, September 2022 | Volume 43 | Article 1604686 Public Health Reviews | Owned by SSPH+ | Published by Frontiers 5 Diet and Adolescent Mental Disorders Dabravolskaj et al. TABLE 5 | Description of studies included in the systematic review (systematic review, all countries, up to 2020). Author(s) and publication year Study design Country Sample size Mean age or age range (at baseline if a cohort study) %females Follow-up years Exposure(s) Outcome(s) Comments and conclusions Arat, 2017 [23] Cross- sectional Botswana 2,197 11–17 years old 55% N/A F, Va: Single-item dietary measure as part of the GSHS “During the past 30 days, how many times per day did you usually eat fruit?” and “During the past 30 days, how many times per day did you usually eat vegetable?” Depression and anxiety assessed by single questions: “During the past 12 months, did you ever feel so sad or hopeless almost every day for 2 weeks or more in a row that you stopped doing your usual activities?” and “During the past 12 months, how often have you been so worried about something that you could not sleep at night?” “. . . higher fruit intake as a risk factor for depression, anxiety (except the United Republic of Tanzania). . . higher vegetable consumption as a risk factor for depression, anxiety (except the United Republic of Tanzania and Zambia). . Quality Assessment .” Kenya 3,691 51.3% Seychelles 1,432 52.2% Uganda 3,215 48.8% Tanzania 2,176 52.1% Zambia 2,257 51.1% Arat, 2015 [24] Cross- sectional United States 10,563 12–18 years old Asian American 52.2%, African American 49.6%, Caucasian 47.7% N/A F, V: Single-item dietary measure as part of the YRBSb “During the past 7 days, how many times did you eat fruit?”); “During the past 7 days, how many times did you eat other vegetables? (Do not count green salad, potatoes, or carrots.)” Depression assessed by a single question: “During the past 12 months, did you ever feel so sad or hopeless almost every day for 2 weeks or more in a row that you stopped doing some usual activities?” No association between F and V intake and depression; however, causal language throughout the article (e.g. “risk factors for depression specific to Asians, and not Caucasians or Africans, was lower carrot consumption”) Hoare et al., 2019 [25] Cross- sectional Greece 2,240 9–13 years old 50% N/A F, V: 24-h recall morning interviews conducted by trained dietitians and nutritionists on 2 consecutive weekdays and 1 weekend day Emotional functioning (i.e., depression) assessed by COOPS/WONCA questionnaire: “During the past 2 weeks, how much were you pre- occupied with emotional problems such as feeling anxious, depressed, irritable or downhearted and sad?” “There were no association observed between the consumption of fruits and vegetables and emotional functioning” Hoare et al., 2014 [26] Cross- sectional Australia 800 11.8–14.9 years old 55% N/A FV: single item dietary measure as part of the ABAKQc “How many servings of fruit/vegetables they consumed on the last school day, including Depression assessed by the SMFQ “neither fruit and vegetable nor takeaway food consumption were related to depressive symptomatology in multivariate analyses.” TABLE 5 | Description of studies included in the systematic review (systematic review, all countries, up to 2020). could not sleep at night?” Arat, 2015 [24] Cross- sectional United States 10,563 12–18 years old Asian American 52.2%, African American 49.6%, Caucasian 47.7% N/A F, V: Single-item dietary measure as part of the YRBSb “During the past 7 days, how many times did you eat fruit?”); “During the past 7 days, how many times did you eat other vegetables? (Continued on following page) Quality Assessment (Do not count green salad, potatoes, or carrots.)” Depression assessed by a single question: “During the past 12 months, did you ever feel so sad or hopeless almost every day for 2 weeks or more in a row that you stopped doing some usual activities?” No association between F and V intake and depression; however, causal language throughout the article (e.g. “risk factors for depression specific to Asians, and not Caucasians or Africans, was lower carrot consumption”) Hoare et al., 2019 [25] Cross- sectional Greece 2,240 9–13 years old 50% N/A F, V: 24-h recall morning interviews conducted by trained dietitians and nutritionists on 2 consecutive weekdays and 1 weekend day Emotional functioning (i.e., depression) assessed by COOPS/WONCA questionnaire: “During the past 2 weeks, how much were you pre- occupied with emotional problems such as feeling anxious, depressed, irritable or downhearted and sad?” “There were no association observed between the consumption of fruits and vegetables and emotional functioning” Hoare et al., 2014 [26] Cross- sectional Australia 800 11.8–14.9 years old 55% N/A FV: single item dietary measure as part of the ABAKQc “How many servings of fruit/vegetables they consumed on the last school day, including those eaten at home?” Depression assessed by the SMFQ “neither fruit and vegetable nor takeaway food consumption were related to depressive symptomatology in multivariate analyses.” (Continued on following page) Public Health Reviews | Owned by SSPH+ | Published by Frontiers September 2022 | Volume 43 | Article 1604686 6 Public Health Reviews | Owned by SSPH+ | Published by Frontiers September 2022 | Volume 43 | Article 1604686 Diet and Adolescent Mental Disorders Dabravolskaj et al. TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). Author(s) and publication year Study design Country Sample size Mean age or age range (at baseline if a cohort study) %females Follow-up years Exposure(s) Outcome(s) Comments and conclusions Hoare et al., 2018 [27] Cross- sectionald United States 3,696 15.9 (1.7) Both males and females were included in the sample, but % females not reported N/A F, V: Single-item dietary measure “How often did you eat fruit or drink fruit juice yesterday?” and the same question for vegetable consumption Depression assessed by the 20- item CES-D “Fruit consumption was cross- sectionally related to reduced odds of depression in adolescence in both males and females, both before and after controlling for covariates. Quality Assessment Author(s) and publication year Study design Country Sample size Mean age or age range (at baseline if a cohort study) %females Follow-up years Exposure(s) Outcome(s) Comments and conclusions Hoare et al., 2018 [27] Cross- sectionald United States 3,696 15.9 (1.7) Both males and females were included in the sample, but % females not reported N/A F, V: Single-item dietary measure “How often did you eat fruit or drink fruit juice yesterday?” and the same question for vegetable consumption Depression assessed by the 20- item CES-D “Fruit consumption was cross- sectionally related to reduced odds of depression in adolescence in both males and females, both before and after controlling for covariates. TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). Hoare et al., 2016 [31] 1) Cross- sectional and 2) prospective cohort Australia 634 13.1 (0.6) 53.3% Wave 1 (May 2012), Wave 2 (May 2014) FV: “How many servings of fruit/ vegetables they consumed on the last school day, including those eaten at home?” as part of the ABAKQ Depression assessed by the SMFQ FVI was not a significant predictor in univariate analysis, hence not entered in further models and not commented on Hong and Peltzer, 2017 [28] Cross- sectional Korea 65,528 12–18 years old (mean age 15.1) 47.8% N/A F, V: single-item dietary measure as part of KYRBSe Participants were asked about the frequency of fruits (excluding fruit juices) and vegetable dishes (excluding Kimchi) over the past 7 days Depression symptoms assessed by a single question: “Have you experienced sadness or despair to the degree that you stopped your daily routine for the recent 12 months?” “Positive dietary behaviours (fruit and vegetable consumption . . . (Continued on following page) “. . .consumption of green vegetables and 1 to 3 servings/ day of fruits was associated with decreased risk of depression” Quality Assessment Vegetable consumption among females was cross- sectionally associated with reduced odds of depression in adolescence” Hoare et al., 2016 [31] 1) Cross- sectional and 2) prospective cohort Australia 634 13.1 (0.6) 53.3% Wave 1 (May 2012), Wave 2 (May 2014) FV: “How many servings of fruit/ vegetables they consumed on the last school day, including those eaten at home?” as part of the ABAKQ Depression assessed by the SMFQ FVI was not a significant predictor in univariate analysis, hence not entered in further models and not commented on Hong and Peltzer, 2017 [28] Cross- sectional Korea 65,528 12–18 years old (mean age 15.1) 47.8% N/A F, V: single-item dietary measure as part of KYRBSe Participants were asked about the frequency of fruits (excluding fruit juices) and vegetable dishes (excluding Kimchi) over the past 7 days Depression symptoms assessed by a single question: “Have you experienced sadness or despair to the degree that you stopped your daily routine for the recent 12 months?” “Positive dietary behaviours (fruit and vegetable consumption . . . ) were negatively associated with perceived stress and depression symptoms” Kim et al., 2015 [35] Case-control Korea 849 15 (1.5) 100% N/A (depressive symptoms were assessed during recruitment, while data on dietary patterns was obtained by FFQ in the past 12 months) F, V: FFQ for the KYRBS; frequency range of the FFQ items in the past 12 months was classified into nine categories (never or seldom, once per month, 2–3 times per month, once per week, 2–4 times per week, 5–6 times per week, once per Depression assessed by the Korean version of the Beck Depression Inventory “. . .consumption of green vegetables and 1 to 3 servings/ day of fruits was associated with decreased risk of depression” TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). Public Health Reviews | Owned by SSPH+ | Published by Frontiers Quality Assessment ) were negatively associated with perceived stress and depression symptoms” Kim et al., 2015 [35] Case-control Korea 849 15 (1.5) 100% N/A (depressive symptoms were assessed during recruitment, while data on dietary patterns was obtained by FFQ in the past 12 months) F, V: FFQ for the KYRBS; frequency range of the FFQ items in the past 12 months was classified into nine categories (never or seldom, once per month, 2–3 times per month, once per week, 2–4 times per week, 5–6 times per week, once per day, twice per day and three times per day) and the portion size was divided into three categories (small, medium and large) Depression assessed by the Korean version of the Beck Depression Inventory “. . .consumption of green vegetables and 1 to 3 servings/ day of fruits was associated with decreased risk of depression” (Continued on following page) Public Health Reviews | Owned by SSPH+ | Published by Frontiers September 2022 | Volume 43 | Article 1604686 7 Diet and Adolescent Mental Disorders Dabravolskaj et al. TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). Author(s) and publication year Study design Country Sample size Mean age or age range (at baseline if a cohort study) %females Follow-up years Exposure(s) Outcome(s) Comments and conclusions Liu et al., 2020 [29] Cross- sectional 25 low- and middle-income countriesf, see below 65,267 12–15 years old Country- specific, ranging between 40.5% and 57.9% N/A F, V, FV: Single- item measure as part of the GSHSg “During the past 30 days, how many times per day did you usually eat fruit, such as apples, bananas, oranges?” and “During the past 30 days, how many times per day did you usually eat vegetables, such as salads, spinach, eggplant, tomatoes, and cucumbers?” Depressive and anxiety symptoms assessed by a single question: “During the past 12 months, did you ever feel so sad or hopeless almost every day for 2 weeks or more in a row that you stopped doing your usual activities?” and “During the past 12 months, how often have you been so worried about something that you could not sleep at night?” When country- specific estimates were combined in a meta-analysis, inadequate vs. (Continued on following page) Public Health Reviews | Owned by SSPH+ | Published by Frontiers FVI (Combined) and Depression intake, and depression symptoms at baseline [32]; and household income, parental marital status, parental education, body weight status, physical activity and geographic area [34]. ( ) p Four studies reported on the association between combined FVI and depression. Two studies were cross-sectional, while two others included both cross-sectional and longitudinal analyses. One cross-sectional study [29] reported statistically significant associations in four of 25 low- and middle-income countries (i.e., Seychelles, Ecuador, Jordan, Kenya). One prospective study reported non-significant associations in univariate analyses: OR = 0.87 (95% CI 0.39; 1.96) for males and OR = 0.85 (95% CI 0.54; 1.34) for females [31]. Another prospective cohort study reported no statistically significant associations following adjustment for covariates: ß = 0.14 (95% CI of −0.15; 0.43) [32]. Quality Assessment adequate FVI was associated with a higher risk of depressive symptoms but not anxiety symptoms” Saint Lucia 1,032 13.7 55.6% Egypt 4,476 13.2 48.5% Saint Vincent and Grenadines 1,124 13.5 54.2% Djibouti 928 14.3 40.5 Morocco 1916 14 47.9 Myanmar 2,212 13.6 50.5 Zambia 1,201 13.9 49.7 United Republic of Tanzania 1712 13 53.9 Venezuela 3,827 13.2 52.8 Grenada 1,244 13.7 57.9 Lebanon 4,415 13.6 53 China 8,313 13.7 49.6 Indonesia 2,979 13.8 50.7 Thailand 2,570 13.6 52.7 Uganda 1839 14.3 52.9 Tunisia 2,474 13.6 50.6 Botswana 1,336 14.3 54.4 Sri Lanka 2,435 13.7 50.5 India 7,120 13.9 42.5 Seychelles 1,095 13.6 50.9 Guyana 1,027 14.1 53.7 Ecuador 4,281 13.4 51.6 Jordan 1,542 14.4 54.5 Argentina 1,475 14.1 54.4 Kenya 2,694 13.9 53.4 McMartin et al., 2012 [34] Prospective cohort Canada 3,757 10–11 years old 52% Wave 1 (2003), Wave 2 (2006) FV: FFQ over the past 12 months; number of daily servings of FV Internalizing disorders that include common symptoms of depression and anxiety assessed by physician diagnosis “none of the food items and nutrients including vegetable and fruit consumption . . . showed a statistically significant association with internalizing disorders.” Park et al., 2018 [30] Cross- sectional Korea 65,528 14.99 (1.74) 48.4% N/A F, V: single-item dietary measure as part of the KYRBS how often students engaged in each dietary behaviour within the past 7 days Depression symptoms assessed by a single question: “In the past 12 months, have you ever felt depression or hopelessness severe enough to compromise your daily activities during 2 weeks or more?” “. . .healthier dietary behaviour [including frequent fruits (1 or more servings a day) and vegetables (3 or more times a day) consumption] was associated with . . . lower odds of perceived stress and depressive mood” (Continued on following page) TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). Public Health Reviews | Owned by SSPH+ | Published by Frontiers Public Health Reviews | Owned by SSPH+ | Published by Frontiers September 2022 | Volume 43 | Article 1604686 Diet and Adolescent Mental Disorders Dabravolskaj et al. TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). F&V Intakes (Measured Separately) and Depression p Eight studies (seven cross-sectional [23–25, 27–30] and one case- control [35]) reported inconsistent associations between fruit and vegetable intakes and depression symptoms. For example, the study by Liu et al. [29] that analyzed the association between depression and fruit and vegetable intakes in 25 low- and middle- income countries reported statistically significant associations. While the associations between fruit intake and depression were statistically significant for some countries (e.g., Tanzania, China, Indonesia, Thailand, India, Seychelles, Ecuador, and Jordan), they were not statistically significant for other low- and middle- income countries (Saint Lucia, Egypt, Saint Vincent and the Grenadines, Djibouti, Morocco, Myanmar, Zambia, Venezuela, Grenada, Lebanon, Uganda, Tunisia, Botswana, Sri Lanka, Guyana, Argentina, and Kenya). Associations in all listed countries were adjusted for different potential confounding factors. Liu et al. pooled data from all studies in a meta- analysis: fruit intake of <2 times and 2 or more times/day versus none was associated with 0.79 (0.73; 0.86) and 0.75 (0.68, 0.82) times lower odds of depression, respectively. Vegetable intake of <3 times/day and 3 or more times per day vs. none was associated with 0.74 (0.67; 0.83) and 0.75 (0.68; 0.84) times lower odds of depression, respectively. Quality Assessment Author(s) and publication year Study design Country Sample size Mean age or age range (at baseline if a cohort study) %females Follow-up years Exposure(s) Outcome(s) Comments and conclusions Winpenny et al., 2018 [32] Prospective cohort (with longitudinal and cross- sectional analysis) United Kingdom 603 14.05 (0.3) 60% Wave 1 (2005–2007), Wave 2 (3 years later) FV: 4 days diet diary, including two weekdays and two weekend days, reporting estimated portion sizes in terms of small, medium or large, household measures or as individual items Depression assessed by the Moods and Feelings Questionnaire “There were no significant associations between . . . fruit and vegetable intake . . . and depressive symptoms at baseline, nor . . . at 3- year follow up, after controlling for covariates” TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). aF, fruit intake; V, vegetable intake; FV, total fruit and vegetable intake. bYouth Risk Behaviour Survey. cABAKQ, Adolescent Behaviours, Attitudes, and Knowledge Questionnaire. dThe study by Hoare et al. [28] also included prospective cohort data with the outcome of interest being adult depression. We omitted this part due to the nature of this systematic review. Moreover, there is an overlap between data used in [27] and data used for cross-sectional analysis in [29]. Both studies are included in this systematic review given that the sample size in [27] was 800 compared to 634 in [29]. eKYRBS, Korea Youth Risk Behaviour Web-based Survey. fSaint Lucia, Egypt, Saint Vincent and Grenadines, Djibouti, Morocco, Myanmar, Zambia, United Republic of Tanzania, Venezuela, Grenada, Lebanon, China, Indonesia, Thailand, Uganda, Tunisia, Botswana, Sri Lanka, India, Seychelles, Guyana, Ecuador, Jordan, Argentina, Kenya. gGlobal School-based Health Survey. CES-D, Center for Epidemiologic Studies Depression Scale; COOPS/WONCA, Dartmouth COOP Functional Health Assessment charts/World Organization of Family Doctors; F, fruit intake, V, vegetables intake, FV, fruit and vegetables combined intake; N/A, not applicable; NR, not reported; SMFQ, Moods and Feelings Questionnaire. Global School based Health Survey. CES-D, Center for Epidemiologic Studies Depression Scale; COOPS/WONCA, Dartmouth COOP Functional Health Assessment charts/World Organization of Family Doctors; F, fruit intake, V, vegetables intake, FV, fruit and vegetables combined intake; N/A, not applicable; NR, not reported; SMFQ, Moods and Feelings Questionnaire. Quality Assessment Author(s) and publication year Study design Country Sample size Mean age or age range (at baseline if a cohort study) %females Follow-up years Exposure(s) Outcome(s) Comments and conclusions Winpenny et al., 2018 [32] Prospective cohort (with longitudinal and cross- sectional analysis) United Kingdom 603 14.05 (0.3) 60% Wave 1 (2005–2007), Wave 2 (3 years later) FV: 4 days diet diary, including two weekdays and two weekend days, reporting estimated portion sizes in terms of small, medium or large, household measures or as individual items Depression assessed by the Moods and Feelings Questionnaire “There were no significant associations between . . . fruit and vegetable intake . . . and depressive symptoms at baseline, nor . . . at 3- year follow up, after controlling for covariates” aF, fruit intake; V, vegetable intake; FV, total fruit and vegetable intake. bYouth Risk Behaviour Survey. cABAKQ, Adolescent Behaviours, Attitudes, and Knowledge Questionnaire. dThe study by Hoare et al. [28] also included prospective cohort data with the outcome of interest being adult depression. We omitted this part due to the nature of this systematic review. Moreover, there is an overlap between data used in [27] and data used for cross-sectional analysis in [29]. Both studies are included in this systematic review given that the sample size in [27] was 800 compared to 634 in [29]. eKYRBS, Korea Youth Risk Behaviour Web-based Survey. fSaint Lucia, Egypt, Saint Vincent and Grenadines, Djibouti, Morocco, Myanmar, Zambia, United Republic of Tanzania, Venezuela, Grenada, Lebanon, China, Indonesia, Thailand, Uganda, Tunisia, Botswana, Sri Lanka, India, Seychelles, Guyana, Ecuador, Jordan, Argentina, Kenya. gGlobal School-based Health Survey. CES-D, Center for Epidemiologic Studies Depression Scale; COOPS/WONCA, Dartmouth COOP Functional Health Assessment charts/World Organization of Family Doctors; F, fruit intake, V, vegetables intake, FV, fruit and vegetables combined intake; N/A, not applicable; NR, not reported; SMFQ, Moods and Feelings Questionnaire. TABLE 5 | (Continued) Description of studies included in the systematic review (systematic review, all countries, up to 2020). DISCUSSION Fruit and vegetables have long been recognized for their beneficial effects on gastrointestinal health, weight management, prevention of cardiovascular and metabolic disorders, respiratory health, and high bone mineral density, among other conditions and diseases [36]. Moreover, FVI has recently been shown to be associated with lower risk of mental disorders in the general population [18, 37]. However, our systematic review did not confirm previous claims for the existing association between FVI and CMDs specifically in youth. Among 12 identified studies, one case-control and some of the cross- sectional studies pointed to significant associations between FVI and CMDs in youth, while none of the three prospective cohort studies showed significant associations after adjusting for confounding factors. First of all, SES is an established confounder linked to both diet and mental disorders and therefore should be controlled for in all studies investigating this focal relationship; SES was measured and adjusted for in two [32, 34] out of three cohort studies included in this systematic review. At the same time, some of the variables (e.g., weight status indicators) that were treated as confounding factors could well be intermediate variables that we should not control for. Additionally, it is important to consider the nature of confounding factors (e.g., time-invariant such as ethnicity, race, sex vs. time-variant such as food security, parental mental health, family functioning), which could inform the choice of analytical methods (e.g., parametric G-formula) other than the standard regression models (e.g., linear and logistic regression models). Employing directed acyclic graphs [46] could help guide these pre-analysis steps and identify appropriate adjustment sets, minimize inappropriate adjustment, and invite external scrutiny to enhance the quality of work. Previously proposed biological mechanisms to explain the association between FVI and CMDs revolve around the high content of fiber, nutrients (e.g., vitamin C), and phytochemicals (e.g., polyphenols, carotenoids) [36] found in vegetables and fruits, which are believed to have beneficial effects on neurotransmitter systems, neuronal plasticity [38], and gut health [39–41]. Although the aforementioned biological mechanisms appear plausible and are supported by studies in adults, the effects of FVI on CMDs may differ in youth due to the rapid brain development during adolescence [42]. Another potential explanation involves one of the existing theoretical models derived from life course epidemiology—i.e., the accumulation of risk model [43]. F&V Intakes (Measured Separately) and Anxiety y Two studies [23, 29] examined the associations between fruit and vegetable intakes and anxiety. Arat [23] reported results for six of the low- and middle-income countries and found statistically significant associations in Botswana, Kenya (for fruit but not vegetable intake as the exposure of interest), Seychelles, Uganda, Tanzania, and Zambia. Another study by Liu et al. [29] used data from the same questionnaire as Arat [23], although for a narrower age range, reporting statistically significant associations between fruit intake and anxiety for Morocco, Tanzania, Venezuela China, Indonesia, Uganda, Tunisia, Sri Lanka, India, Ecuador, Jordan, Argentina, Kenya; and between vegetable intake and anxiety in Saint Vincent and the Grenadines, Djibouti, Lebanon, China, Seychelles, and Ecuador. When the September 2022 | Volume 43 | Article 1604686 Public Health Reviews | Owned by SSPH+ | Published by Frontiers 9 Diet and Adolescent Mental Disorders Dabravolskaj et al. epidemiology is warranted. Methodological challenges, discussed below, could also explain our findings. measures of association were combined in a meta-analysis, the fruit intake of <2 times/day and 2 or more times a day compared to no intake was associated with 0.60 (0.54; 0.67) and 0.61 (0.54, 0.68) times lower odds of anxiety, while vegetable intake of <3 times/day and 3 or more times a day versus no intake was associated with 0.71 (0.63; 0.81) and 0.87 (0.73; 1.03) times lower odds of having anxiety symptoms. Neither of the studies reported on the association between the combined FVI and anxiety. Consistent with other literature on the diet-mental health relationship [15, 18, 44], cross-sectional study design was the one most commonly used. While the cross-sectional study design can help generate hypotheses, in respect to the diet-mental health relationship this task has already been fulfilled. The inability to determine the temporal order of diet and mental disorders makes this study design of limited value to any etiological inferences [45]. Moreover, cross-sectional studies identified in this systematic review provided inconsistent conclusions, potentially due to adjusting for different confounding factors. As for the case-control study [35], the authors excluded those with pre-existing mental disorders, thus partly tackling the issue of reverse causality, but we cannot exclude the possibility of recall bias. Both exposure and outcome were assessed at the same time, and the study did not report whether those who completed the dietary assessment were blinded to participants’ outcomes or the research question itself. F&V Intakes (Measured Separately) and Anxiety Given the aforementioned limitations inherent to cross-sectional and case-control study designs and in the absence of prevention trials (in part due to ethical and feasibility concerns), attention and efforts should be redirected to planning and conducting rigorous prospective cohort studies. We identified three prospective cohort studies and, given the incremental nature of research, more prospective cohort studies that address the methodological issues outlined below would be of value. FVI (Combined) and Depression and Anxiety (Combined) One study [34] concluded that there was no statistically significant association between FVI and internalizing disorders when comparing second tertile to first tertile (IRR 1.04, 95% CI 0.71; 1.53) and third tertile to first tertile (IRR 1.25, 95% CI 0.8; 1.99). Analyses were adjusted for energy intake, gender, household income, parental marital status and education, body weight status, physical activity, and geographical area. Public Health Reviews | Owned by SSPH+ | Published by Frontiers REFERENCES 12. Kessler RC, Bromet EJ. The Epidemiology of Depression across Cultures. Annu Rev Public Health (2013) 34:119–38. doi:10.1146/annurev-publhealth-031912- 114409 1. National Institute of Mental Health. Mental Illness Definitions (2022). Available from: https://www.nimh.nih.gov/health/statistics/mental-illness. shtml (Accessed August 8, 2022). 13. McDaid D, Park AL, Wahlbeck K. The Economic Case for the Prevention of Mental Illness. Annu Rev Public Health (2019) 40(1):373–89. doi:10.1146/ annurev-publhealth-040617-013629 2. Vigo D, Thornicroft G, Atun R. Estimating the True Global burden of Mental Illness. Lancet Psychiatry (2016) 3(2):171–8. doi:10.1016/S2215-0366(15) 00505-2 14. O’Neil A, Quirk SE, Housden S, Brennan SL, Williams LJ, Pasco JA, et al. Relationship between Diet and Mental Health in Children and Adolescents: a Systematic Review. Am J Public Health (2014) 104(10):e31–42. doi:10.2105/ AJPH.2014.302110 3. World Health Organization. Global burden of Mental Disorders and the Need for a Comprehensive, Coordinated Response from Health and Social Sectors at the Country Level (2012). Available from: https://apps.who.int/iris/bitstream/ handle/10665/78898/A65_10-en.pdf?sequence=1&isAllowed=y (Accessed October 9, 2019). 15. Khalid S, Williams CM, Reynolds SA. Is There an Association between Diet and Depression in Children and Adolescents? A Systematic Review. Br J Nutr (2016) 116(12):2097–108. doi:10.1017/S0007114516004359 16. World Health Organization. Healthy Diet (2020). Available from: https://www. who.int/news-room/fact-sheets/detail/healthy-diet (Accessed September 20, 2021). 4. Kessler RC, Berglund P, Demler O, Jin R, Merikangas KR, Walters EE. Lifetime Prevalence and Age-Of-Onset Distributions of DSM-IV Disorders in the National Comorbidity Survey Replication. Arch Gen Psychiatry (2005) 62(6):593–602. doi:10.1001/archpsyc.62.6.593 17. Garriguet D. Diet Quality in Canada. Health Rep (2009) 20(3):41–52. 18. Saghafian F, Malmir H, Saneei P, Milajerdi A, Larijani B, Esmaillzadeh A. Fruit and Vegetable Consumption and Risk of Depression: Accumulative Evidence from an Updated Systematic Review and Meta-Analysis of Epidemiological Studies. Br J Nutr (2018) 119(10):1087–101. doi:10.1017/S0007114518000697 5. Merikangas KR, He JP, Burstein M, Swanson SA, Avenevoli S, Cui L, et al. Lifetime Prevalence of Mental Disorders in U.S. Adolescents: Results from the National Comorbidity Survey Replication--Adolescent Supplement (NCS-A). J Am Acad Child Adolesc Psychiatry (2010) 49(10):980–9. doi:10.1016/j.jaac. 2010.05.017 19. Veritas Health Innovation. Covidence Systematic Review Software. Melbourne, Australia: Veritas Health Innovation (2020). 20. Wells GA, Shea B, O’Connell D, Peterson J, Welch V, Losos M, et al. The Newcastle-Ottawa Scale (NOS) for Assessing the Quality of Nonrandomised Studies in Meta-analyses (2008). Available from: http://www.ohri.ca/ programs/clinical_epidemiology/oxford.asp (Accessed August 15, 2019). 6. Thapar A, Collishaw S, Pine DS, Thapar AK. Depression in Adolescence. Lancet (2012) 379(9820):1056–67. doi:10.1016/S0140-6736(11)60871-4 7. Johnson D, Dupuis G, Piche J, Clayborne Z, Colman I. CONFLICT OF INTEREST The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This systematic review showed that while inconsistent associations between FVI and CMDs in youth were reported in cross-sectional and case-control studies, no association was detected in prospective cohort studies. This evidence differs from what has recently been concluded in a systematic review on the association between FVI and depression in adults [18], which can be explained by the accumulation of risk theoretical model of the development of mental disorders and/or methodological challenges outlined in the paper. DISCUSSION This model states that every additional year of exposure is associated with an increased risk of poor outcomes: this could explain why the association between FVI and CMDs becomes apparent later in life. Further research looking at the diet-mental health relationship through the lens of life course In addition, there are measurement errors associated with both self-reported diet and mental disorders. Given the potential for recall and social desirability biases associated with self-report measures, sensitivity analyses to delineate the effects of measurement errors on the focal relationship are needed [47]. Adjustment for total energy intake is another strategy that has been strongly recommended to partially correct for the measurement error associated with self- reported dietary intake. Moreover, fruit juice is excluded from recent healthy eating recommendations due to excess free sugars they contain [48]; for this reason, consumption of fruit juice should not count toward FVI. In addition, validated questionnaires, as opposed to single-item September 2022 | Volume 43 | Article 1604686 Public Health Reviews | Owned by SSPH+ | Published by Frontiers 10 Dabravolskaj et al. Diet and Adolescent Mental Disorders SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.ssph-journal.org/articles/10.3389/phrs.2022.1604686/ full#supplementary-material AUTHOR CONTRIBUTIONS screener questions, should be preferred for the assessment of mental health disorders. Lastly, despite pronounced sex differences in both the prevalence of mental disorders [49] and eating behaviours and diet [50, 51], sub-group analysis was done in less than half of the included studies, and further exploration of potential effect modification is of value. JD conceptualized the idea for the systematic review and is the guarantor of the review. SC executed searches in all databases. SM was the second reviewer. All authors contributed to the initial draft and reviewed the subsequent drafts and the final version. REFERENCES Best O, Ban S. Adolescence: Physical Changes and Neurological Development. Br J Nurs (2021) 30(5):272–5. doi:10.12968/bjon.2021.30.5.272 27. Hoare E, Hockey M, Ruusunen A, Jacka FN. Does Fruit and Vegetable Consumption during Adolescence Predict Adult Depression? A Longitudinal Study of US Adolescents. Front Psychiatry (2018) 9:581. doi:10.3389/fpsyt.2018.00581 43. Evans GW, Li D, Whipple SS. Cumulative Risk and Child Development. 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September 2022 | Volume 43 | Article 1604686 Public Health Reviews | Owned by SSPH+ | Published by Frontiers PHR is edited by the Swiss School of Public Health (SSPH+) in a partnership with the Association of Schools of Public Health of the European Region (ASPHER)+ REFERENCES Six-month Prevalence of Mental Disorders and Service Contacts Among Children and Youth in Ontario: Evidence from the 2014 Ontario Child Health Study. Can J Psychiatry (2019) 64(4):246–55. doi:10.1177/0706743719830024 34. McMartin SE, Kuhle S, Colman I, Kirk SF, Veugelers PJ. Diet Quality and Mental Health in Subsequent Years Among Canadian Youth. Public Health Nutr (2012) 15(12):2253–8. doi:10.1017/S1368980012000535 50. Caine-Bish NL, Scheule B. Gender Differences in Food Preferences of School- Aged Children and Adolescents. J Sch Health (2009) 79(11):532–40. doi:10. 1111/j.1746-1561.2009.00445.x 35. Kim TH, Choiyoung J, Lee HH, Park Y. Associations between Dietary Pattern and Depression in Korean Adolescent Girls. J Pediatr Adolesc Gynecol (2015) 28(6):533–7. doi:10.1016/j.jpag.2015.04.005 51. Keller KL, Kling SMR, Fuchs B, Pearce AL, Reigh NA, Masterson T, et al. A Biopsychosocial Model of Sex Differences in Children’s Eating Behaviors. Nutrients (2019) 11(3):682. doi:10.3390/nu11030682 36. Dreher ML. Whole Fruits and Fruit Fiber Emerging Health Effects. Nutrients (2018) 10(12):E1833. doi:10.3390/nu10121833 Copyright © 2022 Dabravolskaj, Marozoff, Maximova, Campbell and Veugelers. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. 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Curr Neuropharmacol (2018) 16(5):559–73. doi:10.2174/1570159X15666170915141036 PHR is edited by the Swiss School of Public Health (SSPH+) in a partnership with the Association of Schools of Public Health of the European Region (ASPHER)+ PHR is edited by the Swiss School of Public Health (SSPH+) in a partnership with the Association of Schools of Public Health of the European Region (ASPHER)+ Public Health Reviews | Owned by SSPH+ | Published by Frontiers September 2022 | Volume 43 | Article 1604686 12
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AtELP4 a subunit of the Elongator complex in Arabidopsis, mediates cell proliferation and dorsoventral polarity during leaf morphogenesis
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AtELP4 a subunit of the Elongator complex in Arabidopsis, mediates cell proliferation and dorsoventral polarity during leaf morphogenesis Sang Eun Jun 1‡, Kiu-Hyung Cho 1†‡, Muhammad Aamir Manzoor 2‡, Tae Young Hwang 3, Youn Soo Kim 3, Raffael Schaffrath 4 and Gyung-Tae Kim 1,3* 1Department of Molecular Genetics, Dong-A University, Busan, South Korea, 2School of Life Sciences, Anhui Agricultural University, Hefei, China, 3Graduate School of Applied Bioscience, Dong-A University, Busan, South Korea, 4Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany The Elongator complex in eukaryotes has conserved tRNA modification functions and contributes to various physiological processes such as transcriptional control, DNA replication and repair, and chromatin accessibility. ARABIDOPSIS ELONGATOR PROTEIN 4 (AtELP4) is one of the six subunits (AtELP1–AtELP6) in Arabidopsis Elongator. In addition, there is an Elongator-associated protein, DEFORMED ROOTS AND LEAVES 1 (DRL1), whose homolog in yeast (Kti12) binds tRNAs. In this study, we explored the functions of AtELP4 in plant-specific aspects such as leaf morphogenesis and evolutionarily conserved ones between yeast and Arabidopsis. ELP4 comparison between yeast and Arabidopsis revealed that plant ELP4 possesses not only a highly conserved P-loop ATPase domain but also unknown plant-specific motifs. ELP4 function is partially conserved between Arabidopsis and yeast in the growth sensitivity toward caffeine and elevated cultivation temperature. Either single Atelp4 or drl1-102 mutants and double Atelp4 drl1-102 mutants exhibited a reduction in cell proliferation and changed the adaxial–abaxial polarity of leaves. In addition, the single Atelp4 and double Atelp4 drl1-102 mutants showed remarkable downward curling at the whole part of leaf blades in contrast to wild-type leaf blades. Furthermore, our genetic study revealed that AtELP4 might epistatically act on DRL1 in the regulation of cell proliferation and dorsoventral polarity in leaves. Taken together, we suggest that AtELP4 as part of the plant Elongator complex may act upstream of a regulatory pathway for adaxial–abaxial polarity and cell proliferation during leaf development. CITATION Jun SE, Cho K-H, Manzoor MA, Hwang TY, Kim YS, Schaffrath R and Kim G-T (2022) AtELP4 a subunit of the Elongator complex in Arabidopsis, mediates cell proliferation and dorsoventral polarity during leaf morphogenesis. Front. Plant Sci. 13:1033358. doi: 10.3389/fpls.2022.1033358 COPYRIGHT © 2022 Jun, Cho, Manzoor, Hwang, Kim, Schaffrath and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. KEYWORDS AtELP4, cell proliferation, DRL1, Elongator complex, leaf polarity TYPE Original Research PUBLISHED 21 October 2022 DOI 10.3389/fpls.2022.1033358 TYPE Original Research PUBLISHED 21 October 2022 DOI 10.3389/fpls.2022.1033358 TYPE Original Research PUBLISHED 21 October 2022 DOI 10.3389/fpls.2022.1033358 Introduction coupled to tRNA modifications, in particular anticodon wobble uridine (U34) bases. The Elongator complex was originally identified as a transcription factor in yeast (S. cerevisiae) (Otero et al., 1999; Wittschieben et al., 1999). Meanwhile, however, it is widely accepted that the primary and conserved role of Elongator from yeast to plants and humans lies with tRNA modification and mRNA translation (Otero et al., 1999; Hawkes et al., 2002; Huang et al., 2005; Mehlgarten et al., 2010; Nelissen et al., 2010; Johansson et al., 2018; Nakai et al., 2019; Abbassi et al., 2020). Accordingly, the Elongator complex (Elp1–Elp6) is key to a pathway that adds 5- carboxy-methyl (cm5) groups to several tRNA anticodons (Selvadurai et al., 2014; Glatt et al., 2016; Lin et al., 2019; Dauden et al., 2019). Among others, these can be further modified to 5-methoxy-carbonyl-methyl (mcm5) groups or 2- thio derivatives (mcm5s2) thereof in concert with U34 methylase and thiolase activities (Schaffrath and Leidel, 2017; Johansson et al., 2018; Sokołowski et al., 2018). Leaf formation in plants requires intricate and multiple mechanisms to determine various factors including leaf size and shape, phyllotaxis, heteroblasty, and structural asymmetry. These mechanisms are managed through genetic programs adapted by leaf function and environmental cues during evolutionary progression. To accomplish leaf development, plants undergo dynamic sequences of regulatory processes: from loss of meristematic stem cell identity, leaf initiation, establishment of leaf polarity (adaxial–abaxial, medial–lateral, and distal–proximal axis), cytoplasmic growth, cell proliferation and expansion, endoreduplication, cell type-specific differentiation such as guard cells, vascular tissue, and trichomes, to mature leaves (Kalve et al., 2014). During adaxial/abaxialization among these processes, leaves construct the adaxial side, which is adjacent to the shoot apical meristem (SAM), the upper sun-exposed side in mature leaves, and the abaxial side, which is farther from SAM, the lower shaded side in mature leaves. Leaf adaxial/abaxialization specializes leaf external and internal structure according to their function; the adaxial portion is built up into compactly packed and chloroplast-rich cells and more trichomes for optimal light capture and defense, whereas the abaxial portion is built up into loosely packed cells and more guard cells and stoma for gas exchange. The vascular bundle has also discrimination between adaxial and abaxial sides, showing different positioning of xylem and phloem cells. Therefore, the acquisition of adaxial/abaxial identity in leaf structure is necessary for the performance of leaf function. Introduction Numerous studies have shown that the expression of class III homeodomain-leucine zipper (HD-ZIP III) genes, PAHBULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTA (REV) determines the identity of the adaxial domain (McConnell et al., 2001), while the expression of YABBY (YAB) and KANADI (KAN) gene families determines the identity of the abaxial domain (Eshed et al., 2004). In addition, the expression of adaxial- or abaxial-determinant genes is regulated not only by each other through negative feedback (Tsukaya, 2013) but also by small RNAs and auxin (Pekker et al., 2005; Alvarez et al., 2006). The Elongator complex consists of two subcomplexes, each comprising three subunits: Elp123 and Elp456 (Dauden et al., 2019). In yeast, there is an additional protein (Kti12/Tot4), which associates with Elongator (Frohloff et al., 2001; Mehlgarten et al., 2017). It binds tRNA and is essential for the tRNA modification activity, which resides in the Elongator catalytic subunit Elp3 (Svejstrup, 2007; Glatt et al., 2016; Krutyhołowa et al., 2019; Abbassi et al., 2020). Elongator components in eukaryotes are structurally conserved (Dauden et al., 2017a; Dauden et al., 2017b), and previous studies had reported that in yeast, two copies each of Elp4, Elp5, and Elp6 form a RecA-ATPase-like ring structure with roles in substrate recognition during Elongator-mediated tRNA modification (Glatt et al., 2012; Glatt et al., 2016; Dauden et al., 2017a; Dauden et al., 2017b). Despite their similarity in structure and tRNA modification activity, Elongator complexes from yeast, mammals, and plants may functionally differ in various physiological processes and contexts (Nelissen et al., 2005; Close et al., 2006; Chen et al., 2009; Nelissen et al., 2010; Nakai et al., 2019). Previously, we isolated and characterized Arabidopsis DEFORMED ROOTS AND LEAVES 1 (DRL1), a gene homologous to yeast Kluyveromyces lactis TOXIN INSENSITIVE 12/TOXIN TARGET 4 (KTI12/TOT4), which encodes an Elongator-associated tRNA binding protein (Mehlgarten et al., 2017; Krutyhołowa et al., 2019). DRL1 shows a partially conserved function to yeast Kti12 and is involved in the regulation of leaf polarity and cell proliferation of leaves of Arabidopsis thaliana. In this study, we characterized AtELP4 functions. Although we could not detect binding between AtELP4 and DRL1 or other individual plant Elongator subunits and DRL1, we found that an Atelp4 mutant showed narrow and abaxialized leaves very similar to those of a drl1-102 mutant. AtELP4 a subunit of the Elongator complex in Arabidopsis, mediates cell proliferation and dorsoventral polarity during leaf morphogenesis No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science 01 frontiersin.org frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 Frontiers in Plant Science Phylogeny relationship, domain, and motif analysis Phylogeny relationship, domain, and motif analysis All ELP4 full-length protein sequences from various eukaryotes identified and verified conserved domain through Pfam (https://pfam.xfam.org/) (Finn et al., 2006), NCBI Conserved Domain Database (CDD) database (https://www. ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), and SMART tool (http://smart.emblheidelberg.de) (Letunic et al., 2012). In addition, identified ELP4 genes were aligned using ClustalX software with default parameters (Thompson et al., 1997). Finally, the phylogeny was constructed by using the neighbor- joining method (NJM). The amino acid sequences of ELP4 proteins used in the alignment were retrieved from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/). The GenBank accession numbers of the amino acid sequences are A. thaliana (Q9C778.1), Ostreococcus tauri (XP_003082942.1), Marchantia polymorpha subsp. ruderalis (BBN04784.1), Physcomitrella patens (XP_024385693.1), Selaginella moellendorffii (XP_024534079.1), Amborella trichopoda (XP_020520905.1), Nymphaea colorata (XP_031482638.1), Zea mays (NP_001131483.1), Sorghum bicolor (XP_021304923.1), O. sativa Japonica Group (XP_015643899.1), Brachypodium distachyon (XP_003563291.1), Phalaenopsis equestris (XP_020577487.1), Macadamia integrifolia (XP_042486942.1), Rosa chinensis (XP_040364853.1), Glycine max (KAH1257688.1), Medicago truncatula (XP_013443887.1), Gossypium hirsutum (XP_016734795.1), Hibiscus syriacus (KAE8729835.1), Vitis vinifera (RVW57326.1), Helianthus a n n u u s ( X P _ 0 3 5 8 3 2 5 6 6 . 1 ) , M a n i h o t e s c u l e n t a (XP_021624621.1), Cucumis sativus (XP_031743242.1), Ipomoea nil (XP_019185341.1), Nicotiana tabacum (XP_016448924.1), Brassica napus (XP_022558390.1), Salvia hispanica (XP_047969651.1), Solanum lycopersicum ( X P _ 0 2 58 8 38 5 8. 1) , S. cere visiae ( N P _ 0 1 52 2 4) , Schizosaccharomyces pombe (Q9USP1), K. lactis (XP_455048), Dictyostelium discoideum (Q54XS0.1), Hydra vulgaris (XP_047135159.1), Caenorhabditis elegans (NP_001370473.1), Strongylocentrotus purpuratus (XP_030844920.1), Aplysia californica (XP_005105912.1), Drosophila melanogaster (Q9VMQ7), Xenopus laevis (NP_001088363.1), Danio rerio (NP_001017638.1), Mus musculus (NP_076365.2), and Homo sapiens (KAI2559206.1). Subsequently, conserved motifs of ELP4 in plants, yeasts, and animals were identified by using the MEME online tool (http://meme-suite.org/tools/meme) with the other default parameters and five maximum numbers of motifs (Bailey et al., 2015). Cis-acting elements and gene ontology annotation analysis The promoter sequences (ATG start codon with 1,500 bp) of ELP4 genes were estimated through the online PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/ plantcare/html/) (Lescot et al., 2002). CELLO2GO was used to identify the gene ontology (GO) annotations of the whole ELP4 genes. The CELLO platform (http://cello.life.nctu.edu.tw/) uses hierarchical vocabularies to connect genes and GO terms. Functional enrichment analysis of ELP4 genes was carried out by using DAVID 6.8 (http://david.ncifcrf.gov/) online tools. The gene ontology enrichment was categorized into three main groups: biological process (BP), cellular component (CC), and molecular function (MF). Materials and methods Phylogeny relationship, domain, and motif analysis Materials and methods Yeast strains, media, and general methods Yeast strains were from S. cerevisiae wild-type BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0) and ELP4/KTI9/TOT7 deletion mutant Y02150 (As BY4741, but elp4D) (Brachmann et al., 1998). At 30°C, yeast strains were consistently cultivated on standard rich (YPD) and minimal (SC minimal) growth media. For the test of thermosensitivity and caffeine sensitivity, yeast stains grew on YPD media with or without caffeine at 30°C or 38°C. Introduction We suggest that AtELP4 as part of the Elongator complex in Arabidopsis plays critical roles in the The growth imbalance between the adaxial side and the abaxial side causes defects in leaf flatness, and leaf epinasty or hyponasty (upward or downward curling, respectively) (Sandalio et al., 2016). These morphological changes are triggered by alteration in auxin accumulation or gradient (Enders and Strader, 2015). However, it is not well known how auxin controls the leaf flatness or the growth difference between the adaxial/abaxial sides. Apart from the differential transcription of protein-encoding genes, an emerging mechanism to regulate protein levels is translational modulation. The latter involves the modification of tunable mRNA transcripts (MoTT) with their decoding Frontiers in Plant Science 02 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 Syntenic relationship analysis establishment of leaf polarity, especially adaxialization through a collaboration with DRL1 and regulation of leaf cell proliferation. Sequence similarity of ELP4 genes between the plant and yeast was carried through circoletto software (http://tools.bat. infspire.org/circoletto/) (Darzentas, 2010). ELP4 gene sequences were used as queries against plants and yeast. The E-value was maintained constant at 1 × 10−10 and displays the sequences that produced hits based on percentage (%) identity (blue color 70%, green color 80%, orange color 90%, and red color 100%) with other default parameters. Yeast complementation test Arabidopsis and yeast ELP4 coding regions were amplified using the EcoRI site at the 5′ end and BamHI site at the 3′ end to validate AtELP4 in yeast elp4D deletion mutant by using the following primers EcoRI-AtELP4‐for (5′‐GGA ATT CCA TGG CTG CAC CAA ACG TTC GTA G‐3′) and BamHI-AtELP4-rev (5′‐CGG GGA TCC CGT CAA AAA TCT AGT GCT CCG G‐ 3′), and DNA fragment was cloned into pBluescript SK(-) vector. The AtELP4 fragments eluted from EcoRI and BamHI digestion Frontiers in Plant Science 03 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 10.3389/fpls.2022.1033358 were subcloned into the EcoRI–BglII site of the pTU1 vector, which were overexpressed under the control of the TDH3 promoter and CMK1 terminator in the pTU1 vector. Yeast elp4D cells were transformed with pTU1 empty vector and AtELP4-harboring pTU1 using electroporation. Transformants were selected on a minimal medium lacking uracil (SD-U) (Jun et al., 2015). The NIH IMAGE software ImageJ was used to analyze the data, and the Statistical Package for the Social Sciences program (SPSS 13.0, SPSS Inc. Chicago) was used for statistical analysis. Plant materials and growth conditions Individual Arabidopsis homozygous T-DNA insertion mutants (Columbia-0 (Col-0) background) in AtELP4 (At3g11220, SALK 079193/Atelp4) and DRL1 (At1g13870, SALK 056915/drl1-102) were obtained from the Arabidopsis Biological Resource Center (ABRC; https://abrc.osu.edu). The homozygote genotype of insertion mutants was confirmed by amplification of T-DNA in T-DNA inserted genomic DNA region using T-DNA and gene-specific primers. The double mutant was germinated by crossing single mutants, isolated in the F2 progeny, and confirmed by the abovementioned PCR- based method. Photosynthesis efficiency was calculated from 10 plants by measuring chlorophyll fluorescence imaging with the Handy FluorCam fluorescence imaging equipment (Photon Systems Instruments, Brno, Czech Republic). Subsequently, Fv/Fm value was estimated from individual plants using FluorCam7 software according to the manufacturer’s instructions (Arena et al., 2020). Plants were grown in a Murashige–Skoog (MS) media containing 0.43% MS medium salt mixture, 2% sucrose, and 0.2% gellan gum (pH 6.3) and transferred to a mixture of soil, vermiculite, and pearlite (2:1:1) under long-day conditions (16-h light/8-h dark, 50–100 mE/m2/s) at 22°C. Seeds were surface- sterilized, vernalized for 3 days at 4°C, and then germinated on MS media (Jun et al., 2015). RNA isolation and qRT-PCR analysis of gene expression Total RNA was isolated from rosette leaf blades of 21-day- old plants and yeast using the RNeasy mini kit (Qiagen, Hilden, Germany), and cDNA was synthesized by reverse transcription using the Reverse TraAce-a-First strand cDNA synthesis kit (TOYOBO, Tokyo, Japan). The quantitative RT-PCR (qRT- PCR) was performed using SYBR® Premix Ex TaqTM II (TAKARA, Otsu, Japan) and Bio-Rad CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA) with three technical replicates. Primers were designed by Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) for qRT- PCR and are listed in Supplementary Table 5. PCR thermocycles were run using the following conditions: denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The normalized expression level of each gene was calculated using the DDCq method and b-tubulin (TUB4) gene for Arabidopsis, and S. cerevisiae ACTIN 1 (ScACT1) gene for yeast was used as a control. Anatomical analyses The mature third rosette leaves were collected from plants on 21 DAS and fixed with fixation solution (5% (v/v) acetic acid, 5% (v/v) formaldehyde, and 45% (v/v) ethanol in water) under a vacuum. Then, they were dehydrated and cleared as described (Jun et al., 2019). For transverse sectional analysis of leaves, the widest region of dehydrated leaves was embedded in Technovit 7100 resin (Kulzer & Co. GmbH, Wehrheim, Germany) and sliced transversely by using a microtome (Jun et al., 2019). Slices were stained with toluidine blue [1% toluidine blue in phosphate-buffered saline (PBS)], dried, and then observed. Leaf length and width were measured using from tip to base and the widest part of the leaf blade from 10 mature third rosette leaves of individual plants on 21 DAS. Palisade cells in cleared leaves and transverse sectional slices were observed by light microscopy (Axioskop2, Carl Zeiss, Germany). Cell numbers were counted in the region from mid-vein to leaf margin of the widest region in leaves, and cell size and intercellular airspace were measured from palisade cells of the central region between mid-vein and leaf margin at the widest region in leaves from six mature third rosette leaves of the individual plant on 21 DAS. Measurement of chlorophyll content and photosynthetic efficiency To estimate total chlorophyll, fresh weight 0.1 g of leaf blade was taken from 28-day-old plants and soaked in 2 ml of 90% aqueous acetone at 25°C in the dark for 12 h until being cleared by centrifugation for 1 min at 5,000 rpm. The absorbance of the supernatant was measured at wavelengths 645, 663, and 750 nm using a spectrophotometer. Chlorophyll a and b contents were estimated according to the formula of Ritchie (Ritchie, 2006). Frontiers in Plant Science frontiersin.org Phylogeny relationship, evolutionary, conserved motif, and shared plant- specific domain analysis As mentioned above, the Elongator complex was originally identified as a transcription factor in yeast (S. cerevisiae) (Otero et al., 1999; Wittschieben et al., 1999). Elongator components in eukaryotes are structurally conserved (Dauden et al., 2017a,b; Otero et al., 1999). Petrakis et al. (2005) suggested a weak, salt- labile interaction between yeast Kti12 and Elp4, although they did not prove the presence of Elp4 in the highly purified Kti12. Based on these findings, we attempted to examine the functions of AtELP4 and its genetic interaction with DRL1, a gene homologous to yeast KTI12. At first initially, we performed an amino acid alignment and phylogenetic analysis of ELP4 from various eukaryotes including yeast, human, and plants using MEGA7 software. ClustalX pairwise alignment of AtELP4 amino acid sequences with ELP4s from several species indicated that AtELP4 proteins displayed approximately 60% identity with plant ELP4 proteins, but only about 20% identity was observed with yeast or animal Elp4 proteins. In detail, AtELP4 was approximately 65% identical with other dicot plant ELP4 proteins (V. vinifera, C. sativus, G. max, and S. lycopersicum) and about 57% identical with monocot plant ELP4 proteins (O. sativa and Z. mays). Moreover, AtELP4 was just 20% identical to yeast or human Elp4 proteins (Supplementary Table 1). DNA ploidy analysis The mature third rosette leaf of plants on 21 DAS was harvested, and nuclei of leaves were extracted in Otto buffer (Otto, 1992; Doležel and Göhde, 1995) by chopping. Isolated nuclei were stained with propidium iodide (PI). Finally, DNA Frontiers in Plant Science 04 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 10.3389/fpls.2022.1033358 MEME online tool was used to uncover unknown motifs among ELP4 from plants, yeast, and animals (http://meme-suite. org/). A minimum of five motifs were used in the motif analysis along with other default parameters. A phylogenetic tree was built in order to better comprehend the makeup of the 27 plants, 3 yeast, and 10 animal species. The phylogeny was classified into three groups (plants, animals, and yeast) (Figure 1). We investigated the conserved motif distribution on ELP4. ELP4 proteins in the three groups share the same conserved motifs, which supports the phylogenetic tree conclusions (Figure 1). However, ELP4 proteins contain a variety of conserved motifs in each group. Motif 1 was identified as conserved motifs and could be traced in all groups (plant, animal, and yeast). Motifs 2 and 3 were found in plants and animals, not in yeast, showing specific features in multicellular organisms. Interestingly, motif 4 and motif 5 were present only in ELP4 of plants, indicating that these families have some unique evolutionary processes and functionalities (Figure 1 and Supplementary Table 2). These results of phylogenic analysis and conserved motif search revealed that ELP4 might play not only representative roles but also species-specific roles according to organisms, although ELP4 homologs are evolutionarily conserved. ploidy was analyzed with the flow cytometer, Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). ploidy was analyzed with the flow cytometer, Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Syntenic relationship between plant and yeast We evaluate the collinearity relationship among plant and yeast species. The 27 plants (A. trichopoda, A. thaliana, B. distachyon, B. napus, C. sativus, G. max, G. hirsutum, H. annuus, H. syriacus, M. integrifolia, M. esculenta, M. polymorpha subsp ruderalis, M. truncatula, N. tabacum, N. colorata, O. sativa, P. equestris, P. patens, R. chinensis, S. hispanica, S. moellendorffii, S. lycopersicum, S. bicolor, V. vinifera, Z. mays, O. tauri, and I. nil) and three yeast (S. pombe, S. cerevisiae, and K. lactis) were subjected to microsynteny analysis to better understand the evolutionary and origin mechanism of ELP4 genes. Synteny analysis in plants and yeast revealed a substantial association between duplication, expression, gene evolution, triplication, and function. A. thaliana gene sequence demonstrated synteny with the B. napus gene sequence, with the red color indicating that these genes have 100% similarities (Figure 2). Furthermore, yeast (S. pombe, S. cerevisiae, and K. lactis) had synteny with each other and among plants, which was identical in orange (90%) and blue (70%) colors (Figure 2). ELP4 genes belonging to plants are mostly connected via red lines, showing more than 100% identity among plants. As yeast is considered the progenitor of the plants, the same thing was supported by our results where some plant sequences were 70% identical to yeast genes and are shown in blue color in Figure 2. Our result suggests that there are collinearity relationships between the However, we also performed the structural analysis of ELP4 using NCBI (Conserved Domain Database) (https://www.ncbi. nlm.nih.gov/cdd) to find conserved motifs of the ELP4 from various eukaryotes. ELP4 from eukaryotes shared PAXNEB domain, which is found in the histone acetyltransferase complex of RNA polymerase II holoenzyme. Since human and mouse PAXNEB genes were identified as yeast ELP4 homolog due to their significant homology (Winkler et al., 2001); PAXNEB domain (accession no. PF05625, https://www.ebi.ac.uk/interpro/entry/ pfam/PF05625/) became a common/representative domain of an RNA polymerase II Elongator protein subunit. PAXNEB domain is known as the P-loop ATPase motif of RecA or RAD families, which catalyze recombination reactions using ATP- dependent DNA binding and DNA-dependent ATP hydrolase activities (Ponting, 2002; Nelissen et al., 2005; Glatt et al., 2012). Especially, PAXNEB domain in plant ELP4 homologs was conserved with KaiC-like domain (accession no. CD01124, https://www.ncbi.nlm.nih.gov/Structure/cdd/cd01124), which was found in KaiC, a circadian clock protein in cyanobacteria and possesses autophosphorylation activity (E-value < 0.1). Frontiers in Plant Science 05 frontiersin.org Jun et al. Cis-acting elements analysis The potential cis-acting factors were identified by using PlantCARE to better comprehend the expression control mechanism of ELP4 genes in plants and yeasts (Supplementary Table 3). These cis-acting elements were categorized mainly into three biological processes: biotic/ abiotic stress responsiveness, phytohormones, and plant growth/development. The majority of ELP4 genes included the following cis-acting elements: ABRE, AAGAA-motif, TCA- element, ARE, TCA, MYB, LTR, I-box, 02-site, MYB-binding site, CAT-Box, TATA-box, G-box, and Sp1 (Figure 3). Stress- related cis-elements MYB, LTRs, and ARE, which are associated with drought, low temperature, and anaerobic induction, respectively, were stress-related cis-elements that were investigated. I-box, Sp1, Myb-binding site, and G-Box were distributed across the promoter regions (responsible for light). Syntenic relationship between plant and yeast 10.3389/fpls.2022.1033358 FIGURE 1 The phylogeny and conserved motif analysis and classified into three groups: plant, yeast, and animal. ELP4 protein motif composition is represented by distinct colored boxes with appropriate motif numbers and figure legends at the top. FIGURE 1 The phylogeny and conserved motif analysis and classified into three groups: plant, yeast, and animal. ELP4 protein motif composition is represented by distinct colored boxes with appropriate motif numbers and figure legends at the top. 06 Frontiers in Plant Science frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 FIGURE 2 The circos tool was used to determine the synteny relationship of ELP4 genes between the plant and yeast. The colors blue, green, orange, and red represent ≤50%, ≤70%, ≤90%, and >90% similarity, respectively. FIGURE 2 The circos tool was used to determine the synteny relationship of ELP4 genes between the plant and yeast. The colors blue, green, orange, and red represent ≤50%, ≤70%, ≤90%, and >90% similarity, respectively. different plant and yeast gene sequences, suggesting a potential evolutionary mechanism. Phytohormone-related cis-elements, such as the TCA- element, TCA, and ABRE, were also found, which are associated with abscisic acid and salicylic acid (Figure 3 and Supplementary Table 3). O2-site (related in zinc metabolic control), AAGAA-motif (development-related), CAT-box (associated to meristem expression), and TATA-box (transcription start core promoter) are in the growth/ development category. Moreover, Sp1 and TATA-box covered the highest portion, while in phytohormonal response, ABRE contributes the maximum portion. These results indicate that ELP4 genes had versatile functionality in plants and yeast. Functional enrichment analysis The putative functions of the ELP4 protein are anticipated by utilizing GO annotation analysis. Depending on amino acid similarities, ELP4 proteins were categorized into 23 distinct classes and split into three major ontologies: cellular component, molecular function, and biological process (Supplementary Table 4). In molecular function annotation, ELP4 protein was predicted to be most functional in transferase activity (51.67%), followed by enzyme regulator Frontiers in Plant Science 07 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 activity (48.33%). In cellular component annotation, ELP4 protein was annotated with protein complex, cytoplasm, organelle, intracellular, nucleus, and cell, with 13.67% followed by nucleoplasm (12.56%) and cytosol (6.68%). Moreover, in biological process annotation, predicted ELP4 protein was annotated with cellular nitrogen compound metabolic process and biosynthetic process along with the same percentage (12.95%), while signal transduction and stress responsiveness contributed equally (11.70%). However, cellular protein modification and chromosomal organization annotated 11.28%, while embryo development, reproduction, and anatomical structure development contributed equally (5.89%). Catabolic, small molecule, and nucleobase-containing compound catabolic processes contribute minimally (1.84%), B A FIGURE 3 Cis-acting elements in ELP4 promoter of various plants and yeast. (A) Cis-acting elements with functional similarity on the upstream region of ELP4 genes are demonstrated by multiple colors. (B) In each category (plant growth development, abiotic/biotic stress, and phytohormone responsive), present cis-acting elements represent with different colors. A B FIGURE 3 Cis-acting elements in ELP4 promoter of various plants and yeast. (A) Cis-acting elements with functional similarity on the upstream region of ELP4 genes are demonstrated by multiple colors. (B) In each category (plant growth development, abiotic/biotic stress, and phytohormone responsive), present cis-acting elements represent with different colors. Frontiers in Plant Science 08 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 rescued the growth defects at 38°C (Figure 5B). However, AtELP4 expression in Y02150 did not absolutely rescue the sensitivity toward caffeine (Figure 5C). These results exhibited that the function of AtELP4 gene is partially conserved in that of yeast ELP4 gene. while the tRNA metabolic process alone contributed 4.98% (Figure 4). ELP4 functions between Arabidopsis and yeast are partially conserved To identify shared functions between yeast and Arabidopsis, we performed a complementation investigation utilizing AtELP4 protein in a yeast elp4D mutant. Yeast elp4D mutant exhibited resistance toward zymocin and g-toxin, slow cell growth, thermosensitivity above 38°C, and hypersensitivity toward caffeine (Jablonowski et al., 2001). To confirm the functional complementation of AtELP4 in yeast elp4D mutant, we constructed the yeast expression vector AtELP4 harboring pTU1 and transformed it into yeast BY4741-derived elp4D mutant, Y02150. Finally, RT-PCR and qRT-PCR were used to validate AtELP4 expression in yeast elp4D mutant (Supplementary Figure 1). Each transgenic elp4D mutant containing an empty vector and overexpressing AtELP4 (Y02150:pTU-empty #1 and Y02150:pTU-AtELP4 #2, respectively) was chosen for further investigation. Compared to the retardation of cell growth of elp4D mutant Y02150 at normal conditions (30°C), AtELP4 expression in Y02150 did not rescue to wild type, BY4741 (Figure 5A). For a complementation assay for thermosensitivity and caffeine sensitivity, yeast lines grew at 38°C or in caffeine-containing media. Even though it did not fully restore the growth rate of the wild type, Y02150 that expressed AtELP4 grew faster at 38°C than Y02150 (Figure 5B). This result indicates that AtELP4 expression in Y02150 partially Caffeine sensitivity of Arabidopsis DRL1 and AtELP4 mutations As mentioned above, ELP4 and KTI12 mutation in yeast triggered the growth defect by caffeine (Jun et al., 2015; Mehlgarten et al., 2017), and caffeine is well known as a cytokinesis inhibitor that disrupts cell plate in dividing cells of plants. To confirm the functional similarity of DRL1 and ELP4 between Arabidopsis and yeast, we observed the alteration of caffeine sensitivity by single and double ELP4 and DRL1 mutants, Atelp4 and drl1-102 in Arabidopsis. We measured the fresh weight of the plant shoot part that grew on the media containing each caffeine concentration series on 14 DAS. All plants of wild type and single and double mutants showed a decrease in fresh weight depending on caffeine concentration (Figure 5D). However, there was a variation in the extent of weight reduction between wild type and mutants. Compared to the normal condition, the fresh weight of each single and Atelp4 drl1-102 double mutants on the 0.5 mM of caffeine decreased to 70%, 55%, and 55%, whereas that of wild type decreased up to 87% (Figure 5D). The hypersensitivity toward caffeine by DRL1 and AtELP4 mutation in Arabidopsis indicates that DRL1 and FIGURE 4 GO enrichment analysis of ELP4 genes. Based on functions, these are classified into three groups (molecular functions, biological processes, and cellular components) and represented through different colors. GO, gene ontology. FIGURE 4 GO enrichment analysis of ELP4 genes. Based on functions, these are classified into three groups (molecular functions, biological processes, and cellular components) and represented through different colors. GO, gene ontology. 09 Frontiers in Plant Science frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 B C D A FIGURE 5 Complementation test of yeast elp4D mutant and caffeine sensitivity of Arabidopsis Atelp4 and drl1-102 single and double mutants. (A–C) Complementation of yeast elp4D mutant, Y02150 by Arabidopsis ELP4 expression in thermosensitivity and caffeine sensitivity. Wild-type BY4741, elp4D mutant Y02150, and the empty vector pTU1 or ELP4-driven pTU1 introduced Y02150 transgenic lines were grown in YPD media. For test of thermosensitivity, yeast lines were grown at (A) 30°C and (B) 38°C. Asterisks indicate significant differences compared to Y02150 at 20 h using a Student’s t-test (**, p-value <0.01). (C) For test of caffeine sensitivity, yeast lines were grown in YPD media containing 7.5 mM of caffeine. Yeast growth was determined by measuring optical densities at 2-h intervals over a period of 20 h using a spectrophotometer at 600 nm. Caffeine sensitivity of Arabidopsis DRL1 and AtELP4 mutations (D) Caffeine sensitivity of Arabidopsis wild type, and Atelp4, drl1-102, and Atelp4 drl1-102 mutants. Plants were grown in MS media containing caffeine concentration in the range of 0 to 5 mM for 21 DAS, and then fresh weight of plants was measured. Ten individuals in each plant were collected, and data are shown as the means with SEs. Asterisks in panels B and D indicate significant differences compared to no treatment of each plant group using a Student’s t-test (*p-value <0.05; **p-value <0.01). A B C D D C D FIGURE 5 Complementation test of yeast elp4D mutant and caffeine sensitivity of Arabidopsis Atelp4 and drl1-102 single and double mutants. (A–C) Complementation of yeast elp4D mutant, Y02150 by Arabidopsis ELP4 expression in thermosensitivity and caffeine sensitivity. Wild-type BY4741, elp4D mutant Y02150, and the empty vector pTU1 or ELP4-driven pTU1 introduced Y02150 transgenic lines were grown in YPD media. For test of thermosensitivity, yeast lines were grown at (A) 30°C and (B) 38°C. Asterisks indicate significant differences compared to Y02150 at 20 h using a Student’s t-test (**, p-value <0.01). (C) For test of caffeine sensitivity, yeast lines were grown in YPD media containing 7.5 mM of caffeine. Yeast growth was determined by measuring optical densities at 2-h intervals over a period of 20 h using a spectrophotometer at 600 nm. (D) Caffeine sensitivity of Arabidopsis wild type, and Atelp4, drl1-102, and Atelp4 drl1-102 mutants. Plants were grown in MS media containing caffeine concentration in the range of 0 to 5 mM for 21 DAS, and then fresh weight of plants was measured. Ten individuals in each plant were collected, and data are shown as the means with SEs. Asterisks in panels B and D indicate significant differences compared to no treatment of each plant group using a Student’s t-test (*p-value <0.05; **p-value <0.01). ELP4 between Arabidopsis and yeast were conserved functionally, in the viewpoint of sensitivity of caffeine. blades with severely decreased size and unclear marginal boundary between blade and petiole; Atelp4 drl1-102 double mutant had a similar phenotype in leaves compared to Atelp4 single mutant, exhibiting a severe reduction in leaf width and serration in leaf margin (Figures 6A, B). The total area of the mature third leaf of Atelp4 and drl1-102 reduced up to 54.2% and 39.8% of that of the wild type (Figure 6C). Caffeine sensitivity of Arabidopsis DRL1 and AtELP4 mutations Similar results were observed for the Atelp4 drl1-102 double mutant (39.2% of that of the wild type) (Figure 6C). The reduction in leaf width (70.8% to 59.3%) was more severe than the decrease in leaf length (82.8% to 75.1% in the mutants), relative to the wild type (Figures 6D, E). Due to more reduced leaf width than leaf length, the leaf index of Atelp4 and drl1-102 single and double mutants was higher (1.7, 1.9, and 1.8, respectively) than that of wild type (1.4) (Figure 6F), indicating narrower leaf blades in mutants. However, there was no significant difference between the three mutants. Our genetic approaches indicate that AtELP4 is epistatic to DRL1. Frontiers in Plant Science Atelp4 mutant had traits similar to drl1- 102 mutant but showed severe phenotype during leaf development We characterized Atelp4 single mutant and Atelp4 drl1-102 double mutant of Arabidopsis to study the Elongator complex’s role in leaf development. In the previous report, we described that the drl1-101 mutant formed trumpet-like and filamentous leaves, which had defects in adaxial–abaxial polarity and cell division (Cho et al., 2007). Compared with the wild type (Col-0), the drl1-102 mutant showed narrow and downward-curling (epinastic) leaf blades and an imprecise boundary between the leaf blade and petiole. Atelp4 mutant also showed narrow leaf Frontiers in Plant Science 10 frontiersin.org 10.3389/fpls.2022.1033358 Jun et al. Atelp4 and drl1-102 mutants are defective in cell proliferation and the adaxial–abaxial polarity patterning during leaf development of wild type and Atelp4 and drl1-102 single and double mutants approximately follows a lognormal distribution with an average of 1,018.0, 767.6, 524.0, and 529.6 mm2 with a standard deviation of 92.6, 189.0, 104.3, and 89.0 mm2, respectively (Figure 7F). In addition, leaves of each single and double mutant had increased intracellular airspace between palisade mesophyll cells of the adaxial subepidermal layer (Figures 7B, E). The proportion of intercellular airspace of Col-0, Atelp4, drl1-102, and Atelp4 drl1-102 was 11.1%, 17.6%, 18.5%, and 23.4% to the area of the central region between leaf mid-vein and margin in the widest part of the leaf blade, respectively, and showed an increment of intercellular airspace in the adaxial subepidermal layer in each single and double mutants (Figure 7E). In detail, intercellular airspace in drl1-102, Atelp4, and Atelp4 drl1-102 increased by 215.4%, 221.6%, and 256.1%, respectively, compared to that of the wild type (Figure 7E). These findings suggested that in mutants, the palisade’s adaxial side was transformed into an abaxial-like structure. We used paradermal images and transverse sections to investigate the cellular phenotype at the widest part of the third leaf on the 21 (DAS). This was performed to mitigate the cellular changes in the leaves (Jun et al., 2019). Leaves of drl1-102 mutant showed reductions in cell number and size, 62.0% and 75.4%, respectively, compared to those of the wild type (Figures 7C, D). Atelp4 mutant showed reduced cell number and size, 61.7% and 51.5% in Atelp4, respectively. Atelp4 drl1- 102 mutant also showed a reduction of cell number and size up to 58.2% and 52.0%, respectively, compared to the wild type (Figures 7A, C, D). These data indicated that DRL1 and AtELP4 positively regulated cell numbers in a similar way. From the viewpoint of cell proliferation and expansion, AtELP4 was epistatic to DRL1. In the transverse section, mutants exhibited downward curling at the whole leaf blade, an increase in intercellular space/distance on the adaxial side, and a larger portion of phloem tissue in vascular bundles, compared to those of the wild type (Figure 7B). Especially, at Atelp4 and drl1-102, each single and double mutant showed remarkable downward curling at the whole part of the leaf blades, whereas the wild type showed downward curling at the marginal part of the leaf blades (Figure 7B). Atelp4 and drl1-102 mutants are defective in cell proliferation and the adaxial–abaxial polarity patterning during leaf development In the distribution of palisade cell size in leaves, the drl1-102 mutant showed wider bell-shaped distribution than the wild type (Figure 7F). These results indicated that the cell size of drl1-102 leaves was more irregular than that of the wild type. However, the distribution of cell size in leaves from Atelp4 and Atelp4 drl1- 102 showed sharper bell-shaped distribution, indicating some suppression processes in cell expansion. The measured cell area B C D E F A FIGURE 6 Plant morphology of Atelp4 and drl1-102 single and double mutants. Morphology of (A) plants on 35 DAS and (B) the mature third rosette leaves on 21 DAS of wild type, and Atelp4, drl1-102, and Atelp4 drl1-102 mutants. Scale bar indicates 1 cm. (C) Total area, (D) width, (E) length, and (F) leaf index (the ratio of length to width of leaf blade) on the mature third rosette leaves in plants on 21 DAS. Leaves were independently collected from 10 plants, and data are shown as the means with SEs. Different lowercase letters in panels C–F indicate significant differences between plants, according to ANOVA and Tukey’s multiple range test (p-value <0.01). A B B B FIGURE 6 Plant morphology of Atelp4 and drl1-102 single and double mutants. Morphology of (A) plants on 35 DAS and (B) the mature third rosette leaves on 21 DAS of wild type, and Atelp4, drl1-102, and Atelp4 drl1-102 mutants. Scale bar indicates 1 cm. (C) Total area, (D) width, (E) length, and (F) leaf index (the ratio of length to width of leaf blade) on the mature third rosette leaves in plants on 21 DAS. Leaves were independently collected from 10 plants, and data are shown as the means with SEs. Different lowercase letters in panels C–F indicate significant differences between plants, according to ANOVA and Tukey’s multiple range test (p-value <0.01). 11 Frontiers in Plant Science 11 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 B C D E F A FIGURE 7 Internal morphology in leaves and statistical analysis of palisade cells from Atelp4 and drl1-102 single and double mutants. (A) Paradermal images of palisade cells in the mature third rosette leaves in wild type, Atelp4, drl1-102, and Atelp4 drl1-102 on 21 DAS. (B) Transverse section of the middle region in the mature third leaves. Scale bar = 100 mm. Atelp4 and drl1-102 mutants are defective in cell proliferation and the adaxial–abaxial polarity patterning during leaf development (C) Cell number, (D) cell size, and (E) proportion of intercellular airspace in the mature third rosette leaves on 21 DAS. (F) Area distribution of palisade cells in the central region between leaf mid-vein and margin in the widest part of leaf blade. Leaves were independently collected from six plants, and data are shown as the means with SEs. Different lowercase letters in panels (C–E) indicate significant differences between plants, according to ANOVA and Tukey’s multiple range test (p-value <0.01). B A B A C D E D E D C F F F F FIGURE 7 Internal morphology in leaves and statistical analysis of palisade cells from Atelp4 and drl1-102 single and double mutants. (A) Paradermal images of palisade cells in the mature third rosette leaves in wild type, Atelp4, drl1-102, and Atelp4 drl1-102 on 21 DAS. (B) Transverse section of the middle region in the mature third leaves. Scale bar = 100 mm. (C) Cell number, (D) cell size, and (E) proportion of intercellular airspace in the mature third rosette leaves on 21 DAS. (F) Area distribution of palisade cells in the central region between leaf mid-vein and margin in the widest part of leaf blade. Leaves were independently collected from six plants, and data are shown as the means with SEs. Different lowercase letters in panels (C–E) indicate significant differences between plants, according to ANOVA and Tukey’s multiple range test (p-value <0.01). Frontiers in Plant Science frontiersin.org Decreased chlorophyll content in Atelp4 and drl1-102 mutants (B) Photosynthesis efficiency (Fv/Fm ratio) (n = 10) measured by FluorCam, (C) phenotypes of plants, and (D) false color image of photosynthesis efficiency (Fv/Fm ratio) captured by FluorCam. The color box in panel D means photosynthesis efficiency from 0 (minimum value, blue) to 1.0 (maximum value, red). Data are shown as means of eight plants with SEs. Different lowercase letters in panels A and B indicate significant differences between plants, according to ANOVA and Tukey’s multiple range test (p-value <0.01). Decreased chlorophyll content in Atelp4 and drl1-102 mutants no variation in Fv/Fm value when compared to wild type (Figures 8B, C, D). Presumably, the residual amount of chlorophyll in mutants was sufficient to support a normal rate of photosynthesis containing light capturing. To explore further evidence of the abaxialization of leaves, we measured chlorophyll concentration and photosynthetic efficiency. Total chlorophyll in 1 mg of leaf in drl1-102, Atelp4, and Atelp4 drl1-102 mutants was reduced to 53.3%, 48.7%, and 59.5%, respectively, compared to wild type (Figure 8A). However, reduction in chlorophyll content had no effect on phenotype and photosynthesis efficiency in mutant leaves, with The statistical analysis revealed that modifications in Atelp4 mutant leaves and cells were more severe than those in drl1-102 single mutant and similar with those in Atelp4 drl1-102 double mutant. These results indicate that DRL1 might act upstream of AtELP4 in two processes: regulation of cell proliferation and adaxial–abaxial polarity establishment. Frontiers in Plant Science 12 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 B C D A FIGURE 8 Photosynthesis efficiency and chlorophyll contents of Atelp4 and drl1-102 single and double mutants. (A) Chlorophyll content. (B) Photosynthesis efficiency (Fv/Fm ratio) (n = 10) measured by FluorCam, (C) phenotypes of plants, and (D) false color image of photosynthesis efficiency (Fv/Fm ratio) captured by FluorCam. The color box in panel D means photosynthesis efficiency from 0 (minimum value, blue) to 1.0 (maximum value, red). Data are shown as means of eight plants with SEs. Different lowercase letters in panels A and B indicate significant differences between plants, according to ANOVA and Tukey’s multiple range test (p-value <0.01). B B A B A C C C D FIGURE 8 Photosynthesis efficiency and chlorophyll contents of Atelp4 and drl1-102 single and double mutants. (A) Chlorophyll content. (B) Photosynthesis efficiency (Fv/Fm ratio) (n = 10) measured by FluorCam, (C) phenotypes of plants, and (D) false color image of photosynthesis efficiency (Fv/Fm ratio) captured by FluorCam. The color box in panel D means photosynthesis efficiency from 0 (minimum value, blue) to 1.0 (maximum value, red). Data are shown as means of eight plants with SEs. Different lowercase letters in panels A and B indicate significant differences between plants, according to ANOVA and Tukey’s multiple range test (p-value <0.01). D Photosynthesis efficiency and chlorophyll contents of Atelp4 and drl1-102 single and double mutants. (A) Chlorophyll content. Frontiers in Plant Science frontiersin.org Conserved functions of ELP4 and DRL1 between Arabidopsis and yeast Elongator in eukaryotes is a highly conserved complex with tRNA modification output that contributes to multiple cellular functions (Nelissen et al., 2005; Nakai et al., 2019). Among the six subunits of the Elongator complex, ELP4 forms a RecA- ATPase-like fold that assembles with ELP5 and ELP6 into a heterohexameric ring (Glatt et al., 2012). Functionally, the yeast (Elp456)2 subcomplex specifically recognizes and interacts with tRNA (Glatt et al., 2012). Mutations of yeast Elongator subunits and its associated protein Kti12 caused growth retardation at 38° C and sensitivity to caffeine treatment (Krogan and Greenblatt 2001; Jun et al., 2015; Mehlgarten et al., 2017). Our results showed that AtELP4 expression partially rescued growth retardation at 38°C of yeast elp4D mutant (Figure 5B). In our previous study, we demonstrated that plant DRL1 expression also partially rescued growth retardation at 38°C of the yeast kti12D mutant (Jun et al., 2015), indicating that the gene functions are partially conserved between plant DRL1 and yeast KTI12. Collectively, these cross-complementation analyses suggested that AtELP4 and DRL1 of Arabidopsis were closely related in function to their yeast homologs. AtELP4 and DRL1 control the expression of genes that regulate leaf polarity and cell proliferation of leaves 2 (AS2), expression was decreased in AtELP4 mutation- dependent manner, while leaf abaxial identity specifying genes, AUXIN RESPONSE FACTOR 4 (ARF4), expression was increased in single and double mutants, compared to wild type (Supplementary Figure 2). We previously described that drl1-101 mutant (No-0 background) showed suppression of cell proliferation and alteration in adaxial–abaxial polarity establishment through RT-PCR. We also analyzed the expression level of regulating genes of cell proliferation. Atelp4 and drl1-102 showed a weak increment in CYCB1;1 and CYCD3;1 expression, which is well known as positive cell cycle markers promoting cell division, compared to wild type (Supplementary Figure 2). Because these expressions were opposite to expected results in cell proliferation in mutants, we examined the expression level of KIP-RELATED PROTEINS (KRPs), which act as a cell cycle inhibitor. Single and double mutants displayed increase in KRP1 and KRP4 transcripts, compared to the wild type (Figure 9). According to the gene expression map of Arabidopsis eFP Brower (www.bar.utoronto. ca, Winter et al., 2007), KRP1 and KRP4 were strongly expressed To verify that Atelp4 leaves underwent abaxilization in the adaxial side and inhibition of cell proliferation, we next examined the expression level of leaf polarity- or cell proliferation-relative genes by using qRT-PCR. Atelp4, drl1-102, and Atelp4 drl1-102 mutants showed higher expression levels of the YAB family and abaxialization marker, KAN genes, and lower expression levels in adaxialization marker genes, HD-ZIP III genes, PHB, PHV, and REV (Figure 9). In addition, leaf polarity specifying gene, ASYMMETRIC LEAVES Frontiers in Plant Science 13 frontiersin.org frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 FIGURE 9 Expression level of leaf polarity- and cell proliferation-relative genes in Atelp4 and drl1-102 single and double mutants. Identical amounts of total RNA isolated from 21-day-old rosette leaves of wild type (Col-0), Atelp4, drl1-102, and Atelp4 drl1-102 were subjected to qRT-PCR experiments to analyze the expression level of leaf polarity- and cell proliferation-relative genes. The expression level of analyzed genes was normalized by that of TUB4. Primer sequences of used genes are described in Supplementary Table 5. FIGURE 9 Expression level of leaf polarity- and cell proliferation-relative genes in Atelp4 and drl1-102 single and double mutants. Identical amounts of total RNA isolated from 21-day-old rosette leaves of wild type (Col-0), Atelp4, drl1-102, and Atelp4 drl1-102 were subjected to qRT-PCR experiments to analyze the expression level of leaf polarity- and cell proliferation-relative genes. AtELP4 and DRL1 control the expression of genes that regulate leaf polarity and cell proliferation of leaves The expression level of analyzed genes was normalized by that of TUB4. Primer sequences of used genes are described in Supplementary Table 5. in leaves, whereas other KRPs expressed much less than KRP1 and KRP4 in leaves. Therefore, we speculated that DRL1 and AtELP4 might take part in the control of the cell cycle through transcript regulation of cell cycle regulators. Frontiers in Plant Science frontiersin.org AtELP4 and DRL1 regulate endoreduplication processes during leaf development During the development of Arabidopsis leaves, the cell cycle for cell division and proliferation is known to transit to the endoreduplication cycle repeating DNA replication without entering mitosis (Jun et al., 2013). To understand the function of the Elongator complex on the development of mesophyll cells, we performed a DNA ploidy analysis to examine the nuclear ploidy in the third leaves of Atelp4 and drl1-102. In 21-day-old wild-type plants (Col-0), DNA ploidy distribution in leaves reached an equal portion between the amount of 2C and 4C and the amount of more than 8C. In contrast, Atelp4 and drl1-102 showed more portion of the amount of 2C and 4C than that of 8C. 2C and 4C represent the G1 and G2 phases of dividing cells, respectively, while the appearance and accumulation of 8C and more DNA ploidy represent the departure from the cell cycle and activation of endoreduplication (Supplementary Figure 3). Hence, this result indicates that endoreduplication is delayed in Atelp4 and drl1-102. In contrast, we found one unknown motif specifically in plant ELP4, although further studies are necessary to prove this motif as a plant-specific binding or catalytic site. Previous research suggested that Kti12 motifs preserved in plant Frontiers in Plant Science 14 frontiersin.org frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 suggested based on ChIP analysis that the Holo-Elongator complex did not operate as a general transcription regulator but rather was involved in the acetylation of target genes. Exactly, they reported histone H3 lysine 14 acetylation in the coding regions of auxin repressor Short Hypocotyl 2 (SHY2; called also IAA3) and auxin influx carrier LAX2 genes to be under the control of Holo-Elongator (Nelissen et al., 2010). These findings showed that Elongator-dependent chromatin remodeling may regulate leaf polarity by activating or suppressing the expression of adaxial–abaxial determining genes via the auxin signaling cascade. Our results showed a reduction in AS1 expression in Atelp4 single and Atelp4 drl1-102 double mutants, suggesting the Elongator complex affects the expression of AS1. Taken together, we suggest that the Elongator complex might act upon a regulatory pathway for the expression of adaxial or abaxial determinant genes. However, the precise mode of Elongator action within the regulation of leaf patterning remains to be shown. ortholog DRL1 may be involved in cofactor binding (Jun et al., 2015). AtELP4 and DRL1 regulate endoreduplication processes during leaf development We revealed that DRL1 is unable to fully replace the function of its yeast counterpart, Kti12, whereas hybrid domain fusion proteins between the two can be based on genetic cross- complementation analysis (Mehlgarten et al., 2017). Non- conserved plant-specific regions in DRL1 have been proposed to facilitate a selective physical or functional interaction with particular Elongator subunits from Arabidopsis. Recently, it was demonstrated that ELP4 mutation impairs tRNA binding and diminishes Elongator activity, causing neurological defects in human cells (Gaik et al., 2022). Structural studies of mammalian Elongator provided evidence that in higher eukaryotes, Elp123 and Elp456 subcomplexes differ in a cell-type specific function (Gaik et al., 2022). This may be in contrast to unicellular yeast models suggesting species- specific sequence variation in Elongator subunits as a cause of evolutionary differences between lower and higher eukaryotes. Elongator may be required for the determination of leaf flatness Our results proclaimed that drl1-102, Atelp4 single, and Atelp4 drl1-102 double mutants had defects in the flatness of leaf blades, showing narrow and epinastic leaves (downward curling/curvature of leaf blade margin to abaxial side; downward curling along to the longitudinal axes) (Figure 7B). Leaf epinasty/hyponasty (downward curling or upward curling, respectively) was caused by the coordinated anisotropy of growth including cell division and expansion in epidermal, mesophyll parenchyma, and vascular tissues in leaves (Sandalio et al., 2016). The curling of leaves is known to be caused by differences or imbalances of cell proliferation or expansion rate between epidermis or mesophyll cell layers of adaxial and abaxial sides in leaves. In our results, abaxialized in the adaxial side of leaves from Atelp4, drl1-102, and Atelp4 drl1- 102 mutants caused not only a reduction in cell number and size of palisade mesophyll cells but also an increase in intercellular airspace in the adaxial subepidermal layer, suggesting an alteration in flexibility/strength of the adaxial side (Figure 7E). Our findings show that the distribution pattern of palisade cell size in drl1-102 mutant leaves is more bell-shaped than in wild type (Figure 7F), indicating that the cell size of drl1-102 leaves was more irregular than that of wild type. In addition, leaves of Atelp4 and drl1-102 single and double mutants had increased intracellular airspace between palisade mesophyll cells of the adaxial subepidermal layer (Figure 7E). Furthermore, our results showed that loss-of-function mutation in AtELP4 or DRL1 induced not only increased expression levels of abaxial determinants, YAB and KAN families, but also decreased, expressing levels of adaxial determinants, AS2 and HD-ZIP III transcription factors, indicating that Elongator complex might regulate both the repression of the transcription of YAB and KAN families and the activation of the transcription of AS1 and HD-ZIP III genes. Nelissen et al. (2005) reported that Atelp3 mutant was abaxialized in the adaxial side of leaves, showing the narrow leaves with less, larger, and more irregularly shaped palisade cells with more intercellular spaces. Kojima et al. (2011) also reported that AtELP3 represses the expression of abaxial determinant genes, although it was unclear how AtELP3 regulates the transcription of abaxial determinant genes. These results supported our suggestion of the regulation of the dorsoventrality function by the Elongator complex. Recently, Nakai et al. (2019) reported that the tRNA modification function of Elongator is not required for the control of leaf abaxial– adaxial polarity. Frontiers in Plant Science Funding This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2021R1A2C1006572), by the Basic Science Research Program through the NRF funded by the Ministry of Education (No. 2020R1A6A1A03047729 and No. 2020R1I1A1A01054294), and by the Green Fusion Technology Program funded by Ministry of Environment. Elongator may be required for the determination of leaf flatness Consistent with the epinastic phenotype of leaves from Atelp4 and drl1-102 mutants, overexpression of YAB1 or YAB3 resulted in narrow and epinastic leaves (Siegfried et al., 1999). Loss of function in AtELP3 also showed narrow and epinastic leaves (Kojima et al., 2011). Additionally, the loss of function of AS1, AS2, or HD-ZIP III and a REV feedback regulator, ZPR overexpression, triggered downward curling with abaxialized leaves (Wenkel et al., 2007). These studies supported our suggestion that the Elongator complex may regulate leaf curling. Activation of YAB1/FIL transcription is shown to require the interaction of a specific mediator, MONOPTEROS (MP/ARF5), and SWI/SNF complex and recruitment on YAB loci at flower primordium initiation (Wu et al., 2015). (Nelissen et al. 2010) In contrast, auxin accumulation and gradients are factors controlling leaf epinasty. Exogenous auxin application or genetic alteration of auxin biosynthesis or signaling genes have been Frontiers in Plant Science 15 frontiersin.org Jun et al. Jun et al. 10.3389/fpls.2022.1033358 reported to present epinastic or hyponastic leaves by the disruption of auxin homeostasis (Keller and Van Volkenburgh, 1997; Keller and Van Volkenburgh, 1998; Kawano et al., 2003). Auxin overproduction caused by a reduction in free IAA or a decrease in auxin responsiveness causes leaf epinasty, according to genetic studies of rooty (King et al., 1995), superroot2 (Delarue et al., 1998), yucca (Zhao et al., 2001), and iaaM (Romano et al., 1995), whereas elevated auxin level or increased auxin response causes leaf hyponasty from the genetic studies of icu6 (Pérez- Pérez et al., 2010) and iamt1-D (Qin et al., 2005). iamt1-D, gain- of-function indole-3-acetic acid (IAA) carboxyl methyltransferase1 (IAMT1), converts free IAA to methyl-IAA in vitro, showing that reduction of free IAA has narrow and hyponastic leaves, whereas RNAi transgenic plants had epinastic leaves (Qin et al., 2005). individual Elongator subunits to elucidate Elongator’s diverse and versatile functions in plants. individual Elongator subunits to elucidate Elongator’s diverse and versatile functions in plants. Elongator function in tRNA modification may be required for proper leaf development Modification at the anticodon wobble position (U34) of tRNALys, tRNAGlu, and tRNAGln is in charge of fine-tuning mRNA translation efficiency and rate (Nakai et al., 2019). It is reported that the plant Elongator cooperates with two UBIQUITIN-RELATED MODIFIER 1 (URM1)-like proteins involved in sulfur modification of tRNA at U34 (Nakai et al., 2019). Lack of URM1-like proteins or AtELP3 exhibited an increase in intercellular airspace, a decrease in chlorophyll contents, and a delay of endoreduplication, suggesting the importance of U34 modification in leaf morphogenesis (Nakai et al., 2019). Similarly, our results showed that Atelp4 and drl1- 102 single and double mutants had increased intercellular airspace and decreased cell number. Atelp4 and drl1-102 mutants also showed reduced chlorophyll contents and a delay of endoreduplication in leaves (Figure 8A and Supplementary Figure 3). Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. In summary, we suggest that AtELP4 as part of the Elongator complex in Arabidopsis plays critical roles in cell proliferation, leaf flatness, and establishment of leaf polarity through a collaboration with DRL1 during leaf morphogenesis. While the tRNA modification function clearly is important for proper leaf morphogenesis, the Elongator complex may also be required to contribute to leaf development in plants. Further studies will have to await the molecular mechanism and mode of action of Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. Author contributions K-HC, RS and G-TK conceived and designed the study. SEJ, MAM, RS, and G-TK wrote the manuscript. SEJ, MAM, TYH, and YSK performed the investigation and SEJ, MAM, and G-TK analyzed data. All authors reviewed the results and approved the final version of the manuscript. Although we do not yet have direct evidence of auxin level changes or response to auxin in leaf morphogenesis in Atelp4 mutant, our results presented that Elongator might be a linker between auxin response and the establishment of leaf polarity. These complicated multiple functions enable the Elongator to play an important role in auxin signaling (Nelissen et al., 2010, Leitner et al., 2015). 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Expression level of AtELP4, DRL1, and other genes by qRT-PCR in Atelp4 and drl1-102 single and double mutants. Identical amounts of total RNA Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fpls.2022.1033358/full#supplementary-material Frontiers in Plant Science 16 frontiersin.org Jun et al. 10.3389/fpls.2022.1033358 10.3389/fpls.2022.1033358 isolated from 21-day-old seedlings of wild type (Col-0), Atelp4, drl1-102, and Atelp4 drl1-102 were subjected to qRT-PCR experiments to analyze the expression level of leaf polarity- and cell proliferation-relative genes. The expression level of analyzed genes was normalized by that of TUB4. Primer sequences of used genes are described in SUPPLEMENTARY TABLE 5. 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Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. Photosynth. Res. 89 (1), 27–41. doi: 10.1007/s11120-006-9065-9 Leitner, J., Retzer, K., Malenica, N., Bartkeviciute, R., Lucyshyn, D., Jäger, G., et al. (2015). Meta-regulation of Arabidopsis auxin responses depends on tRNA maturation. Cell Rep. 11 (4), 516–26. doi: 10.1016/j.celrep.2015.03.054 Romano, C. P., Robson, P. R. H., Smith, H., Estelle, M., and Klee, H. (1995). Transgene-mediated auxin overproduction in arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin- resistant mutants. Plant Mol. Biol. 27, 1071–1083. doi: 10.1007/BF00020881 Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., et al. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30 (1), 325– 327. doi: 10.1093/nar/30.1.325 Sandalio, L. M., Rodrı́guez-Serrano, M., and Romero-Puertas, M. C. (2016). Leaf epinasty and auxin: A biochemical and molecular overview. Plant Sci. 253, 187– 193. doi: 10.1016/j.plantsci.2016.10.002 Letunic, I., Doerks, T., and Bork, P. (2012). SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res. 40 (D1), D302–D305. doi: 10.1093/nar/gkr931 Schaffrath, R., and Leidel, S. A. (2017). Wobble uridine modifications – a reason to live, a reason to die?RNA Biol. 14, 1209–1222. doi: 10.1080/ 15476286.2017.1295204 Lin, T.-Y., Abbassi, N. E. H., Zakrzewski, K., Chramiec-Głąbik, A., Jemioła- Rzemińska, M., and Glatt, S. (2019). The elongator subunit Elp3 is a non-canonical tRNA acetyltransferase. Nat. Commun. 10, 625. doi: 10.1038/s41467-019-08579-2 Selvadurai, K., Wang, P., Seimetz, J., and Huang, R. H. (2014). Archaeal Elp3 catalyzes tRNA wobble uridine modification at C5 via a radical mechanism. Nat. Chem. Biol. 10, 810–812. doi: 10.1038/nchembio.1610 McConnell, J. R., Emery, J., Eshed, Y., Bao, N., Bowman, J., and Barton, M. K. (2001). Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411 (6838), 709–713. doi: 10.1038/35079635 Siegfried, K. R., Eshed, Y., Baum, S. F., Otsuga, D., Drews, G. N., and Bowman, J. References doi: 10.3389/fpls.2014.00362 Delarue, M., Prinsen, E., Onckelen, H. V., Caboche, M., and Bellini, C. (1998). Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis. Plant J. 14 (5), 603–611. doi: 10.1046/j.1365- 313x.1998.00163.x Kawano, N., Kawano, T., and Lapeyrie, F. (2003). Inhibition of the indole-3- acetic acid-induced epinastic curvature in tobacco leaf strips by 2,4- dichlorophenoxyacetic acid. Ann. Bot. 91 (4), 465–471. doi: 10.1093/aob/mcg043 Doležel, J., and Göhde, W. (1995). Sex determination in dioecious plants melandrium album and m. rubrum using high-resolution flow cytometry. Cytometry 19, 103–106. doi: 10.1002/cyto.990190203 Keller, C. P., and Van Volkenburgh, E. (1997). Auxin-induced epinasty of tobacco leaf tissues (A nonethylene-mediated response). Plant Physiol. 113 (2), 603–610. doi: 10.1104/pp.113.2.603 Enders, T. A., and Strader, L. C. (2015). Auxin activity: Past, present, and future. Am. J. Bot. 102 (2), 180–196. doi: 10.3732/ajb.1400285 Keller, C. P., and Van Volkenburgh, E. (1998). Evidence that auxin-induced growth of tobacco leaf tissues does not involve cell wall acidification. Plant Physiol. 118 (2), 557–564. doi: 10.1104/pp.118.2.557 Eshed, Y., Izhaki, A., Baum, S. F., Floyd, S. K., and Bowman, J. L. (2004). Asymmetric leaf development and blade expansion in Arabidopsisare mediated by KANADI and YABBY activities. Development 131, 12, 2997–3006. doi: 10.1242/ dev.01186 King, J. J., Stimart, D. P., Fisher, R. H., and Bleecker, A. B. (1995). Mutation altering auxin homeostasis and plant morphology in arabidopsis. Plant Cell 7 (12), 2023–2037. doi: 10.1105/tpc.7.12.2023 Finn, R. D., Mistry, J., Schuster-Böckler, B., Griffiths-Jones, S., Hollich, V., Lassmann, T., et al. (2006). Pfam: clans, web tools and services. Nucleic Acids Res. 34, D247–D251. doi: 10.1093/nar/gkj149 Kojima, S., Iwasaki, M., Takahashi, H., Imai, T., Matsumura, Y., Fleury, D., et al. (2011). ASYMMETRIC LEAVES1 and elongator, a histone acetyltransferase Frontiers in Plant Science 17 frontiersin.org frontiersin.org Jun et al. Jun et al. 10.3389/fpls.2022.1033358 complex, mediate the establishment of polarity in leaves of Arabidopsis thaliana. Plant Cell Physiol. 52, 1259–1273. doi: 10.1093/pcp/pcr083 the chromatin-associated Kti12 protein. J. Biol. Chem. 280, 19454–19460. doi: 10.1074/jbc.M413373200 Krogan, N. J., and Greenblatt, J. F. (2001). Characterization of a six-subunit holo-elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae. Mol. Cell Biol. 21 (23), 8203–8212. doi: 10.1128/ MCB.21.23.8203-8212.2001 Ponting, C. P. (2002). Novel domains and orthologues of eukaryotic transcription elongation factors. Nucleic Acids Res. 30, 3642–3652. References (1999). Elongator, a multisubunit component of a novel RNA polymerase II holoenzyme for transcriptional elongation. Mol. Cell 3 (1), 109–118. doi: 10.1016/ S1097-2765(00)80179-3 Winter, D., Vinegar, B., Nahal, H., Ammar, R., Wilson, G. V., and Provart, N. J. (2007). An “Electronic fluorescent pictograph” browser for exploring and analyzing large-scale biological data sets. PloS One 2, e718. doi: 10.1371/ journal.pone.0000718 Otto, F. (1992). “Preparation and staining of cells for high-resolution DNA analysis,” in Flow cytometry and cell sorting. Ed. A. Radbruch (Berlin: Springer), 101–104. doi: 10.1007/978-3-662-02785-1_8 Wittschieben, B. O., Otero, G., de Bizemont, T., Fellows, J., Erdjument-Bromage, H., Ohba, R., et al. (1999). A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol. Cell. 4, 123–128. doi: 10.1016/ s1097-2765(00)80194-x Pekker, I., Alvarez, J. P., and Eshed, Y. (2005). Auxin response factors mediate arabidopsis organ asymmetry via modulation of KANADI activity. Plant Cell 17, 2899–2910. doi: 10.1105/tpc.105.034876 Wu, M. F., Yamaguchi, N., Xiao, J., Bargmann, B., Estelle, M., Sang, Y., et al. (2015). Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate. eLife 4, e09269. doi: 10.7554/eLife.09269 Pérez-Pérez, J. M., Candela, H., Robles, P., López-Torrejón, G., del Pozo, J. C., and Micol, J. L. (2010). A role for AUXIN RESISTANT3 in the coordination of leaf growth. Plant Cell Physiol. 51 (10), 1661–1673. doi: 10.1093/pcp/pcq123 Zhao, Y., Christensen, S. K., Fankhauser, C., Cashman, J. R., Cohen, J. D., Weigel, D., et al. (2001). A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 291 (5502), 306–309. doi: 10.1126/science.291.5502.306 Petrakis, T. G., Søgaard, T. M. M., Erdjument-Bromage, H., Tempst, P., and Svejstrup, J. Q. (2005). Physical and functional interaction between elongator and Frontiers in Plant Science 18 frontiersin.org
https://openalex.org/W4287825064
https://zenodo.org/record/8120748/files/4317ijcax01.pdf
English
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TRACE LENGTH CALCULATION ON PCBS
Zenodo (CERN European Organization for Nuclear Research)
2,020
cc-by
1,897
2. PROBLEM STATEMENT Before defining the problem of trace length calculation, understanding the board terms is important. 1. INTRODUCTION The design of the Printed Circuit Board (PCB) can be done using any of the CAD tools. Computer-aided design (CAD) can be defined as the use of computer systems to assist in the creation, modification and analysis or optimization of a design. (Computer Aided Design and Manufacturing, K.Lalit et al., 2008) PCB Designer speeds designs from placement through routing till manufacturing with powerful features such as partitioning of design, RF design capabilities, and interconnection of design planning. This PCB design solution is highly scalable and production-proven to increase productivity and help the users to quickly ramp up to volume production. SKILL is the scripting language used to build its ECAD tools. KEYWORDS Trace length calculation, Board design, SKILL script, python script Shlaghya S Vasista Shlaghya S Vasista M.Tech CSE, BMS College of Engineering, India International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 ABSTRACT The design of silicon chips in every semiconductor industry involves the testing of these chips with other components on the board. The platform developed acts as power on vehicle for the silicon chips. This Printed Circuit Board design that serves as a validation platform is foundational to the semiconductor industry. The manual/repetitive design activities that accompany the development of this board must be minimized to achieve high quality, improve design efficiency, and eliminate human-errors. One of the time consuming tasks in the board design is the Trace Length matching. The paper aims to reduce the length matching time by automating it using SKILL scripts. A. Abbreviations and Acronyms • Schematics: Schematics for electronic circuits are designed by design engineers using EDA (electronic design automation) tools called as schematic capture tools. They are drawn as a series of lines and symbols representing the circuit. (Engineering Drawing and Design, David.A et al.) These apparatuses go past straightforward drawing of DOI:10.5121/ijcax.2017.4301 1 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 devices and associations. They are integrated into the IC design flow for validation and simulation of the circuit. devices and associations. They are integrated into the IC design flow for validation and simulation of the circuit. • Part number: Each of the elements on the Schematic design is associated with a part number. The schematics use logical part numbers while the physical part numbers specific to organizations are different. • Part number: Each of the elements on the Schematic design is associated with a part number. The schematics use logical part numbers while the physical part numbers specific to organizations are different. • Symbols: An electronic symbol is a pictogram used to symbolize electrical and electronic devices like wires, resistors, batteries and transistors in a schematic diagram of a circuit. These symbols can vary between countries but are mostly standardized internationally. • Footprint: A footprint or a pattern is the arrangement of pads (in surface-mount technology) or throughholes (in through-hole technology) used to electrically connect and physically attach components to PCB. The pattern on a circuit board matches the arrangement of leads on a component • Library: A central database that stores all the symbol and footprints of different boards • Part Table File (.ptf extension): CAD tool’s file format for Schematic parts • Net: A net is a conductor that interconnects 2 or more component terminals • Microstrip and Stripline length: The length of the connection on external layers of the circuit is called microstrip while the length in between the layers is called stripline length • Microstrip and Stripline length: The length of the connection on external layers of the circuit is called microstrip while the length in between the layers is called stripline length 3. PROPOSED SOLUTION The solution to this problem is the development of a SKILL script that calculates the distance between 2 components for all the nets in the board. The SKILL script starts the length calculation from point1 and stops when point2 is reached. This eliminates the inclusion of the dangling length. It also separately calculates the length of the net as microstrip and stripline. The execution of this script produces a text report that contains the net names, total lengths, micro strip and strip line lengths. The script also provides the break point in the net if there exists any in the case of a T junction. It provides separate lengths to each end point from the breakpoint observed. This will help to calculate the dangling length but does not include it in the total length calculation. B. Trace Length Calculation When a board design is in process, there are rules that need to be followed. One of the rules is that the total length of high speed signal routings must not exceed a threshold value. The micro strip and the strip line lengths must be extracted separately from the board to verify the lengths. When a connection is made from a component U1 to component U2, the connection ends with a resistor. This length is called as termination length or the dangling length. To calculate lengths, the CAD tool provides in built reports which are drawn for a given board. There is a layer by layer length report that can be extracted from this tool. This provides the To calculate lengths, the CAD tool provides in built reports which are drawn for a given board. There is a layer by layer length report that can be extracted from this tool. This provides the 2 2 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 length of each connection on multiple layers. The total length of a connection and the dangling lengths must be manually extracted from the report. length of each connection on multiple layers. The total length of a connection and the dangling lengths must be manually extracted from the report. The board design will be followed by the board tape out. At each stage of the design, it must be checked for its correctness. This means that the length reports from the CAD tool must be extracted and the manual calculations must be done each time there is a change in the board. This is a tedious task and consumes precious time during board tape outs. 4. IMPLEMENTATION In electronic design, a netlist is a description of the connectivity of an electronic circuit. A single netlist is a collection of several related lists. A much simpler definition states that a netlist consists of a list of the terminals (pins) of the electronic components in a circuit and a list of the electrical conductors that interconnect the terminals. A net is a conductor that interconnects two or more component terminals. Branches are edges of a circuit. Branches are edges of a circuit. Each branch has children. Children can be “paths”, “vias” or “pins”. A path consists of line segments or arc segments. 3 3 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 The below code snippet traverses the entire path, from Net Name till the segments. It obtains the start and end points of the segments. The line segments can be horizontal or vertical or odd as shown in the figure below. The length of horizontal and vertical segments can be calculated by subtracting the x and y locations of the end points. However the odd line length must be calculated using Pythagoras theorem. The line segments can be horizontal or vertical or odd as shown in the figure below. The length of horizontal and vertical segments can be calculated by subtracting the x and y locations of the end points. However the odd line length must be calculated using Pythagoras theorem. 4 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 The below code snippet shows the implementation of the line types. The below code snippet shows the implementation of the line types. The below code snippet shows the implementation of the line types. The length of the line segments can be easily calculate d. These segments might also contain arcs whose length might be crucial. The length of the line segments can be easily calculate d. These segments might also contain arcs whose length might be crucial. The arc length must be calculated separately as shown below in the code snippet. The arc length must be calculated separately as shown below in the code snippet. 5 5 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 5. RESULTS The SKILL code is saved with a .il extension. The code can be loaded on the PCB Editor using the command: Load(“TLC.il”) Once the script is loaded, the function name must be called to execute the script. The function accepts inputs of component 1 and 2. The Netlist file can also be provided as an input. In this solution, the netlist name and location are hardcoded in the program. The output file is created in the same location where the script and the input files are stored. The output file consists of the lengths as below. The output file is created in the same location where the script and the input files are stored. The output file consists of the lengths as below. The output file is created in the same location where the script and the input files are stored. The output file consists of the lengths as below. 6 6 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 6. CONCLUSION AND FUTURE WORK The SKILL script developed helps calculate the lengths in minutes which earlier was taking more than 1 working day. The script works significantly if the board design is accurate. It also works if the board contains vias that are misplaced at the terminating end of the connection. However, if the board contains vias that are misplaced at the connection beginning, the SKILL script fails to extract the net length as it does not find the right pin in the beginning. The way to find out the misplaced via and its length extraction when the board is not accurate is the future work of this script. ACKNOWLEDGEMENT I would like to thank Intel India Pvt Ltd for having given me the opportunity to learn and develop my skills in the area of board design and automation. Learning the CAD tool and its scripting language is an added advantage to my career. I would like to thank my manager and the guiding force behind this project, Mr.Suresh Subramanyam, Engineering Manager, Client Research and Development, Intel India Pvt Ltd. I would also like to thank my mentors Ramesh Pradeep, Intel India Pvt Ltd and C Yogasundram, Intel India Pvt Ltd for their constant guidance in the project. I would like to thank my college BMS College of Engineering for having given me the opportunity to publish this paper. My sincere thanks to Dr.B.G.Prasad, Professor and HoD, CSE, BMS College of Engineering, Bangalore. REFERENCES [1] Engineering Drawing and Design by David.A. Madsen, David.P.Madsen, 2012, P 811 [2] Computer Aided Design and Manufacturing by K.Lalit Narayan, K.Mallikarjun Rao, M.M.M.Sarcar [2] Computer Aided Design and Manufacturing by K.Lalit Narayan, K.Mallikarjun Rao, M.M.M.Sa [3] http://www.intel.com [4] http://library.intel.com 7 International Journal of Computer- Aided Technologies (IJCAx) Vol.4, No.3, July 2017 [5] https://intelpedia.intel.com [6] http://www.extremetech.com [7] https://en.wikipedia.org [8] https://www.tutorialspoint.com [7] https://en.wikipedia.org 8 8
https://openalex.org/W4283322848
https://ejournal.um-sorong.ac.id/index.php/metode/article/download/1696/934
Indonesian
null
ANALISIS TINGKAT KETELITIAN, KECEPATAN, DAN KONSTANSI KERJA PADA KARYAWAN PEMOTONG IKAN
Metode
2,022
cc-by-sa
4,115
Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Abstract Every industry needs employees as workers who carry out every activity in the company's organization and are the most important assets that have a huge influence on the success of a company. This study aims to determine the level of accuracy, speed, and work constancy of fish cutter employees. Respondents in this study were employees of the same fish cutter on the morning and evening shifts totaling 6 people. This study uses the Bourdon Wiersma Test method and data analysis using Univariate and Multivariate Analysis with the Mann Whitney Test. The results showed that there was a difference in the level of accuracy of work on fish cutter employees in the morning and evening shifts (ρ = 0.005 < 0.05). There is no difference in the level of work speed for fish cutter employees in the morning and evening shifts (ρ = 1 > 0.05). There is no difference in the level of work constant for fish cutter employees in the morning and evening shifts (ρ = 0.57 > 0.05). Factors that affect employees such as age, gender, Body Mass Index, and years of service are not too dominant, because the number of employees is still small in fish slaughter, which is 6 people in each work shift. Keywords: Shift Work. Level of Accuracy, Speed, and Consistency of Work. Bourdon Wiersma Test. Keywords: Shift Work. Level of Accuracy, Speed, and Consistency of Work. Bourdon Wiersma Test. ANALISIS TINGKAT KETELITIAN, KECEPATAN, DAN KONSTANSI KERJA PADA KARYAWAN PEMOTONG IKAN (PT. CITRA RAJA AMPAT CANNING) Irman Amri1, Sanny Hahury2, Muhammad Fandi Marsulan3 123)Jurusan Teknik Industri, Fakultas Teknik, Universitas Muhammadiyah Sorong Jl. Pendidikan No 27 Malaingkedi Kota Sorong, Papua Barat. Telp : (0951)322383/Fax : (0951)326162 Korespondensi Penulis, E-mail : irmanamri@um-sorong.ac.id, sanny0557@gmail.com, fmarsulan@gmail.com Abstrak Setiap industri membutuhkan karyawan sebagai tenaga kerja yang menjalankan setiap aktivitas yang ada dalam organisasi perusahaan dan merupakan asset terpenting yang memiliki pengaruh sangat besar terhadap kesuksesan sebuah perusahaan. Penelitian ini bertujuan untuk mengetahui tingkat ketelitian, kecepatan, dan konstansi kerja pada karyawan pemotong ikan. Responden dalam penelitian ini adalah karyawan pemotong ikan yang sama pada shift pagi dan malam berjumlah 6 orang. Penelitian ini menggunakan metode Bourdon Wiersma Test dan analisis data menggunakan Analisis Univariat dan Multivariat dengan Uji Mann Whitney. Hasil penelitian menunjukkan ada perbedaan terhadap tingkat ketelitian kerja pada karyawan pemotong ikan shift pagi dan malam (ρ = 0.005 < 0.05). Tidak ada perbedaan tingkat kecepatan kerja pada karyawan pemotong ikan shift pagi dan malam (ρ = 1 > 0.05). Tidak ada perbedaan tingkat konstansi kerja pada karyawan pemotong ikan shift pagi dan malam (ρ = 0.57 > 0.05). Faktor – faktor yang mempengaruhi karyawan seperti usia, jenis kelamin, Indeks Massa Tubuh, dan masa kerja tidak terlalu dominan, karena jumlah karyawan masih sedikit pada pemotongan ikan, yaitu berjumlah 6 orang pada setiap shift kerja. Kata Kunci : Shift Kerja. Tingkat Ketelitian, Kecepatan, dan Konstansi Kerja. Bourdon Wiersma Test. Keywords: Shift Work. Level of Accuracy, Speed, and Consistency of Work. Bourdon Wiersma Test. 2.1 Tahap Pengolahan Data Langkah-langkah dalam pengolahan data sebagai berikut : Langkah-langkah dalam pengolahan data sebagai berikut : 1. Scoring, Yaitu dilakukan dengan pemberian skor pada setiap item jawaban dari variabel – variabel yang diteliti Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Data yang terkumpul kemudian dijadikan sebagai landasan teori dari penelitian ini. 1. Pendahuluan setengah jadi atau barang jadi menjadi barang yang bermutu tinggi dalam penggunaannya, termasuk kegiatan rancang bangun dan perekayasaan industri. Salah satu industri Industri adalah kegiatan ekonomi yang mengolah bahan mentah, bahan baku, barang 21 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 karyawan yang bekerja pada tahap pemotongan ikan baik shift pagi dan malam. Pada tahap ini ada 6 karyawan yang sama dan dibagi waktu kerja pada pagi hari pukul 08.00 – 16.00 dan malam hari pukul 22.00 – 06.00. manufaktur yang berada di Kota Sorong yaitu PT. Citra Raja Ampat Canning. PT. Citra Raja Ampat Canning merupakan perusahaan yang bergerak di bidang industri pengalengan dengan jenis produk ikan kaleng yang beroperasi di Kota Sorong. Di dalam proses produksi ikan kaleng memiliki tahap dari penyortiran, penyimpanan bahan baku, pemotongan, processing dan penyimpanan bahan baku. Masing-masing tahap memiliki karyawan yang bekerja. Dari hasil penelitian terdahulu yang penulis lakukan terdapat beberapa karyawan kurang teliti dalam pembersihan sirip dan organ ikan pada shift malam. Terkait kecepatan dalam menyelesaikan pekerjaan dari kedua shift durasi kerja tidak ada perbedaan, tetapi terdapat 4 jam nganggur pada shift malam Penulis ingin menganalisis tingkat ketelitian, kecepatan, dan konstansi kerja sehingga pekerjaan terlihat terburu – buru. Oleh sebab itu, penulis tertarik untuk mengangkat masalah mengenai tingkat ketelitian, kecepatan dan konstansi kerja pada karyawan pemotong ikan pada shift pagi dan shift malam. terhadap objek yang diteliti yaitu karyawan pemotong ikan dengan orang yang sama pada shift pagi dan malam dengan jumlah 6 orang menggunakan Bourdon Wiersma Test terhadap objek yang diteliti yaitu karyawan pemotong ikan dengan orang yang sama pada shift pagi dan malam dengan jumlah 6 orang menggunakan Bourdon Wiersma Test terhadap objek yang diteliti yaitu karyawan pemotong ikan dengan orang yang sama pada shift pagi dan malam dengan jumlah 6 orang menggunakan Bourdon Wiersma Test 2. Wawancara, Data yang terkumpul merupakan data pendukung atau data sekunder. Indikator dalam wawancara yaitu a. Usia Dari uraian masalah yang telah dikumpulkan maka peneliti mencoba mengangkat hal ini yang sebagai kajian dengan mengambil judul “Analisis Tingkat Ketelitian, Kecepatan, dan Konstansi Karyawan Pemotong Ikan Terhadap Shift Kerja (PT. Citra Raja Ampat Canning).” b. Jenis kelamin c. Durasi Kerja d. Indeks Massa Tubuh e. Masa kerja d. Indeks Massa Tubuh 3. Studi Kepustakaan, Pengumpulan data dari berbagai literatur dan referensi buku yang ada kaitannya dengan masalah yang diteliti. Data yang terkumpul kemudian dijadikan sebagai landasan teori dari penelitian ini. Tujuan dari penelitian ini adalah untuk mengetahui berapa tingkat ketelitian, kecepatan, dan konstansi pada karyawan pemotong ikan di PT. Citra Raja Ampat Canning dan mengetahui faktor-faktor dominan yang menyebabkan tingkat ketelitian, kecepatan, dan konstansi kerja pada karyawan pemotong ikan di PT. Citra Raja Ampat Canning. 2. Metode Penelitian 2. Coding, merupakan pemberian kode – kode untuk menambahkan proses pengolahan data Tahapan yang dilaksanakan dalam penelitian ini terdiri dari : Tahapan yang dilaksanakan dalam penelitian ini terdiri dari : 1. Observasi, Pengumpulan data dilakukan dengan pengamatan langsung dilapangan 1. Observasi, Pengumpulan data dilakukan dengan pengamatan langsung dilapangan 22 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 3. Entry, memasukkan data pada komputer untuk diolah menggunakan software Microsoft Excel setelah selesai melakukan pemberian kode-kode. Data yang dimasukkan meliputi shift kerja, ketelitian, kecepatan, konstansi, usia, IMT, jenis kelamin, dan masa kerja. 3. Entry, memasukkan data pada komputer untuk diolah menggunakan software Microsoft Excel setelah selesai melakukan pemberian kode-kode. Data yang dimasukkan meliputi shift kerja, ketelitian, kecepatan, konstansi, usia, IMT, jenis kelamin, dan masa kerja. 2. Analisis Multivariat, Merupakan analisis terhadap banyak variabel atau memiliki lebih dari dua variabel yang merupakan pengembangan yang dilakukan secara bersamaan. Analisis multivariate dilakukan untuk mengetahui perbedaan antara variable bebas yaitu shift kerja dengan variable terikat yaitu tingkat ketelitian, kecepatan, dan konstansi kerja. Analisis data menggunakan perangkat lunak computer dengan uji statistic Mann Whitney dengan taraf kepercayaan 95%. 4. Tabulating, yaitu pengelompokan data sesuai dengan variabel yang diteliti guna mempermudah analisis data. 4. Tabulating, yaitu pengelompokan data sesuai dengan variabel yang diteliti guna mempermudah analisis data. Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 20 Tabel 1. Scoring dan Coding Variabel Independent dan Dependent Variabel Independent dan Dependent Scoring Coding Shift Kerja Shift Pagi 1 Shift Malam 2 Tingkat Ketelitian Baik (1) 1 Cukup Baik (2 - 3) 2 Cukup (4 - 12) 3 Ragu - ragu (13 - 31) 4 Kurang (32 - >59) 5 Tingkat Kecepatan Baik (0 - 9.6") 1 Cukup Baik (9.7 - 11.1") 2 Cukup (11.2 - 14.6") 3 Ragu - ragu (14.7 - 20.0") 4 Kurang (20.1 - >25.5") 5 Tingkat Konstansi Baik (0 - 1.9") 1 Cukup Baik (2.0 - 3.2") 2 Cukup (3.3 - 6.7") 3 Ragu - ragu (6.8 - 15.0") 4 Kurang (15.1 - >26.0") 5 Tabel 2. Scoring dan Coding Variabel Kategori Responden Kategori Responden Scoring Coding Usia Remaja Akhir (17 - 25 Tahun) 1 Dewasa Awal (26 - 35 Tahun) 2 Dewasa Akhir (36 - 45 Tahun) 3 Lansia Awal (46 - 55 Tahun) 4 Lansia Akhir (56 - 65 Tahun) 5 JenisKelamin Laki – laki 1 Perempuan 2 IMT Underweight (< 18.5) 1 Normal (18.5 - 25.0) 2 Overweight (> 25.0) 3 MasaKerja < 5 Tahun 1 > 5 Tahun 2 Tabel 1. Scoring dan Coding Variabel Independent dan Dependent Variabel Independent dan Dependent Scoring Coding Shift Kerja Shift Pagi 1 Shift Malam 2 Tingkat Ketelitian Baik (1) 1 Cukup Baik (2 - 3) 2 Cukup (4 - 12) 3 Ragu - ragu (13 - 31) 4 Kurang (32 - >59) 5 Tingkat Kecepatan Baik (0 - 9.6") 1 Cukup Baik (9.7 - 11.1") 2 Cukup (11.2 - 14.6") 3 Ragu - ragu (14.7 - 20.0") 4 Kurang (20.1 - >25.5") 5 Tingkat Konstansi Baik (0 - 1.9") 1 Cukup Baik (2.0 - 3.2") 2 Cukup (3.3 - 6.7") 3 Ragu - ragu (6.8 - 15.0") 4 Kurang (15.1 - >26.0") 5 Tabel 2. Scoring dan Coding Variabel Kategori Responden Kategori Responden Scoring Coding Usia Remaja Akhir (17 - 25 Tahun) 1 Dewasa Awal (26 - 35 Tahun) 2 Dewasa Akhir (36 - 45 Tahun) 3 Lansia Awal (46 - 55 Tahun) 4 Lansia Akhir (56 - 65 Tahun) 5 JenisKelamin Laki – laki 1 Perempuan 2 IMT Underweight (< 18.5) 1 Normal (18.5 - 25.0) 2 Overweight (> 25.0) 3 MasaKerja < 5 Tahun 1 > 5 Tahun 2 Tabel 2. Scoring dan Coding Variabel Kategori Responden 22 3. Hasil dan Pembahasan Langkah – langkah dalam analisis data : 1. Analisis Univariat, Dilakukan untuk menjelaskan dan mendeskripsikan setiap variable penelitian yang disajikan dalam bentuk distribusi frekuensi ataupun presentase. Hasil dari analisis ini berupa nilai terendah, nilai tertinggi, standar deviasi, dan distribusi frekuensi yang diteliti. PT. Citra Raja Ampat Canning merupakan industri manufaktur yang bergerak dalam bidang industri pengalengan dengan hasil produksi ikan kaleng. Dalam tahap pemotongan ikan terdapat dua shift kerja, yaitu shift pagi dan malam yang masing-masing shift kerja memiliki 6 karyawan. Karyawan yang sama pada setiap shift ditahap ini dijadikan sebagai responden Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Dewasa Akhir (36 - 45 tahun) 1 16.67 Lansia Awal (45 - 56 tahun) 1 16.67 Jumlah 6 100 Standar Deviasi 1.17 Jenis Kelamin Laki – Laki 6 100 Perempuan 0 0 Jumlah 6 100 Standar Deviasi 0 Indeks Massa Tubuh Underweight 1 16.67 Normal 5 83.33 Overweight 0 0 Jumlah 6 100 Standar Deviasi 0.41 Masa Kerja < 5 tahun 3 50 > 5 tahun 3 50 Jumlah 6 100 Standar Deviasi 0.55 Dari hasil penelitian distribusi frekuensi responden dapat dilihat pada Tabel 4 distribusi frekuensi karakteristik responden menurut usia dalam kategori remaja akhir dan dewasa awal yaitu masing – masing sebanyak 2 orang (33.33%). Responden yang berjenis kelamin laki – laki sebanyak 6 orang (100%). Untuk Indeks Massa Tubuh (IMT) yang dihitung berdasarkan Berat Badan (BB) responden banding Tinggi Badan (TB2) dalam meter (m) berada pada kategori normal sebanyak 5 orang (83.33%). Frekuensi responden masa kerja <5 tahun sebanyak 3 orang (50%), sedangkan masa kerja >5 tahun sebanyak 3 orang (50%). Dalam tes ini yang dinilai yaitu 1) Ketelitian adalah jumlah kesalahan kerja (banyaknya kelompok 4 titik yang terlewati atau salah mencoret); 2) Kecepatan yaitu waktu rata-rata (mean) yang dipakai oleh 25 baris (dari baris ke-3 sampai baris ke-27); 3)Konstansi yaitu perbandingan rasio antara jumlah kuadrat dari deviasi dan waktu rata-rata. Makin kecil perbedaan antara jumlah kuadrat dari deviasi dan waktu rata-rata, makin konstan hasil kerja seseorang. Sebaliknya, makin besar perbedaan antara jumlah kuadrat dari deviasi dan waktu rata-rata , makin tidak konstan hasil kerjanya. 1. Menghitung Ketelitian Untuk menghitung ketelitian diperiksa setaip baris kelompok 4 tititk yang dilompati atau salah mencoret yang dicoret bukan kelompok 4 titik. Jumlah kesalahan menetukan ketelitian. 1. Menghitung Ketelitian Untuk menghitung ketelitian diperiksa setaip baris kelompok 4 tititk yang dilompati atau salah mencoret yang dicoret bukan kelompok 4 titik. Jumlah kesalahan menetukan ketelitian. 3.1 Karakteristik Responden Tabel 3. Karakteristik Responden Tabel 3. Karakteristik Responden No Nama Umur Kategori Umur IMT Jenis Kelamin Masa Kerja 1 Ahmad Mustari 36 tahun Dewasa Akhir 23.18 kg/m2 Pria 6 Tahun 2 Suraya 47 tahun Lansia Awal 21.1 kg/m2 Pria 8 Tahun 3 Edmon 27 tahun Dewasa Awal 17.56 kg/m2 Pria 5 Tahun 4 Gilbert 20 tahun Remaja Akhir 22.63 kg/m2 Pria 1 Tahun 5 Frandeil Latuloma 19 tahun Remaja Akhir 19.4 kg/m2 Pria 1 Tahun 6 Jhon Charles 27 tahun Dewasa Awal 18.91 kg/m2 Pria 3 Tahun Tabel 4. Distribusi Frekuensi Karakteristik Responden Karakteristik Responden Distribusi Frekuensi N Presentase (%) Usia Remaja Akhir (17 - 25 tahun) 2 33.33 Dewasa Awal (26 - 35 tahun) 2 33.33 Tabel 3. Karakteristik Responden Tabel 4. Distribusi Frekuensi Karakteristik Responden Karakteristik Responden Distribusi Frekuensi N Presentase (%) Usia Remaja Akhir (17 - 25 tahun) 2 33.33 Dewasa Awal (26 - 35 tahun) 2 33.33 Tabel 4. Distribusi Frekuensi Karakteristik Responden 23 3.1 Tingkat Ketelitian, Kecepatan, dan Konstansi 2. Menghitung Kecepatan Mean = ∑fX N . . . . . . . . . . . . . . . . . . .(1) 3. Menghitung Konstansi Konstansi = ∑fX2 M . . . . . . . . . . . . . (2) Tes Bourdon Wiersma salah satu tes kognitif yang dikembangkan pada tahun 1982, merupakan tes objektif dari kelelahan. Tes ini dipakai untuk mengevaluasi konsentrasi, kecepatan bekerja dalam tugas yang monoton, ketelitian kerja, dan daya tahan saat bekerja. 24 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Tabel 5. Klasifikasi Variabel Independent dan Dependent Variabel Independent dan Dependent Klasifikasi Variabel Shift Kerja Shift Pagi Shift Malam Tingkat Ketelitian Baik (1) Cukup Baik (2 - 3) Cukup (4 - 12) Ragu - ragu (13 - 31) Kurang (32 - >59) Tingkat Kecepatan Baik (0 - 9.6") Cukup Baik (9.7 - 11.1") Cukup (11.2 - 14.6") Ragu - ragu (14.7 - 20.0") Kurang (20.1 - >25.5") Tingkat Konstansi Baik (0 - 1.9") Cukup Baik (2.0 - 3.2") Cukup (3.3 - 6.7") Ragu - ragu (6.8 - 15.0") Kurang (15.1 - >26.0") Tabel 5. Klasifikasi Variabel Independent dan Dependent Variabel Independent dan Dependent Klasifikasi Variabel Shift Kerja Shift Pagi Shift Malam Tingkat Ketelitian Baik (1) Cukup Baik (2 - 3) Cukup (4 - 12) Ragu - ragu (13 - 31) Kurang (32 - >59) Tingkat Kecepatan Baik (0 - 9.6") Cukup Baik (9.7 - 11.1") Cukup (11.2 - 14.6") Ragu - ragu (14.7 - 20.0") Kurang (20.1 - >25.5") Tingkat Konstansi Baik (0 - 1.9") Cukup Baik (2.0 - 3.2") Cukup (3.3 - 6.7") Ragu - ragu (6.8 - 15.0") Kurang (15.1 - >26.0") Tabel 5. Klasifikasi Variabel Independent dan Dependent Tabel 6. Tingkat Ketelitian, Kecepatan, dan Konstansi Pada Karyawan Shift Pagi No Nama Ketelitian Kecepatan Konstansi 1 Ahmad Mustari 2 7.96" 10.6" 2 Suraya 8 8.2" 12.4" 3 Edmon 5 9.6" 7.9" 4 Gilbert 4 12.9" 24.7" 5 Frandeil Latuloma 7 9.12" 31.0" 6 Jhon Charles 7 9.16" 20.6" Tabel 6. Tingkat Ketelitian, Kecepatan, dan Konstansi Pada Karyawan Shift Pagi Tabel 7. Tingkat Ketelitian, Kecepatan, dan Konstansi Pada Karyawan Shift Malam No Nama Ketelitian Kecepatan Konstansi 1 Ahmad Mustari 14 11.12" 11.9" 2 Suraya 30 9.88" 29.8" Tabel 7. Tingkat Ketelitian, Kecepatan, dan Konstansi Pada Karyawan Shift Malam No Nama Ketelitian Kecepatan Konstansi 1 Ahmad Mustari 14 11.12" 11.9" 2 Suraya 30 9.88" 29.8" Tabel 7. Tingkat Ketelitian, Kecepatan, dan Konstansi Pada Karyawan Shift Malam No Nama Ketelitian Kecepatan Konstansi 1 Ahmad Mustari 14 11.12" 11.9" 2 Suraya 30 9.88" 29.8" 25 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 3 Edmon 19 9.56" 9.5” 4 Gilbert 11 9.96" 20.4" 5 Frandeil Latuloma 27 8.12" 19.5" 6 Jhon Charles 21 9.4" 38.1" 3.2 Analisis Univariat 3.2 Analisis Univariat Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 malam mengalami perbedaan secara kategori, dimana shift pagi dalam kategori cukup baik berjumlah 1 orang (16,67%) dan kategori cukup berjumlah 5 orang (83,33%), sedangkan shift malam dalam kategori cukup berjumlah 1 orang (16,67%) dan kategori ragu – ragu berjumlah 5 orang (83,33%). Analisis multivariat digunakan untuk mengetahui ada atau tidaknya perbedaan antara variabel dependent (tingkat ketelitian, kecepatan, dan konstansi) dengan variabel independent (shift pagi dan malam) dengan menggunakan uji Mann Whitney dengan taraf kepercayaan 95% atau α = 0.05 a. Jika nilai p-value < 0,05 maka hasil dinyatakan signifikan, H0 ditolak. Sehingga ada perbedaan antara variabel bebas dengan variabel terikat. Untuk pengukuran tingkat kecepatan kerja pada shift pagi dan malam tidak mengalami perbedaan secara kategori. Karena kedua shift memiliki jumlah kategori yang sama, dimana kedua shift dalam kategori baik berjumlah 3 orang (50%), kategori cukup baik berjumlah 2 orang (33.33%), dan kategori cukup berjumlah 1 orang (16,67%). g b. Jika nilai p-value > 0,05 maka hasil dinyatakan tidak signifikan, H0 diterima. Sehingga tidak ada perbedaan antara variabel bebas dengan variabel terikat. 1. Tingkat Ketelitian Berdasarkan hasil Uji Mann Whitney pada Tingkat Ketelitian diketahui Nilai Probabilitas 0.005 < Nilai Taraf Kepercayaan (0.05), maka disimpulkan terdapat perbedaan Tingkat Ketelitian yang signifikan antara Shift Pagi dan Shift Malam Sedangkan pengukuran tingkat konstansi pada shift pagi dan malam hampir mengalami perbedaan secara kategori, dimanashift pagi dalam kategori ragu – ragu dan kurang yaitu sama berjumlah 3 orang (50%), sedangkan shift malam dalam kategori ragu – ragu berjumlah 2 orang (33%) dan kategori kurang berjumlah 4 orang (67%). 2. Tingkat Kecepatan Berdasarkan hasil Uji Mann Whitney pada Tingkat Kecepatan diketahui Nilai Probabilitas 1 > Nilai Taraf Kepercayaan (0.05), maka disimpulkan terdapat perbedaan Tingkat Kecepatan yang signifikan antara Shift Pagi dan Shift Malam 2. Tingkat Kecepatan Berdasarkan hasil Uji Mann Whitney pada Tingkat Kecepatan diketahui Nilai Probabilitas 1 > Nilai Taraf Kepercayaan (0.05), maka disimpulkan terdapat perbedaan Tingkat Kecepatan yang signifikan antara Shift Pagi dan Shift Malam Akan tetapi, kesimpulan dalam analisis univariat belum tentu benar karena tidak dibuktikan secara signifikan berdasar taraf kepercayaan yaitu 95% melalui analisis multivariat dengan menggunakan uji Mann- Whitney. 3.2 Analisis Univariat 3.2 Analisis Univariat Tabel 8. Analisis Univariat Tingkat Ketelitian, Kecepatan, dan Konstansi Variabel Kategori N % Shift Pagi Tingkat Ketelitian Kerja Cukup Baik 1 16.67 Cukup 5 83.33 Jumlah 100 Standar Deviasi 0.41 Tingkat Kecepatan Kerja Baik 3 50 Cukup Baik 2 33.33 Cukup 1 16.67 Jumlah 100 Standar Deviasi 0.82 Tingkat Konstansi Kerja Ragu – ragu 3 50 Kurang 3 50 Jumlah 100 Standar Deviasi 0.55 Shift Malam Tingkat Ketelitian Kerja Cukup 1 16.67 Ragu – ragu 5 83.33 Jumlah 100 Standar Deviasi 0.41 Tingkat Kecepatan Kerja Baik 3 50 Cukup Baik 2 33.33 Cukup 1 16.67 Jumlah 100 Standar Deviasi 0.82 Tingkat Konstansi Kerja Ragu – ragu 2 33 Kurang 4 67 Jumlah 100 Standar Deviasi 0.52 Tabel 8. Analisis Univariat Tabel 8. Analisis Univariat Tingkat Ketelitian, Kecepatan, dan Konstansi Hasil univariat digunakan untuk melihat distribusi frekuensi shift kerja, tingkat ketelitian, kecepatan, dan konstansi di PT. Citra Raja Ampat Canning. Berdasarkan Tabel 43 dapat diketahui bahwa hasil pengukuran tingkat ketelitian kerja pada shift pagi dan 26 2022 1. Didapatkan hasil tingkat ketelitian kerja pada shift pagi dan malam dengan p = 0.005 < 0.05, maka dapat disimpulkan bahwa tingkat ketelitian pada pekerja shift pagi dan malam ada perbedaan yang signifikan. Hasil tingkat kecepatan kerja pada shift pagi dan malam dengan p = 1 > 0.05, maka dapat disimpulkan bahwa tingkat kecepatan pada pekerja shift pagi dan malam tidak ada perbedaan yang signifikan. Sedangkan hasil tingkat konstansi kerja pada shift pagi dan malam dengan p = 0.57 > 0.05, maka dapat disimpulkan bahwa tingkat konstansi pada pekerja shift pagi dan malam tidak ada perbedaan yang signifikan baik berjumlah 2 orang (33,33%), dan cukup berjumlah 1 orang (16,67%), hal ini tidak menutup kemungkinan yang terjadi di tempat penelitian. Waktu mulai kerja antara shift pagi dan malam berbeda, hal ini menyebabkan pada shift malam kerjanya yang terburu – buru karena tidak adanya pengawasan pada shift malam. Hasil pengolahan data tingkat konstansi kerja dengan menggunakan uji Mann-Whitney antara shift pagi dan malam didapatkan p = 0,57 > 0,05 yang artinya tidak ada perbedaan untuk tingkat konstansi kerja shift pagi dan malam pada pekerja pemotongan ikan di PT. Citra Raja Ampat Canning. Hubungan tingkat konstansi pada perbedaan shift kerja selaras dengan tingkat kecepatan dan ketelitian pada setiap shift kerja. 2. Faktor – faktor yang mempengaruhi tingkat ketelitian, kecepatan, dan konstansi kerja karyawan seperti usia, jenis kelamin, Indeks Massa Tubuh, dan masa kerja tidak terlalu dominan, karena jumlah karyawan masih sedikit pada pemotongan ikan, yaitu berjumlah 6 orang pada setiap shift kerja. 3.3 Analisis Multivariat 3. Tingkat Konstansi Berdasarkan hasil Uji Mann Whitney pada Tingkat Konstansi diketahui Nilai Probabilitas 0.57 > Nilai Taraf Kepercayaan (0.05), maka disimpulkan terdapat perbedaan Tingkat Konstansi yang signifikan antara Shift Pagi dan Shift Malam 3. Tingkat Konstansi Berdasarkan hasil Uji Mann Whitney pada Tingkat Konstansi diketahui Nilai Probabilitas 0.57 > Nilai Taraf Kepercayaan (0.05), maka disimpulkan terdapat perbedaan Tingkat Konstansi yang signifikan antara Shift Pagi dan Shift Malam kategori ragu – ragu berjumlah 5 orang (83,33%) jelas sangat berbeda secara signifikan terhadap shift pagi kategori cukup baik berjumlah 5 orang (83,33%). Kurangnya tingkat ketelitian pada shift malam dipengaruhi karena kurangnya waktu libur dalam pergantian waktu shift untuk mengembalikan ritme biologis, dinginnya tangan karena pekerjaan bersifat basah dan tidak menggunakan sarung tangan karet, dan kerja yang terburu – buru. Hal ini jelas sangat berbeda dengan pekerja pada shift pagi 3.4 Perbedaan Tingkat Ketelitian, Kecepatan, dan Konstansi Kerja Terhadap Shift Kerja Hasil pengolahan data tingkat kecepatan kerja dengan menggunakan uji Mann-Whitney antara shift pagi dan malam didapatkan p = 1> 0,05, yang artinya tidak ada perbedaan untuk tingkat kecepatan kerja shift pagi dan malam pada pekerja pemotongan ikan di PT. Citra Raja Ampat Canning. Walaupun pada kedua shift memiliki kategori yang sama yaitu baik berjumlah 3 orang (50%), cukup Berdasarkan hasil pengolahan data dengan menggunakan uji Mann-Whitney antara shift pagi dan malam didapatkan p = 0,005 < 0,05, yang artinya ada perbedaan untuk tingkat ketelitian kerjashift pagi dan malam pada pekerja pemotong ikan di PT. Citra Raja Ampat Canning. Secara kategori, tingkat ketelitian pekerja pada shift malam 27 4. Kesimpulan Berdasarkan hasil pengumpulan data, pengolahan, dan analisa data yang dilakukan maka dapat disimpulkan sesuia dengan tujuan penelitian sebagai berikut : Referensi Fatoni Muhammad, Kusmindari Christofora Desi, Mellita Dina. 2019. Tingkat Kelelahan Kerja Mental Dengan Menggunakan Metode Bourdon Wiersma Terhadap Perbedaan Shift Kerja. Jurnal. Universitas Bina Darma. Palembang Bambang Prasetyo, M. Si. 2016. Pengantar Statistik Sosial. Penerbit Universitas Terbuka. Banten Bambang Prasetyo, M. Si. 2016. Pengantar Statistik Sosial. Penerbit Universitas Terbuka. Banten Diah Rahayu Wulandari. 2010. Pemakaian Alat Pelindung Diri Pekerja Di Pabrik Tahu ECO. Jurnal. Universitas Negeri Semarang. Semarang Himawan Santoso, Mira Rahayu, Ilma Mufidah. 2015. Kelelahan Kerja Menggunakan Bourdon Wiersma Untuk Mengevaluasi Kelelahan Kerja Pada Pekerja Gudang Finished Good Mengger Bandung Studi Kasus PT. Papamdayan Cocoa Industries. E–Proceeding of Engineering (3). Universitas Telkom. Bandung Dita Meireza. 2019. Analisis Sistem Kerja Shift Terhadap Tingkat Kelelahan Kerja Operator SPBU Menggunakan Metode Bourdon Wiersma. Jurnal. Universitas Diponegoro. Semarang DR. Suma’mur. P.K., MSc. 2009. Higiene Perusahaan Dan Kesehatan Kerja (Hiperkes). Penerbit Sagung Seto. Jakarta Http://m.youtube.com/watch?v=IYKBOGmPU cw Http: //www. statkomat .com Dr. Riduwan, M.B.A., M.Pddan Prof. Dr. Akdon, M.Pd. 2013. Rumusdan Data dalam Aplikasi Statistik. Penerbit Alfabeta. Bandung 28 Ridhoni Dian Rahmawati. 2020. Perbedaan Tingkat Ketelitian, Kecepatan, dan Konstansi Kerja Pada Pekerja Shift Pagi, Siang, Dan Metode Jurnal Teknik Industri Vol. 8 (1): 21-30 2022 Ir. Eko Nurmianto, M. Eng.Sc., DERT. 2008. Ergonomi, Konsep Dasar Dan Aplikasinya (Edisi Kedua). Penerbit Guna Widya. Surabaya Malam Di PT. Dan Liris Sukoharjo. Skripsi. Universitas Muhammadiyah Surakarta.Surakarta Suyanto dan Prana Ugiana Gio. 2017. Statistika Nonparametrik Dengan SPSS, MINITAB, dan R. E–Book. Penerbit USU Prees. Medan Suyanto dan Prana Ugiana Gio. 2017. Statistika Nonparametrik Dengan SPSS, MINITAB, dan R. E–Book. Penerbit USU Prees. Medan Suyanto dan Prana Ugiana Gio. 2017. Statistika Nonparametrik Dengan SPSS, MINITAB, dan R. E–Book. Penerbit USU Prees. Medan Kevin RA Joelian, Mira Rahayu, Ilma Mufidah. 2015. Pengukuran Kelelahan Kerja Menggunakan Metode Bourdon Wiersma Untuk Mengurangi Kelelahan Kerja Pada Perawat Di Rumah Sakit Paviliun Anak. E– Proceeding of Engineering (2). Universitas Telkom. Jakarta Winda Cipta Puspita Rini. 2016. Penerapan Waktu Istirahat Pendek Terhadap Produktivitas dan Kelelahan Pada Sistem Kerja Shift. Jurnal.Universitas Pembangunan Nasional Veteran.Yogyakarta Winda Cipta Puspita Rini. 2016. Penerapan Waktu Istirahat Pendek Terhadap Produktivitas dan Kelelahan Pada Sistem Kerja Shift. Jurnal.Universitas Pembangunan Nasional Veteran.Yogyakarta Ridhoni Dian Rahmawati. 2020. Perbedaan Tingkat Ketelitian, Kecepatan, dan Konstansi Kerja Pada Pekerja Shift Pagi, Siang, Dan 29
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The quest for environmental justice in China: citizen participation and the rural–urban network against Panguanying’s waste incinerator
Sustainability science
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The EJAtlas: Ecological Distribution Conflicts as Forces for Sustainability The EJAtlas: Ecological Distribution Conflicts as Forces for Sustainability Abstract Environmental distribution conflicts (EDCs) related to the construction and operation of waste incinerators have become commonplace in China. This article presents a detailed case study of citizen opposition to an incinerator in the village of Panguanying, Hebei Province. Drawing on in-depth fieldwork, we show how this case was notable, because it transcended the local arena to raise bigger questions about environmental justice, particularly in relation to public participation in sit- ing decisions, after villagers exposed fraudulent public consultation in the environmental impact assessment. An informal network between villagers and urban environmental activists formed, enabling the Panguanying case to exert influence far beyond the village locality. This network was critical in creating wider public debate about uneven power and substandard public participation in siting disputes, a central feature in many Chinese EDCs. By transcending local specificities and expos- ing broader, systemic inadequacies, this case became instrumental in supporting “strong sustainability”. Keywords  Environmental justice · Sustainability · Environmental networks · China · Waste incinerat Keywords  Environmental justice · Sustainability · Environmental networks · China · Waste incineration · Protest Sustainability Science (2018) 13:733–746 https://doi.org/10.1007/s11625-018-0545-6 The quest for environmental justice in China: citizen participation and the rural–urban network against Panguanying’s waste incinerator Thomas Johnson1   · Anna Lora‑Wainwright2 · Jixia Lu3 Received: 9 March 2017 / Accepted: 20 February 2018 / Published online: 16 March 2018 © The Author(s) 2018 3 College of Humanities and Development Studies, China Agricultural University, 17 Qinghua E Road, Haidian Qu, Beijing Shi 100083, People’s Republic of China * Thomas Johnson Thomas.R.Johnson@Sheffield.ac.uk Handled by Federico Demaria, icta uab, Spain. 2 School of Geography and the Environment, Oxford University Centre for the Environment, University of Oxford, South Parks Road, Oxford OX1 3QY, UK Original research article (Scheidel et al. 2017; Temper et al. 2018; Herrero and Vilella 2017; Camisani 2018). They are driven by changes in social metabolism, namely “the manner in which human societies organize their growing exchanges of energy and materials with the environment” (Martinez-Alier et al. 2010: 153). Although such changes benefit certain groups, others suffer from falling livelihoods, environmental degradation, and worsening public health (Martinez-Alier et al. 2010). Recent transformations in China’s industrial, economic, and social structures have spawned a wide range of EDCs, from large-scale urban protests over facilities such as chemical plants and waste incinerators, to protracted struggles over industrial pollution in rural areas (Lora-Wainwright et al. 2012, 2017; Steinhardt and Wu 2016). Environmental distribution conflicts (EDCs), defined as “mobilizations by local communities against particular eco- nomic activities whereby environmental impacts are a key element of their grievances” (Temper et al. 2015: 261–2), have become widespread in China. Whilst they are often centred on community efforts to uphold social justice and protect their local environments, EDCs can also be impor- tant by contributing to broader sustainability transitions Handled by Federico Demaria, icta uab, Spain. Handled by Federico Demaria, icta uab, Spain. * Thomas Johnson Thomas.R.Johnson@Sheffield.ac.uk * Thomas Johnson Thomas.R.Johnson@Sheffield.ac.uk * Thomas Johnson Thomas.R.Johnson@Sheffield.ac.uk In China, EDCs centred on the construction and opera- tion of waste incinerators have become particularly com- monplace. Since 2004 China has been the world’s biggest waste generator, and by 2030 is predicted to generate double the amount of municipal solid waste produced in the United States (Hoornweg and Bhada-Tata 2012). An impending 1 Department of Politics, University of Sheffield, Elmfield Building, Northumberland Road, Western Bank, Sheffield S10 2TU, UK 1 Department of Politics, University of Sheffield, Elmfield Building, Northumberland Road, Western Bank, Sheffield S10 2TU, UK 2 School of Geography and the Environment, Oxford University Centre for the Environment, University of Oxford, South Parks Road, Oxford OX1 3QY, UK 2 School of Geography and the Environment, Oxford University Centre for the Environment, University of Oxford, South Parks Road, Oxford OX1 3QY, UK :(0123 1 3456789) 3 Sustainability Science (2018) 13:733–746 734 legitimacy. Yet, access to the media is not easily available to the majority of communities (Cai 2010). This greatly increases the difficulty of obtaining information on these cases so that they can be added to the EJ Atlas database. It is likely, therefore, that many disputes, particularly unsuccess- ful cases, remain under the radar. Original research article Through our on-going work on the EJ Atlas project, we identified 54 anti-incinerator EDCs in China. Most of these were identified through an internet search and from information provided by Chinese environmental NGO activ- ists. At time of writing, 11 cases had been entered into the EJ Atlas database (http://ejatl​as.org), a collaborative project whose participants collect and upload case studies of social conflict surrounding environmental issues from around the world. These 11 cases are drawn from Beijing (Asuwei, Liulitun), Wuhan (Guodingshan), Guangdong Province (Luoding, Likeng, Panyu, Boluo, Qingshuihe), Hangzhou (Yuhang), Fujian Province (Qingpuling), and Hebei Prov- ince (Panguanying). Contention over incinerators in these cases occurred between 2005 and 2014. In eight of these cases, citizen opposition was aimed at preventing incinera- tors from being constructed, whilst in three of the cases, citizens fought existing facilities. In seven of these cases, community activists forged links (albeit to varying degrees) with Chinese environmental NGOs, particularly Beijing- based Nature University, discussed in more detail below. In more than half of the cases, communities had been suffering from serious pollution for several years, mostly in the form of landfills that existed long before the incinerators. And, seven of the cases resulted in the incinerators being relocated or indefinitely halted, and might, therefore, be considered “successful” cases for local residents. This high success rate is not representative of incinerator-related EDCs in China, however. As Yongshun Cai (2010) has demonstrated, there is often a positive relationship between media coverage of contention and its likelihood of success, because media reporting can expose local injustices to a wider audience and compel higher levels to intervene to shore up regime This article contributes to the debate surrounding “why, through whom, how, and when” EDCs result in social jus- tice and environmental sustainability (Scheidel et al. 2017) through a detailed study of a Chinese anti-incinerator cam- paign centred on the village of Panguanying in Hebei Prov- ince. This EDC was notable, because more than any other case in our database, it transcended the local arena to raise bigger questions about environmental justice—particularly in relation to public participation in siting decisions—and challenged the desirability of incineration more generally. Crucial to this, was the (strategic) focus on procedural jus- tice and on legal avenues adopted by the key village cam- paigners. 1  The city of Shenzhen is currently constructing the world’s biggest waste-to-energy incinerator, which will burn 5,000 tonnes of waste every day (South China Morning Post 2011). The 2008 Trial Meas- ures on Environmental Information Disclosure enable citizens to apply for disclosure of certain types of information. Original research article Indeed, studies by Chinese scholars suggest that most cases of citizen contention are unsuccessful, and that this is particularly so in rural areas (e.g., Chen 2014; Zhang 2009). “garbage crisis” (laji weiji) has resulted in major restructur- ing of solid waste disposal. This includes a surge in con- struction of “waste-to-energy” (WTE) incinerators, some of which can burn thousands of tonnes of waste per day (John- son 2017).1 The Chinese state’s 12th 5-Year Plan targeted a tripling of the number of incinerators, from 103 in 2010 to over 300 by the Plan’s end in 2015 (Johnson 2013: 357). As of February 2016, 231 incinerators were operational in China (Wuhu Ecology Center and Friends of Nature 2016). g g Anti-incinerator conflicts—just like EDCs more broadly—are usefully analysed through the concept of environmental justice. This concept is premised upon the assumption that “generalised social injustices are manifest in environmental conditions, among other ways” (Schlos- berg 2013: 40). Early applications of an environmental jus- tice framework focused on the uneven distribution of toxic pollution to racial and ethnic minority and low-income communities in the United States (see for instance Bullard 1990). Since then, the concept has been applied to a growing range of issues, including climate change and biodiversity conservation, and has encompassed aspects of recognition and procedural justice (Schlosberg 2007; Sze and London 2008; Walker 2009). In addition, environmental justice frameworks have increasingly been applied beyond West- ern liberal democracies (see, for example, McDonald 2002; Williams and Mawdsley 2006; Carruthers 2008; Özkaynak et al. 2015), although rarely so in the case of China (for exceptions see Lora-Wainwright 2017; Ma 2010; Xie 2011). These studies show that the specific contexts in which envi- ronmental justice movements develop strongly impacts their ability to trigger broader sustainability transformations (Martinez-Alier et al. 2016; Schneidel et al. 2018). In the United States, for example, disparate campaigns in the foot- steps of the infamous Love Canal case scaled up to become a powerful environmental justice movement (Szasz 1994). WTE incineration is a lucrative sector in China. It fea- tures a mixture of state-owned enterprises, Chinese private companies, and multinational corporations and benefits from preferential policies and tax breaks (Johnson 2017). Yet, there has been substantial opposition from communi- ties that bear the brunt of incinerators detrimental to qual- ity of life including, in some cases, public health (Balkan 2012). Original research article This was facilitated by high levels of interaction between villagers and members of a Beijing-based network of environmental activists comprising NGO activists, legal professionals, academics and journalists, who visited the vil- lage and provided support. We know from interviews that the Panguanying case generated a particularly high level of 1 3 3 Sustainability Science (2018) 13:733–746 735 outsider support, which was partly due to the village’s prox- imity to the capital and the perceived significance of the case according to urban activists. Networks have been analysed as important organisational structures that can help further environmental justice claims and facilitate EDCs, which play a crucial role in promoting sustainability transitions (Schlos- berg 1999; Scheidel et al. 2017). Recently, their importance has been highlighted also in the Chinese context (see below for more details). In the case of Panguanying, we show how networking with actors beyond the village helped open legal channels otherwise beyond most ordinary villagers’ reach, and brought the case to a wider audience. forums designed for information sharing between mem- bers; informal networks that may combine organisational and individual members; and personal networks that exist between individuals. Although the diversity found in networks can be a source of strength, it can also create tension that undermines col- lective action (Heyman 2011). Existing studies of environ- mental activism have highlighted tension between the envi- ronmentalism of the wealthy—sometimes linked to Ronald Inglehart’s post-materialist thesis (Inglehart 1977), whereby people only start to care about the environment when they are not concerned with basic material needs—and the “envi- ronmentalism of the poor”, where conflicts are localised and rooted in the inequitable distribution of ecological costs and benefits (Guha and Martinez-Alier 2013). Scholarship has also illustrated potential and related tension between locally focused campaigns often aimed at securing compensation (or “weak sustainability”) and broader social movements concerned with “strong sustainability” and premised on the value of the environment per se (see Martinez-Alier 1998, cited in; Scheidel et al. 2017). Harvey, for instance, viewed the “militant particularism” of poor groups as a barrier to social movement formation (Harvey 1996), and Piller argued that mutual suspicion between NGOs and community activ- ists constrained network formation and scale shift (Piller 1991). g In line with environmental justice literature, the Pan- guanying case shows how EDCs do not just arise out of distribution of ecological harms, but also due to the uneven distribution of power and participation. Original research article Indeed, lack of effec- tive public participation and transparency are regularly con- tested in Chinese EDCs, and improving them is a key focus of environmental NGO activity. In the Panguanying case, as we shall see, campaigners uncovered evidence of system- atic fraud concerning the public participation element of the environmental impact assessment (EIA). This discovery, and the legal contestation surrounding it, was vital to the campaign’s success. Consequently, Chinese environmen- tal activists viewed Panguanying as an exemplary case in highlighting widespread problems associated with the EIA process for waste incinerators and with limited public par- ticipation to a wider (national) audience. By transcending local specificities and shining a light on broader, systemic inadequacies, this case became instrumental in supporting “strong sustainability” (Scheidel et al. 2017). But, as the case of Panguanying will illustrate, diversity in aims and environmental discourses initially embraced is not always an obstacle; indeed, it can be transcended in pro- ductive ways. Some studies have shown how broad, cross- cultural coalitions that combine, or “hybridize”, different types of environmental discourse can bridge divides between campaigns (Pirkey 2012). Crucially, such encounters do not simply involve a one-way transfer of resources from privi- leged to poor (as a resource mobilisation approach might imply). Rather, new dynamics and understandings of issues can form through “friction” (Tsing 2005) produced where diverging groups and interests meet. For example, Gottlieb (2001) showed how tension between different manifesta- tions of environmental concern in the United States stimu- lated the adoption of new tactics and concerns, resulting in the rejuvenation of an environmental movement viewed as overly centralised, hierarchical, and lacking diversity (see also Schlosberg 1999). The next section reviews the social movement literature on environmental networks, showing how this relates to what we know about environmental activism in China. We then examine how and why Chinese urban environmental activists have started to forge links with grassroots commu- nities faced with serious pollution threats. The substantive body of the article presents a close and systematic analysis of the Panguanying case. Finally, in the “Discussion” and “Conclusion” sections, the article builds on this original data to elucidate the relationship between EDCs, environmental justice, and sustainability transitions in China. Environmental networks One group of studies on the latter (strong sustainability) has focused on “environ- mentalist” NGOs (Johnson 2010), particularly in relation to the issues of nature conservation (Economy 2004; Sun and Zhao 2008) and anti-hydropower campaigning (Liu 2013; Mertha 2008), and in terms of their relationships with the Party-state (Ho and Edmonds 2008). Meanwhile, another body of work has examined how ordinary citizens respond to localised pollution (Jing 2000; Lora-Wainwright 2017; Stern 2013; Tilt 2010; Van Rooij 2010; Van Rooij et al. 2012), how grassroots communities play a role in conservation efforts (Coggins 2003; Hathaway 2013; Herrold-Menzies 2009), and about how communities in ethnic minority areas engage with environmental issues (Yeh 2009). Together, these two groups of studies have (sometimes only implicitly) shown how activism occurs at different points on a spectrum bookended by “embedded in the state” NGOs on the one hand (Ho and Edmonds 2008), and rural pollution victims, who are often “isolated” from intermediary support, on the other (Van Rooij 2010). The Panguanying case requires and enables a different type of analysis from these previous studies, one that com- bines attention to grassroots sensibilities (in this case, those of the villagers), the networks that they became part of, and the ways in which they intersected and interacted. These encounters did not engender tensions but rather the grad- ual transcendence of the campaign’s significance from the locality itself to a broader anti-incineration network, as their aims converged. Conversely, our article documents mutual feedback between local campaigners and a wide spectrum of urban activists (see Martinez-Alier et al. 2014). The lan- guage, focus and strategies used by village-based campaign- ers drew from insights they gleaned from other successful urban campaigns, but their success also provided crucial support to the agenda of professional environmentalists and environmental lawyers. The case also furnished lessons about the importance of public participation and procedural justice in the quest for strong sustainability. j While early studies of Chinese environmental activism often made ENGOs their unit of analysis, recent work has focused more on individual activists and networks. Such studies have examined activists’ participation in transna- tional and regional networks (Wells-Dang 2012; Hathaway 2013; Wu 2013), and how they forge mutually beneficial connections with elites, particularly those within the Party- state (Ho and Edmonds 2008; Spires 2011). Environmental networks China’s political and social context diverges significantly from countries that have been the subject of most environ- mental justice scholarship, including the United States. For many years, environmental protection was relegated to secondary importance behind economic development. In the past decade, however, environmental issues have risen up the political agenda and now form a crucial element of officials’ evaluation processes (Kostka 2015). The Minis- try of Environmental Protection (and its predecessor, the Environmental networks are manifested in a multitude of different forms, ranging from enduring, formal alliances, to “temporary coalitions” between environmental organisations and “not in my backyard” (NIMBY) campaigners (Saunders 2013: 28). In the Chinese context, Wells-Dang (2012) dis- tinguishes between four network types: formal “coalitions” featuring hierarchical structures and large memberships; 1 3 1 3 Sustainability Science (2018) 13:733–746 736 State Environmental Protection Administration), has found common ground with environmental activists committed to improving environmental governance within China’s one-party system (Johnson 2014). This includes efforts to promote greater public participation and transparency as a means to hold local officials accountable for not follow- ing environmental laws and regulations. Despite this, many party-state officials remain ambivalent at best to environ- mental activists, fearing that they may undermine local eco- nomic growth or social stability. involved in a campaign against construction of an incinerator at Asuwei in Beijing. They highlight the large discrepancy in capabilities and resources between these two groups, which resulted in limited cooperation, and advocate closer links between communities and environmental NGOs in future (Tan and Ren 2017). In contrast, we show how community and NGO activists worked together closely in the Panguany- ing case.i Whilst existing analyses present a significant step for- ward in identifying potential crossovers between weak and strong sustainability and highlighting the complex inter- actions between a range of actors, few studies, in Chinese or English, have examined the dynamics of environmental networks in China. Most studies of environmental activism that aims to prevent construction of potentially polluting projects focus on case studies in urban areas (e.g., Chen 2012; Lang and Xu 2013; Johnson 2013). One exception, a study by Bondes and Johnson (2017) which also examines the Panguanying case, pays attention to networks but does not explain in detail how they formed, or explicitly examine their potential contribution to sustainability transitions.f Initially, studies on environmentalism in China also examined quests for weak and strong sustainability as though they existed in separation. 3 Environmental networks Mertha (2008) showed how China’s highly fragmented policy-making process provided a structural opportunity for the forma- tion of anti-hydropower coalitions involving state and non- state actors (Mertha 2008). Wells-Dang (2012), who also examined anti-hydropower campaigning in China, similarly focused on the role of “urban elites”. The crucial role that elite allies can play in environmental contention in China has also prominently featured in the Chinese literature including in relation to anti-incinerator campaigns (e.g., Guo and Chen 2011), but little research has been done on the dynamics of network formation between elites and community activists. One partial exception is Tan and Ren’s (2017) comparison of an environmental NGO and a community action group both Our analysis below is based on in-depth interviews with key members of the urban environmental network that we conducted periodically between 2012 and 2017. We also visited Panguanying on several occasions between 2012 and 2016. Johnson interviewed several key village-based cam- paigners in 2012 after learning about the case from Zhao Zhangyuan, a retired Chinese Academy of Sciences pro- fessor and outspoken critic of incineration who had visited Panguanying previously to offer support to villagers (more details below). In 2013, all three co-authors went to Pan- guanying to conduct further interviews with key campaign- ers. We employed a snowballing technique and interviewed several other villagers involved in the campaign. However, our efforts to interview a wider range of villagers including 3 Sustainability Science (2018) 13:733–746 737 environmental activism. An organisation in its own right, Nature University has also become a hub, or “clearing house”, for environmentalists in Beijing, for example, through holding regular seminars on environmental issues. Nature University has begun offering help to communities contesting waste incinerators and other forms of pollution. Once contact with a local community is established, Nature University sends staff members to the site with journalists and other “people who care about the issue”. A key aim of this approach is to attract external publicity, or, in other words, to transcend the local context. This serves two main purposes—pressurizing officials into resolving problems through attracting the attention of higher levels and the wider public, and protecting local activists from retribu- tion at the hands of local officials. Instead of advocating disruptive tactics sometimes associated with NIMBY (not in my backyard) campaigns, Nature University encourages communities to utilize “legal weapons” as fully as possi- ble. Pathways to network formation: the urban perspective Informal networking, based on personal as opposed to insti- tutional linkages, is increasingly recognized as an important feature of China’s environmental movement (Wells-Dang 2012). Beijing is a key hub in this regard. It is home to an informal environmental network (hereinafter referred to as the “Beijing environmental network”) comprising NGO staff, journalists, lawyers, and academics that has advo- cated, among other things, greater environmental transpar- ency, public participation, and more sustainable solutions to China’s waste challenge. Whilst Chinese NGOs were previously believed to shun links with pollution victims (Ho and Edmonds 2008), in recent years, Beijing environmental network members have reached out to local communities opposed to incinerators and other forms of pollution. Steinhardt and Wu (2016) claim that media commercialisation and the spread of the internet and social media, declining risks associated with protest, and improved NGO capacity have all facilitated this growing collaboration between policy advocates and protes- tors. In addition, we find that this development was partly due to frustration among certain environmental activists concerning NGOs’ lack of community engagement, which was part of their “self-imposed censorship” deemed neces- sary for organisational survival in China’s one-party-state (Ho and Edmonds 2008). In the words of one NGO leader, “my biggest gripe was that NGOs didn’t dare to engage with real issues” (NGOs bu gan jieru xianshi). Another impor- tant development was the state’s promotion of governance reforms nominally designed to empower the public to hold polluters to account (Johnson 2010). By focusing on pro- cedural deficiencies including perceived lack of official transparency and public consultation, urban middle class protestors shared similar ground with NGOs, yet the latter remained largely disconnected from protests (Johnson 2010). I thi t t i t l j li t F Y f Aside from helping pollution victims, Nature University and other activists involved in the Beijing Environmental Network have a more normative goal. In the early 2000s, China’s Ministry of Environmental Protection (MEP) intro- duced policies to stimulate public participation and trans- parency, including in the EIA process (Johnson 2014). In helping local residents assert their “right to information, right to supervision, right to participation” (zhiqing quan, jiandu quan, canyu quan), network members hope to pro- mote a more participatory form of environmental govern- ance, which addresses the root causes of problems that cause EDCs in the first place. Environmental networks For example, in another anti-incinerator case at Hai’an in Jiangsu Province, Nature University helped villager Xie Yong, whose son Xie Yongkang was born with cerebral palsy. Xie Yong blamed this on the incinerator located only a couple of hundred metres from his home. Nature Univer- sity helped Xie Yong contact the Centre of Legal Assistance for Pollution Victims (CLAPV), which represented him in an unsuccessful lawsuit against the incinerator company. Throughout this process, Nature University also helped Xie and his fellow villagers (unsuccessfully) apply for disclosure of the incinerator’s pollution data to use as legal evidence.2 As of May 2013, Nature University had filed up to twenty such requests, and sued the Guangzhou Municipal Govern- ment when it refused to disclose information about the city’s Likeng incinerator. some who may not have been directly involved in the case were undermined when local officials in Panguanying told us to cease our fieldwork there. While, as a Chinese researcher, Lu was able to carry out follow-up research and interview a wider range of stakeholders, particularly through employing the help of students, Johnson and Lora-Wainwright had to abandon plans for subsequent fieldwork and limit themselves to interviews with a small number of key villagers who met with us in a nearby city. Our analysis is also based on more than 40 documents, including petition letters and court deci- sions, which we obtained from leading village activists. 2  The 2008 Trial Measures on Environmental Information Disclosure enable citizens to apply for disclosure of certain types of information. Pathways to network formation: the urban perspective As one NGO leader put it: We aim to play the role of mediator and be a com- munication bridge [between the public and the gov- ernment]… We hope that, after an ignorant appeal In this context, environmental journalist Feng Yongfeng established the NGO Nature University (Ziran Daxue), which has pioneered a more “grassroots” approach to 1 3 3 738 Sustainability Science (2018) 13:733–746 2009, with the project having been approved “in principle” by various local government departments, inspection trips to other incinerators operated by Weiming, the private com- pany in charge of constructing and operating the Panguany- ing incinerator, were arranged. Although the Hebei Provin- cial Environmental Protection Bureau (EPB) subsequently claimed that these visits had successfully dispelled any fears concerning incineration that residents might have had, par- ticipants only comprised a handful of local officials who were likely to approve of the project or at least not oppose it. The only village representative included on these trips was Panguanying Party Secretary Qiao Yanli, who had been accused of corruption and was deeply unpopular amongst a significant portion of villagers. (bu ming bu bai de shangfang), we can examine why the appeal was ignorant, how to enable public partici- pation, and how to resolve this problem. We need to return to the situation before the appeal took place, and see whether this project can continue. If it can continue, should there be more compensation? Should the site be changed? Is there a need to improve super- vision of the facility? Only if these issues are properly addressed can these NIMBY cases result in a gradual improvement (emphasis added). Another strand of Nature University’s work involves enabling pollution activists to share their experiences and to network with similar people from other locations. For example, in 2012, a workshop was held on “NIMBY cases in China” that resulted in an NGO report documenting over 20 such “NIMBY” cases.3 The recent popularisation of social media and micro blogging—especially Weibo—has also facilitated communication between grassroots communities and urban activists (Bondes and Johnson 2017). Through this, otherwise “isolated” (Van Rooij 2010: 76), activists become networked into a broader activist community where they can share knowledge and experience. One NGO inform- ant described Weibo as a “small command centre” (xiaoxing zhihuibu) that serves as a platform for sharing information and breaking issues out of local confines. 4  One key stakeholder that we interviewed insisted that officials had covertly altered the land designation from basic agricultural land to yuandi in order that these procedures could be completed. Pathways to network formation: the urban perspective i The Panguanying incinerator only became common knowledge in mid-April 2009 when Qiao led a group of workers to measure the land for the project. He was approached by Pan Zuofu, a member of one of Panguany- ing’s “teams” (the smallest administrative designation which refers to a sub-village unit), which controlled 14 mu (a little less than a hectare) of farmland designated for the incinera- tor. Pan Zuofu became involved in a heated discussion about compensation levels, which he found thoroughly inadequate. Qiao retored that, with higher levels backing the incinerator project, any resistance would be “futile”. p j y Pan Zuofu enlisted support from his uncle Pan Qing- wen, who belonged to the same farming team and whose interests were, therefore, also under threat, as well as other sympathetic villagers. They appealed at multiple levels via the letters and visits (xinfang) system that provides an institutionalised channel through which citizens can lodge complaints against officials, claiming that the land expro- priation was illegal and that the decision-making process contained procedural flaws. They claimed that their land was “basic farmland” (jiben nongtian), and not, as local offi- cials claimed yuandi (literally, “garden land”). This mattered because according to Chinese law expropriation of the for- mer requires central government approval whilst the latter does not. However, local officials rebuffed the men’s com- plaints, and the Hebei EPB approved the project’s EIA in May 2009, allowing construction to begin. Undeterred, the villagers repeatedly contacted the National Land Resources Ministry, which in September 2009 compelled the local county government to halt the project until proper proce- dures related to the land issue had been followed. Shortly thereafter those procedures were completed and construc- tion resumed.4 i Network formation between urban and rural activists is not just a top–down process. It is contingent upon sev- eral local level factors, including grassroots leadership. We examine these factors next through a case study of the Pan- guanying anti-incinerator campaign. 3  On file with the authors. Reframing opposition: from land to health problem’s resolution. In response, the MEP called for the project’s suspension pending further investigation, and the planned incinerator was later relocated (Johnson 2013). The incinerator issue was soon reframed from a land dis- pute involving a handful of affected villagers to a major public health issue. First, a villager formerly employed as an environmental protection officer for a local paper mill warned that burning waste could cause the release of diox- ins, a harmful pollutant. Then, on 1 September 2009, China Central Television (CCTV) aired an episode of Half Hour Economy (Jingji Ban Xiaoshi) entitled Dioxins are Tor- menting China (Er’eying Kunrao Zhongguo).5 A major international conference on dioxins had recently been held in Beijing, where several recent anti-incinerator protests motivated in part by fears concerning dioxin emissions had occurred (Johnson 2013). The Half Hour Economy episode dramatized the link between dioxins and cancer, and interviewed Professor Zhao Zhangyuan. The episode also featured interviews with anti-incinerator campaigners from Liulitun, a Beijing suburb where residents had been opposing a planned incinerator since 2006. Through this, Panguanying villagers discovered that other communities were also struggling against incinerators. Importantly, the realization that the incinerator could threaten public health inspired a third villager, Pan Zhizhong, to join the campaign. He distributed copies of Dioxins are Tormenting China and other materials from the internet to local people to raise awareness. This resulted in 1500 villagers (from Panguany- ing and surrounding villages) signing a petition against the incinerator (Shang 2013). Pan Zhizhong then visited every village within a 5-km radius of the incinerator site and col- lected handwritten statements from 37 village heads express- ing opposition to the project.6 p Reflecting on the Half Hour Economy episode, one lead- ing campaign participant said, Lots of national experts said that it’s best to keep incinerators to a minimum because they are harmful to health… We subsequently saw the [Liulitun report], and downloaded it from the Internet…we saw that Liulitun residents had filed a lawsuit, and there was a lawyer, Xia Jun. We decided to also contact a lawyer, because [in relation to the Liulitun Report] we can’t write this kind of thing, we don’t understand environ- mental law, so we contacted Xia Jun. The Panguanying anti‑incinerator campaign In 2008, the Qinhuangdao Municipal Government decided to construct a waste incinerator in the village of Panguanying, approximately 35 km away from the city yet only 200 metres from the nearest homes. Villagers were excluded from the decision-making process and denied even the most basic information about the project. Instead they were viewed as potential obstacles to be overcome rather than as partici- pants in the decision-making process. The decision-making process was deliberately structured to guarantee support from village leaders and neutralize potential opposition from local people through denying them a voice. Although local officials advertised a public comment period in line with EIA Law requirements, they did this through placing small notices in the county government, which were not seen (and not meant to be seen) by villagers. Then, in early 1 3 Sustainability Science (2018) 13:733–746 739 5  The online link to this programme no longer works. It is possible to find an abridged, five-minute version on the Internet (available at http://my.tv.sohu.com/us/52230​36/36287​61.shtml​). 6  After coming under pressure from the Township government, two village heads revoked their complaints (Shang 2013). 7  On file with the authors. Fraudulent public participation: the smoking gun The China Meteorological Association (Qixiang Ju) (CMA) had conducted the EIA. In evidence submitted to the MEP during the 2010 administrative review process, the provin- cial EPB claimed results from 100 questionnaires issued to local residents as part of the EIA process indicated “strong public support” (jun tongyi) for the project. Residents sus- pected foul play, but lacked evidence to verify their suspi- cions. Amazingly, however, this changed when, during the evidence-collecting period for the administrative litigation lawsuit, the plaintiffs obtained the full EIA report from the EPB. According to Xia Jun, the court was known to be relatively strict, and would likely not allow either side to add evidence after proceedings began. He speculated that this explained why the EPB, perhaps unaware of any prob- lems with the EIA, submitted the full version as evidence. After obtaining the EIA, the anti-incinerator campaigners quickly determined that the questionnaire survey had been completely falsified, providing them with an incontrovert- ible “smoking gun” that proved procedural malpractice. For example, some survey responses had been attributed to vil- lagers who had died or left the village long ago. The 64 “respondents” still living in the village denied participating in the survey. They signed statements indicating that they opposed the incinerator, and that the questionnaires were fake. Upon receiving this news, villagers notified Nature University, which wrote to the MEP and contacted a Phoenix Weekly journalist who visited Panguanying and reported on the case (Bondes and Johnson 2017).8 y The Hebei EPB’s revoking of the EIA did not, there- fore, spell an end to the EDC. In March 2011, villagers challenged the MEP to hold the CMA accountable for the shoddy EIA, and attempted to sue the MEP when it refused to do so. However, the court case was not accepted. Several Beijing-based NGOs were also committed to improving the performance of units conducting EIAs. For example, after discovering similar problems with public consultation for another EIA—also conducted by the CMA—in relation to the Sujiatuo incinerator in suburban Beijing, five NGOs issued an open letter calling for the CMA’s licence for con- ducting EIAs to be stripped. This call was not heeded. Vil- lagers also challenged Weiming’s application to conduct an initial public offering (IPO). Companies are required to obtain approval from the MEP before launching an IPO to prevent highly polluting companies from being listed; Weiming had requested MEP approval in December 2010. The aftermath: negotiating a stalemate EPB’s decision to approve the EIA. The case was accepted and both sides submitted evidence in advance of the court case. During that process, villagers stumbled on a discovery that transformed the case. Whilst the halting of the incinerator project was regarded as a major victory for campaigners, the half-built structure still literally and figuratively loomed over the village. Cam- paigners worried that the EIA would be rectified, allowing the project to resume as planned. Rather than claiming an outright victory then, campaigners’ efforts had resulted in an uneasy stalemate (jiangju). Reframing opposition: from land to health In June 2010, Panguanying campaigners produced their own ten-page report.7 It resembled the Liulitun document, including scientific assertions that incinerators pose a health hazard and claims that, by excluding the public, the siting decision contravened laws and regulations. Then, in sum- mer 2010, the Pans travelled to Beijing to meet with Zhao Zhangyuan and Xia Jun. Both men, together with several Nature University activists, subsequently visited Panguany- ing to provide technical information about the dangers of incineration and advise the Pans about how to contest the incinerator. Lawyer Xia agreed to represent the villagers, and in September 2010 helped them file an administrative review application challenging the Hebei EPB’s decision to approve the incinerator project’s EIA on five grounds: • The project had not been included in municipal govern- ment plans; • The project contravened national government policies, including protection of groundwater and arable land, and of a nearby scenic zone; The Liulitun case strongly influenced Panguanying vil- lagers. There, campaigners had articulated persuasive, facts- based arguments against the incinerator project (Johnson 2013). Liulitun residents produced a detailed 44-page report [hereinafter referred to as the “Liulitun Report”] outlining their rationale and strategies for opposing the incinerator, and uploaded it to the internet. They also fully utilised legal channels, challenging the project based on errors in the sit- ing process such as factual mistakes and lack of public par- ticipation (Johnson 2013). They did this through hiring envi- ronmental lawyer Xia Jun, who helped campaigners contest the project’s EIA through an administrative review applica- tion. They also peacefully surrounded MEP headquarters in Beijing on World Environment Day 2007 to demand the • There was no evidence about how dioxin emissions would be kept within safe standards; • The EIA did not consider, among other things, incinera- tor sludge and ash treatment, and monitoring of dioxins; • The EIA violated the law because public opinion wasn’t solicited and there was insufficient information disclo- sure. The MEP accepted the administrative review application but ultimately upheld the EPB’s decision. Villagers contin- ued to file administrative reviews into other aspects of the case, including the land designation issue, yet these efforts were also rebuffed. Then, in 2011, again with Xia’s help, they filed an administrative litigation lawsuit at the Shiji- azhuang Qiaoxi District People’s Court against the Hebei 1 3 740 Sustainability Science (2018) 13:733–746 8  See http://news.ifeng​.com/fhzk/detai​l_2011_03/01/49057​32_0.shtml​. Discussion Several factors contributed to the formation of a loose net- work between village-based activists and a range of anti- incineration activists and experts, which in turn enabled the case to transcend its local significance and become regarded as a ‘classic’ case that activists could learn from. Most basically, local campaigners’ focus on participation and procedural justice allowed their interests to merge with those of professional anti-incineration campaigners. Framing environmental justice claims in the language of procedural justice is a well-practiced strategy for Chinese environmental activists, and belongs to a broader tendency to depoliticise environmental politics (Ho and Edmonds 2008). Indeed, much contention in China relies upon identifying and drawing attention to misimplementation of laws and regulations and taking advantage of cleavages within the state (O’Brien and Li 2006). Official state sup- port for public consultation in relation to siting decisions enabled villagers to challenge the project on procedural grounds, since they had not been adequately consulted. This was also vital in supporting the development of this rural–urban anti-incineration network. Legal reforms promoting public participation are enabling NGOs to out- grow their reliance on personal connections with party- state officials, or, in Ho and Edmonds’ (2008) words, their “embeddedness” and to work more closely with affected communities. Contingent political circumstances also pre- sented a timely opportunity: activists attributed the Shi- jiazhuang court decision in favour of the villagers to the prior departure of the Provincial EPB Head. This meant that the EIA approval could be suspended without causing him to lose face. fi The charisma and strength of character of the people involved are crucial to the survival of any campaign, let alone its success and the possibility of networking with other campaigners. Although Pan Qingwen was incapacitated by a stroke early in the campaign, Pan Zhizhong and Pan Zuofu resolutely maintained their opposition. This incurred a high personal cost. They were threatened by local thugs and Pan Zuofu’s windows were smashed. Local officials also (unsuc- cessfully) attempted to persuade the three men to drop their campaign through applying pressure through relational ties, something that Deng and O’Brien (2013) refer to as “rela- tional repression”. For example, one county leader alleg- edly contacted Pan Zhizhong through a relative, and offered him alternative accommodation outside of the village. Pan Zuofu’s cousin, a civil servant, advised him against con- tinuing his opposition. But, both men refused to listen to these offers/threats. Fraudulent public participation: the smoking gun Villagers claimed that the fraudulent EIA rendered Weim- ing unfit to launch its IPO. However, the MEP announced that, because the IPO application had occurred before the problems with the Panguanying case emerged, Weiming was eligible to proceed with its IPO. These examples highlight the difficulties associated with translating local case-based issues into wider struggles. Back in Panguanying, in 2012, Pan Zhizhong stood for election as village head after villagers had forced Qiao Yanli to step down. Pan believed that this would enable him to deal a fatal blow to the project. Several environmental activ- ists went to Panguanying to witness the election and offer protection to Pan, who was coming under severe pressure from pro-incinerator forces. However, this election, and a subsequent one, was disrupted by local thug allegedly linked to Township officials, leaving the village without a head for several years. Evidence concerning the fake questionnaires proved decisive in stopping construction of the incinerator for a second time. In March 2011, the Hebei EPB halted the pro- ject and announced that it would not approve any EIAs in Qinhuangdao until the new incinerator EIA had been passed with proper public consultation. On 27 May, the Hebei EPB officially revoked the EIA, and 2 weeks later the court con- firmed that villagers had withdrawn their lawsuit. As of October 2017, construction has not resumed, and villagers employed by Weiming to watch over the half-built incinera- tor have begun to cultivate vegetables again within its walls. In late 2012, in a clear signal that they wanted to resume the project following 2 years of inaction, Weiming attempted to negotiate with Pan Zhizhong and Pan Zuofu. The two men agreed to have lunch with Weiming representatives, during which Pan Zhizhong reasserted his intention to stand for vil- lage head and oppose the incinerator (Shang 2013). Weiming also invited members of the environmental network—includ- ing Mao Da, Chen Liwen, Feng Yongfeng, Zhao Zhangy- uan, and Xia Jun—to visit an incinerator in Jiangsu Prov- ince, which they accepted. However, scope for cooperation 8  See http://news.ifeng​.com/fhzk/detai​l_2011_03/01/49057​32_0.shtml​. 1 3 741 Sustainability Science (2018) 13:733–746 between the two sides was limited. When Weiming asked Mao Da if network members could mediate between it and the villagers, he insisted that the format should be a waste management forum where all issues could be openly debated and covered by the media. Fraudulent public participation: the smoking gun However, Weiming refused, and mediation failed, leaving the issue unresolved. public participation is being incorrectly carried out, so that when other places build something they have to consider public participation, obtain everyone’s agreement. We don’t want money, we’re interested in justice. Similarly, despite limited opportunities for networking with other communities, leading campaigners in the Pan- guanying case publicly shunned the “NIMBY” label and acknowledged the wider (strong) sustainability issues con- cerning waste management. One campaigner stated that he wanted the half-built incinerator to be transformed into an environmental protection museum. And, in August 2017, a Nature University activist involved in the Panguanying case revealed she was discussing the possibility of using the village as a site for a pilot project in waste reduction and sorting with residents. Overall, as the Panguanying case shows, resorting to discourses of sustainability and justice serves as an increasingly desirable strategy for local campaigners to avoid the stigmatised NIMBY label. It also strengthens opportunities for collaboration with urban- based, professional environmentalists including Beijing anti-incineration network members. Conversely, members of the network performed the role of ‘elite allies’ shining a spotlight on the village and exposing official malfeasance. Discussion For example, Xia Jun had a vested interest in promoting the resolution of environmental disputes via legal means to expand the role of lawyers in environmental governance.9 Recounting a conversation with Peking University law professor Wang Jin, he stated, we had been discussing law and public participation, we wanted to find some cases, then [the Panguany- ing] case came along, it was very “classic” (dianx- ing).” I asked him why can’t lawyers help construc- tors [of projects with potential environmental impact] with [conducting] public participation, which has been very problematic? But we feel it’s difficult, because the MEP and EPBs tend to reject lawyers, they think that the EIA system can handle everything, including legal service. We didn’t have a good example case to show our perspective. But the Panguanying case was one such case, it’s very classic, down to the level of fabrication [of the questionnaires], there was serious illegality. we had been discussing law and public participation, we wanted to find some cases, then [the Panguany- ing] case came along, it was very “classic” (dianx- ing).” I asked him why can’t lawyers help construc- tors [of projects with potential environmental impact] with [conducting] public participation, which has been very problematic? But we feel it’s difficult, because the MEP and EPBs tend to reject lawyers, they think that the EIA system can handle everything, including legal service. We didn’t have a good example case to show our perspective. But the Panguanying case was one such case, it’s very classic, down to the level of fabrication [of the questionnaires], there was serious illegality. While the formation and persistence of environmental networks requires and fosters some shared ground between the parties involved, it does not necessarily entail that they should share all their aims. Village campaigners were not primarily concerned with the kind of normative govern- ance goals prioritised by Beijing Environmental Network members. As one NGO activist with close ties to the case commented, “[the villagers] just want to pull this project down once and for all”. In addition, apart from focusing on procedural issues, Panguanying villagers also framed their grievances in the language of distribution and recognition. This was evident in their ten-page report, the final section of which was titled “Common People’s Opinions” (Baix- ing Yijian). 9  For more information on the mixed motives of environmental law- yers, see Stern 2013 Chap. 6. 10  http://zhaoz​hangy​uan.blog.sohu.com/16552​1477.html. Discussion Meanwhile, the Qinhuangdao Govern- ment offered to find work for Pan Qingwen’s five daughters, and to pay for treatment for his sick wife—he also refused this offer. When Pan Qingwen’s application for minimum living guarantee was turned down, his daughters claimed that it was because of his involvement in the anti-incinerator campaign. Closely related to this convergence of focus is a con- vergence of language between village activists and profes- sional environmentalists who collaborated with them. Very much in this vein, village-based campaigners in Panguany- ing adopted some of the discourses of justice and recogni- tion employed by members of the Beijing anti-incineration network. For example, one villager stated that: Opportunities for contact and mutual learning were central to supporting and protecting village-based activ- ists, and in turn to the formation of this rural–urban net- work. Technology and infrastructure played a key role in this regard. The proximity to Beijing (2 h by high-speed When we sued the government, we didn’t want money, we wanted accountability. Even if they give me one Yuan, I don’t mind, as long as the govern- ment loses. We can ring an alarm bell in China, that 1 742 Sustainability Science (2018) 13:733–746 rail), facilitated several face-to-face interactions between villagers and ENGO activists. The media, particularly the CCTV documentary, was also instrumental in supporting the rural–urban network. First and foremost, it made villag- ers realise that theirs was not an isolated struggle. Second, it sounded alarm bells about the potential health effects of incineration in their vicinity, enabling their opposition to go beyond initial economistic concerns about loss of land and livelihood and to include concerns with health too. The latter provided a crucial shared ground with professional activists. Third, it provided a roadmap for their opposition to the incinerator based on the experience of Liulitun campaign and allowed them to identify and contact potential allies (such as lawyer Xia Jun). one of the leading campaigners to ask rhetorically “are we not people too?” In contrast, professional environmentalists seek to use grassroots cases to shape an ambivalent and contested regulatory landscape (Van Rooij et al. 2016). Discussion This prompted 1 3 Sustainability Science (2018) 13:733–746 743 to public participation in siting disputes, something viewed as an endemic issue in China. organised by brokers and urban professional activists is cen- tral to the establishment and strengthening of networks. An important way in which individual, local campaigns become scaled up to broader significance is in their role as precedents. Several precedents were set in the Panguany- ing case, including the release of the full EIA report and the decision to revoke the EIA approval after the discovery of malpractice in relation to the solicitation of public com- ments. Given that public participation requirements are fre- quently overlooked—something that is not helped by weakly worded legislation—Nature University, Xia Jun and other network members hoped that this case could highlight this problem and stimulate broader institutional reforms. Rural–urban networks like the one which emerged around Panguanying’s incinerator can go far beyond environmen- tal issues in challenging multiple forms of domination (Schlosberg 1999). In the Panguanying case, the battle for recognition was rooted in the wider issue of citizenship and participation. These features of environmental justice are closely linked—as Schlosberg (2007: 25) noted, “if you are not recognized, you do not participate; if you do not par- ticipate, you are not recognized”. Decision makers strived to render villagers invisible—they were not informed of the project and were completely excluded from the decision- making process, culminating in the forged questionnaires. By fabricating locals’ approval, the EIA attempted to silence villagers while pretending that they had in fact been given a voice. Conversely, efforts by village campaigners and their allies in Beijing aimed to unmask injustices, both in terms of the lack of recognition of villagers’ views and potential effects on their health and livelihoods. Whilst the network analysed in this article contributed crucially to the relatively “successful” conclusion of this case from the activists’ perspective, it was not the only fac- tor. Panguanying’s proximity to Beijing, just two hours away by train, facilitated the involvement of environmental activ- ists and the media in this case. There was also an element of luck—for example, the EPB’s decision to hand over the entire EIA report came as a surprise to Xia Jun (Bondes and Johnson 2017). Discussion It drew a sharp distinction between urban areas as producers of waste that were being beautified, and rural areas as dumping grounds populated by irrelevant and dis- posable people. One passage stated that, Similarly, a Nature University staff member repeatedly suggested that Panguanying was a good “case” (anzi) from which to learn and for highlighting shortcomings in the EIA process and encourage other communities to also use legal channels to resolve EDCs. One of the main goals of urban activists is to raise societal awareness by enabling cases to enter the public sphere (jinru shehui de shijiao). The story of the faked questionnaires was highly newsworthy. Several media outlets, including the People’s Daily, reported on the fake questionnaires in an article entitled “EIA Unit Actu- ally Engages in This Kind of Forgery” (Huanping jigou jing zheyang zaojia) after environmental activists tipped them off, and Zhao Zhangyuan wrote a blistering criticism of the project’s EIA process.10 In addition, Pan Zhizhong and Pan Zuofu have participated in seminars organised by Beijing activists. The first time this happened was in 2010, after Xia Jun introduced the case to other network members. The two men travelled to Beijing and shared their story with activists and other people fighting against pollution. In 2013, shortly after our field visit, the two men participated in a small con- ference in Beijing on “NIMBY in China”, organised by the NGO Nature University. Their story was written up, along with 20 other cases, and included in the conference pro- ceedings. Participation by local campaigners in such events Villagers aren’t a group of fools, we also know to stand up and fight when our survival is threatened. Waste incineration came about because of cities. It cleaned city spaces whilst polluting vulnerable villag- ers. Almost every aspect [of incinerators sited in rural areas] affects nearby villagers’ livelihoods, sacrificing their health in the short term to alleviate the waste problems that accompany urban development… We also want to survive, we also want environmental pro- tection, and we also want to live with dignity. Villagers’ examination of official documents concerning the siting decision revealed that Panguanying was chosen, because it was downwind from urban (chengzhen) areas and “some distance” (yiding juli) from residential areas, which was desirable in limiting “disturbance” (ganrao). Yet, according to villagers, almost 30,000 rural residents lived within five kilometres of the incinerator site. Discussion And, one NGO activist who had participated in the case told us that waste disposal was not a particularly pressing issue in the region, and that local officials were, therefore, not in a rush to construct an incinerator, something which contributed to the stalemate described above. Finally, the courage and determination of key village activists cannot be underestimated. All of these factors contributed to the outcome in this case. f In discussing the potential for EDCs to contribute to strong sustainability, Scheidel et al. (2017) differentiate between intermodal and intramodal conflicts. The for- mer emerge within an established pattern of resource use between different social groups, whereas the latter defends a particular mode of resource use against industrial society’s attempts to transform it. The Panguanying case straddled these two categories. On the one hand, the village campaign- ers’ primary goal was to prevent an incinerator being con- structed in Panguanying, indicating an intermodal conflict. Yet, in doing so, they became increasingly aware about the dangers of incineration in general, and about shortcomings in the EIA process that robbed them of any agency. This intramodal aspect of the Panguanying case was also influ- enced by strategic imperatives and by urban activists whose concerns about incineration and environmental governance went far beyond the village itself. Engagement of activists such as the Pans can be both inter and intramodal, depending on the context—the two are not mutually exclusive. Conclusion Let us return to one of the questions leading this special issue: “why, through whom, how and when” do EDCs result in social justice and environmental sustainability (Scheidel et al. 2017) considering the Panguanying case and the net- work which formed around it. Rural–urban environmental networks like the one surrounding Panguanying are a new form of movement organising in China. Schlosberg (1999: 142) argued that “networks expand the notion of environ- mental locality, as they expose the similarities shared by communities in disparate places”. Similarly, in the Pan- guanying case, their most crucial effect is enabling what began as a place-based EDC to transcend the local level, giving it wider significance and potential to become a prec- edent for subsequent campaigns. From the view point of local activists, the rural–urban network amplified their voice and helped them challenge unjust decisions. Conversely, this network served the interests of professional activists by creating wider public debate surrounding the misimple- mentaion of environmental regulations, mainly in relation The Panguanying case is an instance in which an EDC supports sustainability. This is not because the incinerator is on hold, but rather because it raises questions about the failings of the EIA process and engages with the question of incineration safety and desirability as a whole. Their proponents claim that waste incinerators represent a clean and efficient way of handling waste, and are preferable to landfill. Yet, studies have cast doubt on this in China due to concerns that incinerators are not managed or regulated to high enough standards (Johnson 2013). One rare study of emissions from 19 Chinese incinerators found considerable variation, with some achieving EU standards and others fail- ing to meet much laxer national standards (Ni et al. 2009). By becoming an exemplary case trumpeted by professional 1 3 1 3 744 Sustainability Science (2018) 13:733–746 Environmental networks face obstacles both against hori- zontal expansion (forging links between affected communi- ties) and vertical expansion (between professional activists and affected communities) (Bondes and Johnson 2017). Regarding the latter, whilst the MEP introduced regulations to increase the scope of non-state involvement, in the Pan- guanying case, it repeatedly turned a deaf ear to villagers’ appeals, choosing instead to side with local officials. Even when the CMA had been guilty of serious wrongdoing on more than one occasion, the MEP did not take formal dis- ciplinary action against it. Conclusion The state’s ambivalence towards non-state actors (Stern 2013)—even when they have legiti- mate demands—limits the potential for improvements in environmental governance within a one-party system. This is unfortunate, especially given that elite allies such as NGOs, journalists and lawyers help soften the boundaries between state and society and generally view their role as mediators instead of agitators. activists and the media, Panguanying highlighted serious problems with the EIA process and showed how the rights and interests of vulnerable populations can be overlooked by planners. And as of late 2017, Nature University activists were in discussion with Panguanying residents about using the village as a pilot site for a rural waste reduction and sort- ing project. At least three other cases in our sample—Aobei, Liulitun and Panyu—also resulted in community efforts in waste sorting and reduction (Johnson 2013). The case, there- fore, supports Scheidel et al. (2017) argument that environ- mental justice success can support wider efforts to improve sustainability.i y Despite Panguanying case’s significance, the prospects for a scaling-up of anti-incinerator activism in China are highly constrained. The professional activist community is small and lacks resources. More importantly, the party-state maintains a strong aversion to linked up activism, particu- larly between disparate affected communities, that may chal- lenge its monopoly on political power. Hence, whilst there appears to be strong demand among grassroots and activ- ist communities for a strong environmental justice move- ment based on incinerators and other pollution problems, the prospects for this materialising appear bleak. The vast majority of EDCs will likely remain rooted in local power struggles. Yet, as the early stages of the Panguanying cam- paign showed, materials related to other communities’ EDC experiences are still readily accessible through the Inter- net. These discourses, which are rooted in broader concerns about the regulation and governance of environmental risks, will likely inform future EDCs even when campaigners fail to network with outside activists. g As Temper et al. (2018) argue, EDCs can result in the creation of new norms and institutional structures that alter power relations resulting in “transformations to sustainabil- ity”. This happens when EDCs coalesce into bigger move- ments that “question the broader structures causing envi- ronmental injustices” and whose approach “is often radical and broad-minded” (Scheidel et al. 2017). In China, any such transformations are most likely to occur when there is strong cooperation between state and non-state actors. References Johnson T (2013) The health factor in anti-waste incinerator campaigns in Beijing and Guangzhou. 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Lang G, Xu Y (2013) Anti-incinerator campaigns and the evolution of t t liti i Chi E i P liti 22(5) 832 848 Lang G, Xu Y (2013) Anti-incinerator campaigns and the evolution of protest politics in China. Environ Politics 22(5):832–848 Cai Y (2010) Collective resistance in China: why popular protests suc- ceed or fail. Stanford University Press, Stanford protest politics in China. Environ Politics 22(5):832–84 Liu J (2013) Defending tiger leaping Gorge. In: Geall Sam (ed) China and the environment: the green revolution. Zed Books, London, pp 203–235 Camisani PB (2018) Sri Lanka: a political ecology of socio-environ- mental conflicts and development projects. Sustain Sci. https​:// doi.org/10.1007/s1162​5-018-0544-7 Lora-Wainwright A (2017) Resigned activism: living with pollution in rural China. MIT Press, Cambridge Mass g Carruthers D (ed) (2008) Environmental justice in Latin America: problems, promise, and practice. MIT Press, Cambridge Lora-Wainwright A, Zhang Y, Wu Y, Van Rooij B (2012) Learning to live with pollution: the making of environmental subjects in a Chinese industrialized village. China J 68:106–124 Chen X (2012) Qu zuzhihua: Yuezhu jiti xingdong de celüe [Go and organise: homeowners’ collective action strategy]. Gonggong Guanli Xuebao 2:67–75 Chinese industrialized village. China J 68:106–124 Ma C (2010) Who bears the environmental burden in China—an analy- sis of the distribution of industrial pollution sources? Ecol Econ 69(9):1869–1876 Chen T (2014) Zhongguo de huanjing kangzheng: yi xiang wenxian yanjiu [Environmental resistance in China: a piece of research based on documents]. Hehai Daxue Xuebao 1:33–43 Martinez-Alier J, Munda G, O’Neill J (1998) Weak comparability of values as a foundation for ecological economics. References Ecol Econ 26:277–286 Coggins C (2003) The tiger and the pangolin: nature, culture, and con- servation in China. University of Hawaii Press, Honolulu Deng Y, O’Brien K (2013) Relational repression in China: using social ties to demobilize protesters. China Q 215:533–552 Martinez-Alier J, Kallis G, Veuthey S, Walter M, Temper L (2010) Social metabolism, ecological distribution conflicts, and valuation languages. Ecol Econ 70:153–158 Economy E (2004) The river runs black: the environmental challenge to China’s future. Cornell University Press, Ithaca Martinez-Alier J, Anguelovski I, Bond P, Del Bene D, Demaria F, Gerber J, Greyl L, Haas W, Healy H, Marín-Burgos V, Ojo G, Porto M, Rijnhout L, Rodríguez-Labajos B, Spangenberg J, Temper L, Warlenius R, Yánez I (2014) Between activism and science: grassroots concepts for sustainability coined by environmental justice organizations. J Political Ecol 21:19–60 Gottlieb R (2001) Environmentalism unbound: Exploring new path- ways for change. MIT Press, Cambridge Guha R, Martinez-Alier J (2013) Varieties of environmentalism: essays North and South. Earthscan Publications, London Guo W, Chen X (2011) Laji chuli zhengce yu gongmin chuangyi yun- dong [Waste treatment policy and citizen advocacy movement]. Zhongshan Daxue Xuebao (Shehui Kexue Ban) 51(4):181–192f Martinez-Alier J, Temper L, Del Bene D, Scheidel A (2016) Is there a global environmental justice movement? J Peasant Stud 43(3):731–755 Harvey D (1996) Justice, nature, and the geography of difference. Blackwell Publishers, Cambridge McDonald D (2002) Environmental justice in South Africa. Ohio University Press, Athens Hathaway M (2013) Environmental winds: making the global in South- west China. University of California Press, Berkeley Mertha A (2008) China’s water warriors: citize change. Cornell University Press, Ithaca Mertha A (2008) China’s water warriors: citizen action and policy change. Cornell University Press, Ithaca Herrold-Menzies M (2009) Peasant resistance against nature reserves. In: Hsing Y, Lee CK (eds) Reclaiming Chinese society: politics of redistribution, recognition and representation. Routledge, London, pp 83–98 Ni Y, Zhang H, Fan S, Zhang X, Zhang Q, Chen J (2009) Emissions of PCDD/Fs from municipal solid waste incinerators in China. Chemosphere 75:1153–1158 pp Herrero A, Vilella M (2017) ‘We have a right to breathe clean air’: the emerging environmental justice movement against waste incinera- tion in cement kilns in Spain. Sustain Sci. https​://doi.org/10.1007/ s1162​5-017-0473-x O’Brien K, Li L (2006) Rightful resistance in rural China. Conclusion Whilst a form of “depoliticised” (Ho and Edmonds 2008) environmental activism is tolerated by the state, this does not include radical movements intent on reconfiguring power structures. For example, the battle to improve public par- ticipation in environmental issues is an iterative process between sympathetic state actors and their policies, and non- state activists who demand that those policies are adhered to. Yet, whilst Chinese leaders have thrown their weight behind energy efficiency and pollution reduction policies, making them key aspects of local official evaluation (see Wang 2013; Kostka 2015), the promotion of public participation as a means for enhancing environmental justice lacks high level political support. Although there are several challenges to EDCs scaling-up, the emergence of networks like the one surrounding Panguanying, however contingent, offers some room for hope in the pursuit of strong sustainability. The level of networking in the Panguanying case exceeded that witnessed in most of the other anti-inciner- ator cases documented so far in the EJ Atlas database. One reason why EDCs so often remain localised is that the rural grassroots level often provides an inhospitable environment for activism, even when it is perfectly legal. One urban envi- ronmental activist recounted his experience in rural China: I’m terrified to death of taking a photo [of pollution]. I don’t even dare to call the pollution reporting hotline. It’s like if you make a phone call you are commit- ting a crime. It’s also not possible to tell the police. Sometimes people give me photos and ask me to report the situation, they are scared that the Public Security Bureau will find them if they publicise the photos, they are scared of being arrested. Open Access  This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat​iveco​ mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Because outside attention can result in local officials being disciplined, they have a strong incentive to limit con- tact between local campaigners and outsiders. The limita- tions we faced whilst carrying out fieldwork may serve as additional illustration of these dynamics. 1 3 3 Sustainability Science (2018) 13:733–746 745 References Pace Environ Law (PELR) Rev 29(3):701–745 i South China Morning Post (2011) Shenzhen plans world’s largest incinerator. 15 September. http://www.scmp.com/artic​le/97905​7/ shenz​hen-plans​-world​s-large​st-incin​erato​r. Accessed 19 Sept 2017 Van Rooij B, SternRE, Fürst K (2016) The authoritarian logic of regu- latory pluralism: understanding China’s new environmental actors. Regul Gov 10(1):3–13 Spires A (2011) Contingent symbiosis and civil society in an authori- tarian state: understanding the survival of China’s grassroots NGOs. Am J Sociol 117(1):1–45 Walker G (2009) Beyond distribution and proximity: exploring the multiple spatialities of environmental justice. Antipode 41(4):614–636 Steinhardt H, Wu F (2016) In the name of the public: environmen- tal protest and the changing landscape of popular contention in China. China J 75(1):61–82 Wang A (2013) The search for sustainable legitimacy: environmen- tal law and bureaucracy in China. Harvard Environ Law Rev 37:365–440 Stern R (2013) Environmental litigation in China: a study in political ambivalence. Cambridge University Press, Cambridge Wells-Dang A (2012) Civil Society networks in China and Vietnam: informal pathbreakers in health and the environment. Palgrave Macmillan, Basingstoke Sun Y, Zhao D (2008) Environmental campaigns. In: O’Brien KJ (ed) Popular protest in China. Harvard University Press, Cambridge, pp 144–162 Williams G, Mawdsley E (2006) Postcolonial environmental justice: government and governance in India. Geoforum 37(5):660–670 Szasz A (1994) EcoPopulism: toxic waste and the movement for envi- ronmental justice. University of Minnesota Press, Minneapolis Wu F (2013) Environmental activism in provincial China: comparative evidence from Guangdong and Guanxi. J Environ Policy Plan 15(1):89–108 Sze J, London J (2008) Environmental justice at the crossroads. Sociol Compass 2(4):1331–1354 Tan S, Ren T (2017) Wo guo huanjing kangzheng zhong shequ yu minjian huanbao NGO zhi bijiao: yi Asuwei laji fenshao xiangmu fanjian shijian wei li [Comparison between environmental NGOs and community environmental resistance in China: taking the Asuwei anti-incinerator case as an example]. Nanjing Gongye Daxue Xuebao (Shehui Kexue Ban) 4:40–48 Wuhu Ecology Center and Friends of Nature (2016) 231 zuo shenghuo laji fenshaochang: xinxi gongkai yu wuranwu paifang baogao (di san qi) (231 waste incinerators: report on information disclosure and pollutant emissions (third phase)). http://www.waste​-cwin. org/sites​/defau​lt/files​/231zu​o_la_ji_fen_shao_han_xin_xi_gong_ kai_bao_gao_zui_zhong​_ban_-email​.pdf. Accessed 8 Mar 2017 Temper L, Del Bene D, Martinez-Alier J (2015) Mapping the fron- tiers and front lines of global environmental justice: the EJAtlas. J Political Ecol 22:255–278 Xie L (2011) Environmental justice in China’s urban decision-making. Taiwan Comp Perspect 3:160–179 Yeh E (2009) Greening western China: a critical view. References Cam- bridge University Press, New York Ö Özkaynak B, Aydın Cī, Ertör-Akyazı P, Ertör I (2015) The Gezi Park resistance from an environmental justice and social metabolism perspective. Capital Nat Social 26(1):99–114i Heyman J (2011) An academic in an activist coalition: Recognizing and bridging role conflicts. Ann Anthropol Pract 35(2):136–153 Piller C (1991) The fail-safe society: community defiance and the end of American technological optimism. BasicBooks, New York l Ho P, Edmonds R (eds) (2008) China’s embedded activism: opportu- nities and constraints of a social movement. Routledge, London Hoornweg D, Bhada-Tata P (2012) What a waste: a global review of solid waste management. Urban Development Series; Knowledge Papers No. 15. The World Bank, Washington, DC Pirkey W (2012) Beyond militant particularisms: collaboration and hybridization in the “contact zones” of environmentalism. Capital Nat Social 23(3):70–91 Inglehart R (1977) The silent revolution: Changing values and politi- cal styles among Western publics. Princeton University Press, Princeton Saunders C (2013) Environmental networks and social movement theory. New York, Bloomsbury Scheidel A, Temper L, Demaria F, Martinez-Alier J (2017) Ecologi- cal distribution conflicts as forces for sustainability: an overview and conceptual framework. Sustain Sci. https​://doi.org/10.1007/ s1162​5-017-0519-0 Jing J (2000) Environmental protests in rural China. In: Perry EJ, Selden M (eds) Chinese society: change, conflict and resistance. Routledge, London, pp 143–160 Johnson T (2010) Environmentalism and NIMBYism in China: pro- moting a rules-based approach to public participation. Environ Politics 19(3):430–448 Schlosberg D (1999) Networks and mobile arrangements: organisa- tional innovation in the US environmental justice movement. Environ Politics 8(1):122–148 1 3 746 Sustainability Science (2018) 13:733–746 Tilt B (2010) Struggling for sustainability in rural China: environmen- tal values and civil society. Columbia University Press, New York Schlosberg D (2007) Defining environmental justice: theories, move- ments, and nature. Oxford University Press, Oxford y Schlosberg D (2013) Theorising environmental justice: the expanding sphere of a discourse. Environ Politics 22(1):37–55 y y Tsing A (2005) Friction: an ethnography of global connection. Prince- ton University Press, Princeton y Van Rooij B (2010) The People vs. pollution: understanding citizen action against pollution in China. J Contemp China 19(63):55–77 Shang H (2013) Juji laji fenshaochang (Waste incinerator ambush). China Weekly, 21 March. http://www.china​weekl​y.cn/benca​ndy. php?fid=63&id=6503. Accessed 15 Mar 2014 Van Rooij B, Lora-Wainwright A, Wu Y, Zhang Y (2012) The compen- sation trap: the limits of community-based pollution regulation in China. References Geoforum 40:884–894 Temper L, Walter M, Rodriguez I, Kothari A, Turhan E (2018) A radi- cal perspective on transformations to sustainability: resistances, movements, alternatives. Sustain Sci. https​://doi.org/10.1007/ s1162​5-018-0543-8 Zhang Y (2009) Ling yi zhong bu pingdeng. Lü Ye 4:28–4 1 3
https://openalex.org/W2784784159
https://aacr.figshare.com/articles/journal_contribution/Figure_S4_from_CCL20_Expression_by_Tumor-Associated_Macrophages_Predicts_Progression_of_Human_Primary_Cutaneous_Melanoma/22539874/1/files/40003207.pdf
English
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CCL20 Expression by Tumor-Associated Macrophages Predicts Progression of Human Primary Cutaneous Melanoma
Cancer immunology research
2,018
cc-by
1,694
VEGF-A CD163 TAM VEGF-A (a.u.) 0 50 100 150 150 100 50 0 R= -0.305 P= 0.335 N= 14 Tumor VEGF-A (a.u.) Primary melanomas Non-metastatic Metastatic C 50 µm M1 48h M1 +TNF 24h M1 +TNF 48h M1 +VEGFA 24h M1 +VEGFA 48h M2 48h M2 +TNF 24h M2 +TNF 48h M2 +VEGFA 24h M2 +VEGFA 48h 70 60 50 40 30 20 10 0 hCCL20 (pg/ml) B TNF CD14 50 40 30 20 10 0 TNFA mRNA MELANOMA SAMPLES TC (CD14- CD3-) TAM (CD14+) In vivo TC and TAM (Immunofluorescence) Ex vivo freshly isolated TC and TAM (qPCR) A #274 #276 TC TAM TC TAM 50 µm #274 #276 VEGF-A CD163 Dapi Supplementary Figure S4. TAM from metastatic primary melanomas express TNF and VEGF-A. A. Melanoma samples (a metastasis and a stage-IV primary tumor) processed in parallel for in vivo (frozen- tissue immunofluorescence) and ex vivo (purification and qPCR) analyses. Left, tissues stained for CD14 (red) and TNF (green) proteins. Right, freshly isolated TAM (CD14+) and TC (CD14/CD3 depleted cells) assessed for TNF mRNA (relative to TBP expression). B. hCCL20 levels of in vitro differentiated macropha- ges M1(GM-CSF) and M2(M-CSF) incubated with 10 ng/ml TNF or 25 ng/ml VEGF-A for 24 and 48 hours. C. Correlation between average TAM-VEGF-A and tumor-VEGF-A MFI values (n=14, Spearman’s coeffi- cient). Representative image of a primary melanoma stained for VEGF-A (green) and CD163 (red). D. Corre- lation between CCL20 and TNF or VEGF-A staining in TAM from metastatic (grey) and non-metastatic (black) primary tumors. Data are given as average TAM MFI values for each tumor sample. Scale bars, 50 µm. TAM TNF (a.u.) TAM VEGF-A (a.u.) 0 50 100 150 0 50 100 150 150 100 50 0 150 100 50 0 R= 0.749 P= 0.001 N= 17 R= 0.612 P= 0.019 N=14 TAM CCL20 (a.u.) TAM CCL20 (a.u.) D Metastatic Non-metastatic TAM TNF (a.u.) TAM VEGF-A (a.u.) 0 50 100 150 0 50 100 150 150 100 50 0 150 100 50 0 R= 0.749 P= 0.001 N= 17 R= 0.612 P= 0.019 N=14 TAM CCL20 (a.u.) TAM CCL20 (a.u.) D Metastatic Non-metastatic M1 48h M1 +TNF 24h M1 +TNF 48h M1 +VEGFA 24h M1 +VEGFA 48h M2 48h M2 +TNF 24h M2 +TNF 48h M2 +VEGFA 24h M2 +VEGFA 48h 70 60 50 40 30 20 10 0 hCCL20 (pg/ml) B TNF CD14 50 40 30 20 10 0 TNFA mRNA MELANOMA SAMPLES TC (CD14- CD3-) TAM (CD14+) In vivo TC and TAM (Immunofluorescence) Ex vivo freshly isolated TC and TAM (qPCR) A #274 #276 TC TAM TC TAM 50 µm #274 #276 M1 48h M1 +TNF 24h M1 +TNF 48h M1 +VEGFA 24h M1 +VEGFA 48h M2 48h M2 +TNF 24h M2 +TNF 48h M2 +VEGFA 24h M2 +VEGFA 48h 70 60 50 40 30 20 10 0 hCCL20 (pg/ml) B 50 40 30 20 10 0 TNFA mRNA S TC (CD14- CD3-) TAM (CD14+) Ex vivo freshly isolated TC and TAM (qPCR) #274 #276 TC TAM TC TAM B A MELANOMA SAMPLES TNF CD14 In vivo TC and TAM (Immunofluorescence) 50 µm #274 #276 In vivo TC and TAM (Immunofluorescence) TAM TNF (a.u.) 0 50 100 150 150 100 50 0 R= 0.749 P= 0.001 N= 17 TAM CCL20 (a.u.) D Metastatic VEGF-A CD163 TAM VEGF-A (a.u.) 0 50 100 150 150 100 50 0 R= -0.305 P= 0.335 N= 14 Tumor VEGF-A (a.u.) Primary melanomas Non-metastatic Metastatic C 50 µm VEGF-A CD163 Dapi TAM TNF (a.u.) TAM VEGF-A (a.u.) 0 50 100 150 0 50 100 150 150 100 50 0 150 100 50 0 R= 0.749 P= 0.001 N= 17 R= 0.612 P= 0.019 N=14 TAM CCL20 (a.u.) TAM CCL20 (a.u.) D Metastatic Non-metastatic TAM VEGF-A (a.u.) 0 50 100 150 150 100 50 0 R= -0.305 P= 0.335 N= 14 Tumor VEGF-A (a.u.) Primary melanomas Non-metastatic Metastatic C C D Figure S4. TAM from metastatic primary melanomas express TNF and VEGF-A. Supplementary Figure S4. TAM from metastatic primary melanomas express TNF and VEGF-A. A. Melanoma samples (a metastasis and a stage-IV primary tumor) processed in parallel for in vivo (frozen- tissue immunofluorescence) and ex vivo (purification and qPCR) analyses. Left, tissues stained for CD14 (red) and TNF (green) proteins. VEGF-A CD163 TAM VEGF-A (a.u.) 0 50 100 150 150 100 50 0 R= -0.305 P= 0.335 N= 14 Tumor VEGF-A (a.u.) Primary melanomas Non-metastatic Metastatic C 50 µm M1 48h M1 +TNF 24h M1 +TNF 48h M1 +VEGFA 24h M1 +VEGFA 48h M2 48h M2 +TNF 24h M2 +TNF 48h M2 +VEGFA 24h M2 +VEGFA 48h 70 60 50 40 30 20 10 0 hCCL20 (pg/ml) B TNF CD14 50 40 30 20 10 0 TNFA mRNA MELANOMA SAMPLES TC (CD14- CD3-) TAM (CD14+) In vivo TC and TAM (Immunofluorescence) Ex vivo freshly isolated TC and TAM (qPCR) A #274 #276 TC TAM TC TAM 50 µm #274 #276 VEGF-A CD163 Dapi Supplementary Figure S4. TAM from metastatic primary melanomas express TNF and VEGF-A. A. Melanoma samples (a metastasis and a stage-IV primary tumor) processed in parallel for in vivo (frozen- tissue immunofluorescence) and ex vivo (purification and qPCR) analyses. Left, tissues stained for CD14 (red) and TNF (green) proteins. Right, freshly isolated TAM (CD14+) and TC (CD14/CD3 depleted cells) assessed for TNF mRNA (relative to TBP expression). B. hCCL20 levels of in vitro differentiated macropha- ges M1(GM-CSF) and M2(M-CSF) incubated with 10 ng/ml TNF or 25 ng/ml VEGF-A for 24 and 48 hours. C. Correlation between average TAM-VEGF-A and tumor-VEGF-A MFI values (n=14, Spearman’s coeffi- cient). Representative image of a primary melanoma stained for VEGF-A (green) and CD163 (red). D. Corre- lation between CCL20 and TNF or VEGF-A staining in TAM from metastatic (grey) and non-metastatic (black) primary tumors. Data are given as average TAM MFI values for each tumor sample. Scale bars, 50 µm. TAM TNF (a.u.) TAM VEGF-A (a.u.) 0 50 100 150 0 50 100 150 150 100 50 0 150 100 50 0 R= 0.749 P= 0.001 N= 17 R= 0.612 P= 0.019 N=14 TAM CCL20 (a.u.) TAM CCL20 (a.u.) D Metastatic Non-metastatic VEGF-A CD163 TAM VEGF-A (a.u.) 0 50 100 150 150 100 50 0 R= -0.305 P= 0.335 N= 14 Tumor VEGF-A (a.u.) Primary melanomas Non-metastatic Metastatic C 50 µm M1 48h M1 +TNF 24h M1 +TNF 48h M1 +VEGFA 24h M1 +VEGFA 48h M2 48h M2 +TNF 24h M2 +TNF 48h M2 +VEGFA 24h M2 +VEGFA 48h 70 60 50 40 30 20 10 0 hCCL20 (pg/ml) B TNF CD14 50 40 30 20 10 0 TNFA mRNA MELANOMA SAMPLES TC (CD14- CD3-) TAM (CD14+) In vivo TC and TAM (Immunofluorescence) Ex vivo freshly isolated TC and TAM (qPCR) A #274 #276 TC TAM TC TAM 50 µm #274 #276 VEGF-A CD163 Dapi Supplementary Figure S4. TAM from metastatic primary melanomas express TNF and VEGF-A. A. Melanoma samples (a metastasis and a stage-IV primary tumor) processed in parallel for in vivo (frozen- tissue immunofluorescence) and ex vivo (purification and qPCR) analyses. Left, tissues stained for CD14 (red) and TNF (green) proteins. Right, freshly isolated TAM (CD14+) and TC (CD14/CD3 depleted cells) assessed for TNF mRNA (relative to TBP expression). B. hCCL20 levels of in vitro differentiated macropha- ges M1(GM-CSF) and M2(M-CSF) incubated with 10 ng/ml TNF or 25 ng/ml VEGF-A for 24 and 48 hours. C. Correlation between average TAM-VEGF-A and tumor-VEGF-A MFI values (n=14, Spearman’s coeffi- cient). Representative image of a primary melanoma stained for VEGF-A (green) and CD163 (red). D. Corre- lation between CCL20 and TNF or VEGF-A staining in TAM from metastatic (grey) and non-metastatic (black) primary tumors. Data are given as average TAM MFI values for each tumor sample. Scale bars, 50 µm. VEGF-A CD163 TAM VEGF-A (a.u.) 0 50 100 150 150 100 50 0 R= -0.305 P= 0.335 N= 14 Tumor VEGF-A (a.u.) Primary melanomas Non-metastatic Metastatic C 50 µm M1 48h M1 +TNF 24h M1 +TNF 48h M1 +VEGFA 24h M1 +VEGFA 48h M2 48h M2 +TNF 24h M2 +TNF 48h M2 +VEGFA 24h M2 +VEGFA 48h 70 60 50 40 30 20 10 0 hCCL20 (pg/ml) B TNF CD14 50 40 30 20 10 0 TNFA mRNA MELANOMA SAMPLES TC (CD14- CD3-) TAM (CD14+) In vivo TC and TAM (Immunofluorescence) Ex vivo freshly isolated TC and TAM (qPCR) A #274 #276 TC TAM TC TAM 50 µm #274 #276 VEGF-A CD163 Dapi Supplementary Figure S4. TAM from metastatic primary melanomas express TNF and VEGF-A. A. Melanoma samples (a metastasis and a stage-IV primary tumor) processed in parallel for in vivo (frozen- tissue immunofluorescence) and ex vivo (purification and qPCR) analyses. Left, tissues stained for CD14 (red) and TNF (green) proteins. Right, freshly isolated TAM (CD14+) and TC (CD14/CD3 depleted cells) assessed for TNF mRNA (relative to TBP expression). B. hCCL20 levels of in vitro differentiated macropha- ges M1(GM-CSF) and M2(M-CSF) incubated with 10 ng/ml TNF or 25 ng/ml VEGF-A for 24 and 48 hours. C. Correlation between average TAM-VEGF-A and tumor-VEGF-A MFI values (n=14, Spearman’s coeffi- cient). Representative image of a primary melanoma stained for VEGF-A (green) and CD163 (red). D. Corre- lation between CCL20 and TNF or VEGF-A staining in TAM from metastatic (grey) and non-metastatic (black) primary tumors. Data are given as average TAM MFI values for each tumor sample. Scale bars, 50 µm. TAM TNF (a.u.) TAM VEGF-A (a.u.) 0 50 100 150 0 50 100 150 150 100 50 0 150 100 50 0 R= 0.749 P= 0.001 N= 17 R= 0.612 P= 0.019 N=14 TAM CCL20 (a.u.) TAM CCL20 (a.u.) D Metastatic Non-metastatic Right, freshly isolated TAM (CD14+) and TC (CD14/CD3 depleted cells) assessed for TNF mRNA (relative to TBP expression). B. hCCL20 levels of in vitro differentiated macropha- ges M1(GM-CSF) and M2(M-CSF) incubated with 10 ng/ml TNF or 25 ng/ml VEGF-A for 24 and 48 hours. C. Correlation between average TAM-VEGF-A and tumor-VEGF-A MFI values (n=14, Spearman’s coeffi- cient). Representative image of a primary melanoma stained for VEGF-A (green) and CD163 (red). D. Corre- lation between CCL20 and TNF or VEGF-A staining in TAM from metastatic (grey) and non-metastatic (black) primary tumors. Data are given as average TAM MFI values for each tumor sample. Scale bars, 50 µm.
https://openalex.org/W3184358838
https://pub.dzne.de/record/162715/files/DZNE-2021-01372.pdf
English
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Role of CGRP pathway polymorphisms in migraine: a systematic review and impact on CGRP mAbs migraine therapy
˜The œJournal of headache and pain
2,021
cc-by
13,527
RESEARCH ARTICLE Open Access Abstract Background: the interest of clinical reaseach in polymorphisms and epigenetics in migraine has been growing over the years. Due to the new era of preventative migraine treatment opened by monoclonal antibodies (mAbs) targeting the signaling of the calcitonin-gene related peptide (CGRP), the present systematic review aims at identifying genetic variants occurring along the CGRP pathway and at verifying whether these can affect the clinical features and the course of disease and the responsiveness of patients to therapy. Methods: the literature search has been conducted consulting the most relevant scientific databases, i.e. PubMed/ MEDLINE, Scopus, Web of Science, the Human Genome Epidemiology (HuGE) Published Literature database (Public Health Genomics Knowledge Base) and Clinicaltrials.gov from database inception until April 1, 2021. The process of identification and selection of the studies included in the analysis has followed the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) criteria for systematic reviews and meta-analyses and the guidance from the Human Genome Epidemiology Network for reporting gene-disease associations. Results: the search has retrieved 800 results, among which only 7 studies have met the eligibility criteria for inclusion in the analysis. The latter are case-control studies of genetic association and an exploratory analysis and two polymorphisms have been detected as the most recurring: the rs3781719 (T > C) of the CALC A gene encoding CGRP and the rs7590387 of the gene encoding the receptor activity-modifying protein (RAMP) 1 (C > G). Only one study assessing the methylation pattern with regard to CGRP pathway has been found from the search. No genetic association studies investigating the possible effect of genetic variants affecting CGRP signaling on the Results: the search has retrieved 800 results, among which only 7 studies have met the eligibility criteria for inclusion in the analysis. The latter are case-control studies of genetic association and an exploratory analysis and two polymorphisms have been detected as the most recurring: the rs3781719 (T > C) of the CALC A gene encoding CGRP and the rs7590387 of the gene encoding the receptor activity-modifying protein (RAMP) 1 (C > G). Only one study assessing the methylation pattern with regard to CGRP pathway has been found from the search. No genetic association studies investigating the possible effect of genetic variants affecting CGRP signaling on the responsiveness to the most recent pharmacological approaches, i.e. anti-CGRP(R) mAbs, gepants and ditans, have been published. Scuteri et al. The Journal of Headache and Pain (2021) 22:87 https://doi.org/10.1186/s10194-021-01295-7 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 https://doi.org/10.1186/s10194-021-01295-7 (2021) 22:87 The Journal of Headache and Pain Scuteri et al. The Journal of Headache and Pain https://doi.org/10.1186/s10194-021-01295-7 Role of CGRP pathway polymorphisms in migraine: a systematic review and impact on CGRP mAbs migraine therapy miana Scuteri1,2, Maria Tiziana Corasaniti3, Paolo Tonin2, Pierluigi Nicotera4 and Giacinto Bagett Damiana Scuteri1,2, Maria Tiziana Corasaniti3, Paolo Tonin2, Pierluigi Nicotera4 and Giacinto Bagetta1* Abstract According to the Human Genome Epidemiology (HuGE) systematic reviews and meta-analyses risk- of-bias score for genetic association studies, the heterogeneity between and across studies and the small sample size do not allow to draw conclusions and prompt future studies. responsiveness to the most recent pharmacological approaches, i.e. anti-CGRP(R) mAbs, gepants and ditans, have been published. According to the Human Genome Epidemiology (HuGE) systematic reviews and meta-analyses risk- of-bias score for genetic association studies, the heterogeneity between and across studies and the small sample size do not allow to draw conclusions and prompt future studies. Conclusions: adequately powered, good quality genetic association studies are needed to understand the impact of genetic variants affecting the pathway of CGRP on migraine susceptibility and clinical manifestation and to predict the response to therapy in terms of efficacy and safety. Keywords: polymorphisms, SNPs, methylation, epigenetic, migraine, CGRP, CALC A, RAMP 1, CLR, RCP, CALCRL, AMYLIN-1, systematic review * Correspondence: g.bagetta@unical.it 1Pharmacotechnology Documentation and Transfer Unit, Preclinical and Translational Pharmacology, Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy Full list of author information is available at the end of the article Rationale and objective Migraine is a primary headache disorder defined as a prevalent neurologic disease characterized by head- aches that can occur with or without aura, consisting of transient focal neurological symptoms (visual, sen- sory, speech and/or language, motor, brainstem and retinal) that usually precede by hours or days, or sometimes accompany, the headache [1]. The social worldwide burden of migraine is noteworthy since it ranks the sixth most prevalent disease and the second cause of disability worldwide [2], accounting for around 7 % of all-cause Years Lived with Disability (YLD) and for 72 % of all YLDs associated to neuro- logical disorders [3]. In fact, according to report from the Global Burden of Disease Headache in 2018, 14.4 % of the global population suffers from migraine making of it the global second leading cause of dis- ability [4]. Migraine belongs to the category of chronic diseases since it is characterized by episodic manifestations (CDEM) [5] that can undergo chronifi- cation in the process of clinical transformation and progression [6]. The prevention of the episodic attacks is fundamental to avoid chronification. The nocicep- tors from the dura mater and periorbital skin project [7] to second-order neurons in the trigeminal nucleus caudalis [8, 9], that can be subjected to sensitization as third-order neurons mainly in the pulvinar of the thalamus. The sensitization of the latter induces cuta- neous allodynia, cephalic at the beginning and gener- alized or extracephalic at later stage [8, 9] and it is involved in chronification. The vasodilatory neuropep- tides are remarkably implicated in the latter dural neurogenic inflammation. Among these, calcitonin- gene related peptide (CGRP) is the the most import- ant player responsible for clinically relevant In particular, the α-CGRP encoded by the CALC A (or CALC I) gene is involved in the pathogenesis of migraine [12]. The effects of CGRP are mediated by its interaction with the CGRP receptor, a Gαs protein- coupled receptor formed by the calcitonin receptor- like receptor (CLR), the receptor activity-modifying protein (RAMP) 1 and the receptor component protein (RCP) [13].CGRP requires also the fusion protein of the extracellular domains of human G protein-coupled receptor calcitonin receptor-like receptor CALCRL to activate the downstream signaling that ends with vaso- dilation [14]. Apart from this canonical receptor, CGRP signal transduction is mediated by the second receptor that is the human amylin subtype 1 receptor (AMY1). © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Scuteri et al. The Journal of Headache and Pain Rationale and objective The genes encoding the latter molecules re- sponsible for CGRP-induced signaling are subjected to genetic variants influencing their activities. Specific anti-migraine drugs for acute treatment of attacks, e.g. triptans, are agonists of 5-HT1B, 5-HT1D and 5-HT1F that finally inhibit CGRP release during migraine at- tacks [12]. Furthermore, novel therapeutic and pre- ventative approaches target the CGRP signaling: these are the gepants, antagonists of CGRP receptor, and the anti-CGRP(R) monoclonal antibodies (mAbs) [15]. Al- though the anti-CGRP(R) mAbs are the first specific preventive therapy which can provide pain relief to difficult-to-treat patients [12], some 40 % of the latter are non responders [16]. Apart from the monogenic forms of migraine and the evidence of rare pathologic genetic variants, several single-nucleotide polymor- phisms (SNPs) have been associated with differences in migraine susceptibility, clinical features and re- sponse to treatment. For instance, genes related to vas- cular modifications and cardiovascular diseases, e.g. Fig. 1 The Calcitonin-gene related peptide (CGRP)-signaling in the trigeminovascular system. The CGRP released from perivascular afferents in the dura, causes dilation of arterial vessels (AV). Nitric oxide (NO) from the vascular endothelium facilitates CGRP release. The CGRP signals to trigeminal ganglion neurons (Aδ/C) CGRP-ergic receptors inducing facilitation of nociceptive transmission to second-order neurons possibly by increasing the release of the excitatory neurotransmitter glutamate (Glu) from neighboring primary afferent terminals. CGRP may also signal directly to second-order neurons (dotted arrows). Adapted with permission from [11]. Fig. 1 The Calcitonin-gene related peptide (CGRP)-signaling in the trigeminovascular system. The CGRP released from perivascular afferents in the dura, causes dilation of arterial vessels (AV). Nitric oxide (NO) from the vascular endothelium facilitates CGRP release. The CGRP signals to trigeminal ganglion neurons (Aδ/C) CGRP-ergic receptors inducing facilitation of nociceptive transmission to second-order neurons possibly by increasing the release of the excitatory neurotransmitter glutamate (Glu) from neighboring primary afferent terminals. CGRP may also signal directly to second-order neurons (dotted arrows). Adapted with permission from [11]. Fig. 1 The Calcitonin-gene related peptide (CGRP)-signaling in the trigeminovascular system. The CGRP released from perivascular afferents in the dura, causes dilation of arterial vessels (AV). Nitric oxide (NO) from the vascular endothelium facilitates CGRP release. The CGRP signals to trigeminal ganglion neurons (Aδ/C) CGRP-ergic receptors inducing facilitation of nociceptive transmission to second-order neurons possibly by increasing the release of the excitatory neurotransmitter glutamate (Glu) from neighboring primary afferent terminals. CGRP may also signal directly to second-order neurons (dotted arrows). Background vasodilation acting on its receptor in the trigeminal ganglion [10] (Fig. 1). Methods SNPs of the gene OMIM encoding the angiotensin converting enzyme (ACE) or SNPs of the methylene- tetrahydrofolate reductase (MTHFR) genetic variants have been implicated in the susceptibility to migraine and aura and in the frequency of migraine attacks. The human leukocyte antigens (HLA) have been associated in heredity for migraine [17] and the HLA Class II DR2 antigen has been found to have a protective role toward migraine with aura [18, 19]. Interestingly, some SNPs have been associated with responsiveness to drugs and, thus, to tendency to chronification with overuse and medications overuse headache (MOH) [20]. Thus, in the future genetic profiling will be fun- damental to foresee the efficacy and safety of therapy, depending on individual genetic variability [21, 22] and to design new drugs tailored on each patient’s genetics [23]. For example, the SNP C825TC of rs5443 in the gene GNB3 coding the G protein β3 subunit in the signaling of 5HT1B/1D is a common genetic variant implicated in the rate of good responsiveness to trip- tans [24, 25]. Other SNPs affecting the destiny of trip- tans are crucial to their pharmacokinetics and pharmacodynamics. The enzymes responsible for trip- tan degradation monoamine oxidase (MAO) A and the cytochrome CYP1A2 influence the response to triptans [25] and the SNP rs4680 of the catechol-O- methyltransferase (COMT) increases the risk of poor response to frovatriptan [26]. Therefore, SNPs occur- ring along the CGRP receptor pathway could affect the clinical evolution of migraine and, thus, might influ- ence to some extent the responsiveness to anti- CGRP(R) mAbs [20]. In particular, for mAbs directed towards CGRP and its receptor the affinity for the functional receptor effectors Fc receptors is fundamen- tal for the maintenance of the antibody-ligand com- plex and for the mAb elimination half-life [27]. FcγRs are responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer therapy [28, 29], e.g. ri- tuximab. In fact, they can influence the affinity be- tween the FcγRs and the IgG [30] and the alteration of this mechanism can be of fundamental importance for the effectiveness of anti-CGRP/CGRP(R) mAbs. Poly- morphisms and gene copy-number variations (CNVs) of FcRs have been associated to the efficacy of mAbs, as it may occur for trastuzumab [31]. Finally, epigen- etic modifications, including DNA methylation and post-translational modifications of the histones tails, have been implicated in modulation of attack fre- quency [32]. Rationale and objective Adapted with permission from [11]. Page 3 of 14 Page 3 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Scuteri et al. The Journal of Headache and Pain Methods The aim of this systematic review is to as- sess whether there are SNPs or methylation patterns along the CGRP pathway that can influence suscepti- bility to migraine, with and without aura, frequency and severity of attacks and responsiveness to treatment. Objectives and protocol j p To our knowledge the present systematic review is the first designed to verify the working hypothesis that the SNPs or methylation patterns occurring along the CGRP pathway can affect the clinical features and the course of disease and the responsiveness of pa- tients affected to anti-migraine therapy. In order to address this PICOS (participants/population, interven- tions, comparisons, outcomes, and study design) ques- tion, the PRISMA recommendations [33, 34] and the guidance from the Human Genome Epidemiology Network for reporting gene-disease associations [35] have been followed. The possibility of SNPs to ac- count for some lack of response to anti-CGRP(R) mAbs, mainly, and to anti-migraine therapy, in gen- eral, is a broad question still representing a lack of knowledge; therefore, this work will provide an over- view of evidence, including all the existing studies in- vestigating direct genetic association, for assessing the consistency of the body of evidence to prompt future research. For this reason, the protocol has not been registered in the International prospective register of systematic reviews PROSPERO. The systematic review and meta-analysis has been conducted in accordance to a protocol established prior to the literature search. The retrieved results have been evaluated and double- checked independently by two researchers. Any con- flicts have been resolved by a third author. Inclusion criteria Th l i i l The analysis included genetic association studies asses- sing the direct genetic association of SNPs or epigenetic modifications affecting genes involved in the CGRP pathway on the following aspects: susceptibility to mi- graine, with and without aura; frequency and severity of attacks; responsiveness to treatment. No filters about study duration or follow-up and no restrictions con- cerned with publication date have been applied. In vitro and in vivo animal studies, narrative or systematic re- views and meta-analysis, abstracts and congress commu- nications, proceedings, editorials and book chapters as well as studies not available in full text and not pub- lished in English have been excluded from the analysis. Search strategy The following Medical Subject Headings (MeSH) terms and modifications have been used as search terms in combination: “migraine”, “CGRP”, ‘‘calcitonin gene- related peptide’’, “CGRP receptor”, “CALC A”, “CALC I”, “RAMP 1”, “CALCRL”, “CLR”, “RCP”, “AMYLIN-1”, “AMYLIN-1 receptor”, “polymorphisms”, “SNP(s)”, “epi- genetic”, “methylation”, “anti-CGRP(R) monoclonal anti- bodies”, “anti-CGRP(R) mAbs”, “triptans”, “gepants”, “ditans”. Data analysis h h The synthesis of the results has been conducted accord- ing to the Cochrane Consumers and Communication Review Group guidelines [37]. The assessment of the risk of bias and of the quality of retrieved studies has been performed in agreement to Human Genome Epi- demiology (HuGE) systematic reviews and meta-analyses risk-of-bias score for genetic association studies [38]. Hence, the latter score ranges from low to unclear and high risk, taking into account the following 4 outcomes rated yes, no or unclear: (1) Information bias – Accuracy of diagnosis of migraine and robustness of genotyping methods; (2) Confounding bias – Population stratifica- tion and other confounder effects; (3) Selective reporting of outcomes – reporting bias; (4) Hardy-Weinberg equi- librium (HWE) – assessment in the control groups. Sample size has been considered. Due to the heterogen- eity of the studies and the difference of polymorphisms and outcomes investigated in the studies meeting the eli- gibility criteria, data concerned with odds ratios for ge- notypes could not be pooled and a genotype-based meta-analysis and assessment of the credibility of cumu- lative evidence through the Venice guidelines [39] have not resulted feasible. The process of identification and selection of the stud- ies is illustrated in Fig. 2. Selection of the studies The search on the databases has retrieved 800 results: 396 records have been obtained form PubMed/MED- LINE, 209 from Scopus, 173 from Web of Science, 19 from the HuGE Published Literature database (Public Health Genomics Knowledge Base) and 3 from Clincal- trials.gov. The 800 records have been searched for dupli- cates. After duplicates removal there were 285 results to screen (also one of the three records obtained from Clin- caltrials.gov has resulted to be a duplicate). The latter have been screened in title and abstract leaving 11 re- sults to assess for eligibility. However, the study by An et al., 2017 [40] was not available in full text and the two records retrieved from Clincaltrials.gov are two studies without results since one is recruiting and the other not yet. In particular, the study INTERROGATE, Biomarker and Genetic Predictors of Erenumab Treatment Re- sponse (NCT04265755) is in the recruitment stage and it aims at exploring the relationship between clinical re- sponse to erenumab and genetic biomarkers, while the purpose of the BIOmarkers of MIGraine (BIOMIGA) proof of concept study (NCT04503083) is to detect bio- markers predictive of response to anti-CGRP(R) mAbs in severe migraineurs using, among others, pharmacoge- netic evaluation and assessment of the methylation levels. Therefore, the full-text articles assessed for eligi- bility are 8 and 7 of them have met the inclusion criteria for qualitative analysis. In fact, the study by Louter et al., that considered the polymorphisms rs2956 of CALC A gene and rs858745 of CALCRL gene possible candidate genes to be implicated in chronification [41] had to be excluded because of its different study design being a three stage genetic association study. Information sources The literature search has been performed consulting the most relevant scientific databases, i.e. PubMed/MED- LINE, Scopus, Web of Science, the Human Genome Epi- demiology (HuGE) Published Literature database (Public Health Genomics Knowledge Base) and Clinicaltrials.- gov. The search could not be conducted also on Embase since it was not freely/institutionally available. No re- striction of publication date has been applied. The Page 4 of 14 Page 4 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Scuteri et al. The Journal of Headache and Pain databases have been searched for records matching the search strings used from their inception to May, 21 2021 that was the date of last search. Studies selection The assessment of the inclusion and exclusion criteria and the determination of eligibility of the studies has been carried out independently by two authors for min- imizing the risk to exclude relevant records. Duplicate records have been eliminated and the following first screening has assessed the title and abstract. Then, the full text has been evaluated for inclusion in qualitative and/or in quantitative synthesis. The references list of the articles has been evaluated in order to extend and refine the search. Complete consensus among all the au- thors has been achieved without relevant conflicts planned to be solved through the Delphi method [36]. Qualitative analysis The 7 articles eligible for analysis are grouped according to the gene of which the polymorphisms have been stud- ied, based on the Cochrane Consumers and Communi- cation Review Group guidelines. A summary of the main characteristics of the studies is reported in Table 1. Polymorphisms of the gene encoding CGRP The gene encoding CGRP has been studied for the first time in the study by Lemos et al., in a European population [42]. The SNP rs1553005 of the latter gene has been found to interact with the variant rs2049046 of the gene encoding brain-derived neuro- trophic factor (BDNF) increasing the risk of migraine [rs1553005(CGRP)*rs2049046(BDNF) = GC*AT – OR 1.88 (95 %CI 1.20–2.93), P = 0.005] [42]. This is sup- ported by the co-expression of the latter Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Page 5 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Fig. 2 PRISMA flow diagram. PRISMA flow diagram reporting the process of identification and selection of the studies eligible for the systematic review and meta-analysis. agram. PRISMA flow diagram reporting the process of identification and selection of the studies eligible for the systematic Fig. 2 PRISMA flow diagram. PRISMA flow diagram reporting the process of identification and selection of the studies eligible for the systematic review and meta-analysis. an Australian population, the study by Sutherland et al., [45] has investigated the possible correlation between the SNPs rs3781719 in the promoter region and rs145837941 in the coding sequence of CALC A and an increased susceptibility to migraine. None of the two polymorphisms have resulted associated to migraine susceptibility or with gender and the rs3781719 has not been associated to increased fre- quency of attacks or to the development of aura. This polymorphism has been studied also by Cargnin et al., in an Italian population for its influence on re- sponse to triptans in patients affected by migraine without aura and it has been tested for association with transformation into MOH, providing no signifi- cant correlation [46]. However, CALC A rs3781719C allele has resulted to increase risk of lack of response to OnabotulinumtoxinA in a female population of Caucasian ethnicity and Spanish origin [OR (95 %CI) neurotransmitter that has been demonstrated in tri- geminal ganglion neurons of rat. Polymorphisms of the gene encoding CGRP N = 284 South Eastern Australian: adult Caucasians of European descent living in Australia, DNA extraction from white blood cells rs3781719 (SNP 624 (T/ C) of the CALC A gene promoter) Investigate the contribution of the 4 selected SNPs in the risk of migraine TT: -migraine47.8 %; -migraine with aura 49.0 % -migraine without aura 45.7 % TC: -migraine44.7 %; -migraine with aura 42.2 %; -migraine without aura 49.4 % CC: -migraine7.5 %; -migraine with aura 8.8 %; -migraine without aura 4.9 % HWE has been verified. Genotypes for SNPs were in HWE in case and control groups No significant association with migraine (P = 0.260), migraine with aura (0.563) and migraine without aura (0.133) rs145837941 (4218T > C base- TT: 94.9 % TC: 4.7 % CC: 0.4 % No significant association with migraine (P = 0.913) Table 1 Main characteristics of the studies included in the analysis. General characteristics of studies included in the analysis. OR = odds ratio; CI = confidence interval; P = P value; HWE = Hardy–Weinberg equilibrium. Study Design Disease (N) Control (N) Ethnicity Sample Variant Outcome Genotype 1 Genotype 2 Genotype 3 HWE Results Lemos et al., 2010 [42] Case-control study. The Authors state that sample size is a limitation of the study not providing enough power to detect a variant with an OR < 1.5. Diagnosis according to the International Headache Society (IHS), using the International Classification of Headache Disorders (ICHD) I (before 2004) and II. N = 188 People with no history of migraine, age- matched to cases N = 287 Portuguese Genomic DNA extraction from peripheral blood leucocytes rs1553005 (CGRP) 1) Investigate the role of BDNF in migraine susceptibility; 2) CGRP, studied for the first time as candidate gene in migraine; 3) possible interaction of BDNF and CGRP genes in migraine’s susceptibility. GG: OR (95 %CI) 1.00 0), p= -; multivariable logistic regression analysis GC: OR (95 %CI )1.40 (0.94–2.08), p = 0.09; multivariable logistic regression analysis CC: OR (95 %CI) 0.86 (0.42–1.77), p = 0.69; multivariable logistic regression analysis Case and control groups in HWE for the polymorphism rs1553005(CGRP)* rs2049046(BDNF) = GC*AT – OR 1.88 (95 %CI 1.20–2.93), P = 0.005 Menon et al., 2011 [43] Case-control study. Polymorphisms of the gene encoding CGRP The study by Menon and collaborators [43] has investigated in an Austra- lian population the role of the polymorphism rs35815751, consisting in a 16 bp deletion in the first intron of the CALC A gene that is a region with trip- let G-run motifs, in the development of migraine with aura: no significant association between rs35815751 and migraine [for genotypes (P = 0.575) nor alleles (P = 0.502)], and migraine with aura (genotypes, P = 0.666; alleles, P = 0.7) or without aura (genotypes, P = 0.325; alleles, P = 0.276) has been found. The study of Guldiken and collaborators [44] has tested on female population the possible influence of the polymorph- ism rs3781719, consisting in T-692 C of CALC A gene, on attack frequency and severity and on the oc- currence of aura, finding no significant association with migraine (P = 0.44) and aura (P = 0.52). Also in Page 6 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Table 1 Main characteristics of the studies included in the analysis. General characteristics of studies included in the analysis. OR = odds ratio; CI = confidence interval; P = P value; HWE = Hardy–Weinberg equilibrium. Study Design Disease (N) Control (N) Ethnicity Sample Variant Outcome Genotype 1 Genotype 2 Genotype 3 HWE Results Lemos et al., 2010 [42] Case-control study. The Authors state that sample size is a limitation of the study not providing enough power to detect a variant with an OR < 1.5. Diagnosis according to the International Headache Society (IHS), using the International Classification of Headache Disorders (ICHD) I (before 2004) and II. N = 188 People with no history of migraine, age- matched to cases N = 287 Portuguese Genomic DNA extraction from peripheral blood leucocytes rs1553005 (CGRP) 1) Investigate the role of BDNF in migraine susceptibility; 2) CGRP, studied for the first time as candidate gene in migraine; 3) possible interaction of BDNF and CGRP genes in migraine’s susceptibility. GG: OR (95 %CI) 1.00 0), p= -; multivariable logistic regression analysis GC: OR (95 %CI )1.40 (0.94–2.08), p = 0.09; multivariable logistic regression analysis CC: OR (95 %CI) 0.86 (0.42–1.77), p = 0.69; multivariable logistic regression analysis Case and control groups in HWE for the polymorphism rs1553005(CGRP)* rs2049046(BDNF) = GC*AT – OR 1.88 (95 %CI 1.20–2.93), P = 0.005 Menon et al., 2011 [43] Case-control study. Polymorphisms of the gene encoding CGRP Power analysis reported Clinical diagnosis by an experienced clinical neurologist based on international criteria N = 278 Individuals with no family history of migraine, age, sex and ethnicity matched to cases N = 322 Australian (east coast) Caucasian:of European descent living in Australia, with ancestors emigrated within the last 160 years from the British Isles and other parts of Europe Genomic DNA extraction from white blood cells rs35815751 (16 bp deletion in intron 1 of the CALC A gene) Invetigate the role of 16 bp deletion in the first intron of the CALCA gene in the risk of migraine II (homozygous, insertion/ insertion); no deletion/ deletion OR (95 %CI) 1.2 (0.74–1.85) in migraine, for genotypes (P = 0.575) and for alleles (P = 0.502); logistic regression analysis ID (heterozygous, insertion/ deletion); no deletion/deletion OR (95 %CI) 1.1 (0.68–1.79) in migraine with aura, (genotypes, P = 0.666; alleles, P = 0.7); logistic regression analysis DD (homozygous, deletion/ deletion); no deletion/ deletion OR (95 %CI) 1.6 (0.70–3.44) in migraine without aura, (genotypes, P = 0.325; alleles, P = 0.276); logistic regression analysis Genotypes in HWE (migraineurs: PHWE=0.73 and controls: PHWE=0.247) No association between rs35815751 and migraine for genotypes (P = 0.575) nor alleles (P = 0.502), and migraine with aura (genotypes, P = 0.666; alleles, P = 0.7) or without aura (genotypes, P = 0.325; alleles, P = 0.276) Guldiken et al., 2013 [44] Case-control study on females. Diagnosis of migraine by neurologist based on the criteria of ICHD-II. N = 134 Healty volunteers, health care personal and postpartum females who were hospitalized in the obstetric clinic. N = 96 Local (Turkish) DNA isolation from peripheral blood rs3781719 (SNP T-692 C of CALC A gene) Investigated the frequency of CALCA T-692 C in migraineurs and its association to migraine attack frequency and severity TT: 42.5 % TC: 42.5 % CC: 14.9 % HWE has been evaluated. No association between the genotype and allele frequency of migraine (P = 0.44), without and with aura (P = 0.52), and the severity and frequency of migraine attacks. Frequency of migraine attacks has been measured as the number of attacks in a month. The severity has been assessed with the visual analog scale Sutherland et al., 2013 [45] Case-control study. Power analysis reported Diagnosis by a clinical neurologist according to the IHS criteria. N = 284 Matched for sex, age (+/−5 years) and ethnicity. Polymorphisms of the gene encoding CGRP other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance CC: (R) OR (95 %CI) 0.80 (0.30–2.11), P = 0.65; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance HWE tested. PHWE rs3781719 = 0.52; PHWE rs3754701 = 0.77; PHWE rs7590387 = 0.41. For only MOH patients: (PHWE rs3781719 = 0.18; PHWE rs3754701 = 1; PHWE rs7590387 = 0.69) No significant association for endpoint 1 [risk of inconsistent response to triptans (being consistent responders defined as patients experiencing a ≥2 point reduction after triptan administration in at least 2 out of 3 consecutive attacks)]; significant association of RAMP1rs7590387GG [OR (95 %CI) (R) 0.27 (0.13– 0.57) P = 0.0002)] for endpoint 2 (risk of trasformation of episodic migraine to MOH) rs3754701 [RAMP 1 (T > A)] TT; (A) OR (95 %CI) 0.90 (0.60–1.37), P = 0.63; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = TA; (D) OR (95 %CI) 0.73 (0.41–1.32), P = 0.30; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of AA; (R) OR (95 %CI) 1.19 (0.53–2.67), P = 0.66; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = le 1 Main characteristics of the studies included in the analysis. General characteristics of studies included in the analysis. OR = odds ratio; CI = confidence interval; P = P e; HWE = Hardy–Weinberg equilibrium. Polymorphisms of the gene encoding CGRP (Continued) y Design Disease (N) Control (N) Ethnicity Sample Variant Outcome Genotype 1 Genotype 2 Genotype 3 HWE Results with ancestors emigrated within the last 160 years from British Isles and other parts of Europe exchange in the coding sequence of CALC A) rs3754701 (SNP in the RAMP1 gene promoter at position − 1166 (T/A)) TT: - migraine 37.4 %; - migraine with aura 42.1 %; - migraine without aura 30.6 % TA: - migraine 49.4 %; - migraine with aura 44.8 %; - migraine without aura 56.1 % AA: - migraine 13.2 %; - migraine with aura 13.1 %; - migraine without aura 13.3 % No significant association with migraine (P = 0.360), migraine with aura (P = 0.276) and migraine without aura (P = 0.260) rs7590387 ((G/C) 1.4 kb downstream of the RAMP1 gene) GG: - migraine 25.7 %; - migraine with aura 23.9 %; - migraine without aura 28.4 % GC: - migraine 54.4 %; - migraine with aura 54.3 %; - migraine without aura 54.5 % CC: - migraine 19.9 %; - migraine with aura 21.7 %; - migraine without aura 17.0 % No significant association with migraine (P = 0.341), migraine with aura (P = 0.566) and migraine without aura (P = 0.299) in 2015 Case-control study. Power calculation reported Diagnosis fulfilling ICHD-II criteria for migraine without aura A for at least 1 year and for medication overuse headache (MOH) or previous or current diagnosis of MOH.Patients with migraine without aura and patients with MOH. N (migraine without aura) = 219; N (MOH) = 130 Matched by age and sex and of same ethnicity. N = 209 Italian (north-west) DNA extraction from peripheral blood rs3781719 [CALC A (T > C)] Investigate the role of the two selected SNPs in: 1) risk of inconsistent response to triptans; 2) risk of trasformation of episodic migraine to MOH TT: (A) OR (95 %CI) 0.96 (0.62–1.48), P = 0.84; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance TC: (D) OR (95 %CI) 1.04 (0.59–1.85), P = 0.88; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. Polymorphisms of the gene encoding CGRP Power analysis reported Clinical diagnosis by an experienced clinical neurologist based on international criteria N = 278 Individuals with no family history of migraine, age, sex and ethnicity matched to cases N = 322 Australian (east coast) Caucasian:of European descent living in Australia, with ancestors emigrated within the last 160 years from the British Isles and other parts of Europe Genomic DNA extraction from white blood cells rs35815751 (16 bp deletion in intron 1 of the CALC A gene) Invetigate the role of 16 bp deletion in the first intron of the CALCA gene in the risk of migraine II (homozygous, insertion/ insertion); no deletion/ deletion OR (95 %CI) 1.2 (0.74–1.85) in migraine, for genotypes (P = 0.575) and for alleles (P = 0.502); logistic regression analysis ID (heterozygous, insertion/ deletion); no deletion/deletion OR (95 %CI) 1.1 (0.68–1.79) in migraine with aura, (genotypes, P = 0.666; alleles, P = 0.7); logistic regression analysis DD (homozygous, deletion/ deletion); no deletion/ deletion OR (95 %CI) 1.6 (0.70–3.44) in migraine without aura, (genotypes, P = 0.325; alleles, P = 0.276); logistic regression analysis Genotypes in HWE (migraineurs: PHWE=0.73 and controls: PHWE=0.247) No association between rs35815751 and migraine for genotypes (P = 0.575) nor alleles (P = 0.502), and migraine with aura (genotypes, P = 0.666; alleles, P = 0.7) or without aura (genotypes, P = 0.325; alleles, P = 0.276) Guldiken et al., 2013 [44] Case-control study on females. Diagnosis of migraine by neurologist based on the criteria of ICHD-II. N = 134 Healty volunteers, health care personal and postpartum females who were hospitalized in the obstetric clinic. N = 96 Local (Turkish) DNA isolation from peripheral blood rs3781719 (SNP T-692 C of CALC A gene) Investigated the frequency of CALCA T-692 C in migraineurs and its association to migraine attack frequency and severity TT: 42.5 % TC: 42.5 % CC: 14.9 % HWE has been evaluated. No association between the genotype and allele frequency of migraine (P = 0.44), without and with aura (P = 0.52), and the severity and frequency of migraine attacks. Frequency of migraine attacks has been measured as the number f tt k i th Page 7 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Table 1 Main characteristics of the studies included in the analysis. General characteristics of studies included in the analysis. Polymorphisms of the gene encoding CGRP OR = odds ratio; CI = confidence interval; P = P value; HWE = Hardy–Weinberg equilibrium. (Continued) Study Design Disease (N) Control (N) Ethnicity Sample Variant Outcome Genotype 1 Genotype 2 Genotype 3 HWE Results with ancestors emigrated within the last 160 years from British Isles and other parts of Europe exchange in the coding sequence of CALC A) rs3754701 (SNP in the RAMP1 gene promoter at position − 1166 (T/A)) TT: - migraine 37.4 %; - migraine with aura 42.1 %; - migraine without aura 30.6 % TA: - migraine 49.4 %; - migraine with aura 44.8 %; - migraine without aura 56.1 % AA: - migraine 13.2 %; - migraine with aura 13.1 %; - migraine without aura 13.3 % No significant association with migraine (P = 0.360), migraine with aura (P = 0.276) and migraine without aura (P = 0.260) rs7590387 ((G/C) 1.4 kb downstream of the RAMP1 gene) GG: - migraine 25.7 %; - migraine with aura 23.9 %; - migraine without aura 28.4 % GC: - migraine 54.4 %; - migraine with aura 54.3 %; - migraine without aura 54.5 % CC: - migraine 19.9 %; - migraine with aura 21.7 %; - migraine without aura 17.0 % No significant association with migraine (P = 0.341), migraine with aura (P = 0.566) and migraine without aura (P = 0.299) Cargnin et al., 2015 [46] Case-control study. Power calculation reported Diagnosis fulfilling ICHD-II criteria for migraine without aura A for at least 1 year and for medication overuse headache (MOH) or previous or current diagnosis of MOH.Patients with migraine without aura and patients with MOH. N (migraine without aura) = 219; N (MOH) = 130 Matched by age and sex and of same ethnicity. N = 209 Italian (north-west) DNA extraction from peripheral blood rs3781719 [CALC A (T > C)] Investigate the role of the two selected SNPs in: 1) risk of inconsistent response to triptans; 2) risk of trasformation of episodic migraine to MOH TT: (A) OR (95 %CI) 0.96 (0.62–1.48), P = 0.84; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance TC: (D) OR (95 %CI) 1.04 (0.59–1.85), P = 0.88; logistic regression analysis adjusted for triptan (frovatriptan vs. Polymorphisms of the gene encoding CGRP A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance CG: (D) OR (95 %CI) 0.84 (0.46–1.54), P = 0.57; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance GG: (R) OR (95 %CI) 1.69 (0.86–3.30), P = 0.12; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance Wan et al., 2015 [48] Exploratory analysis. Limitations reported by the Authors: small sample, the analysis could not be conducted on the specific tissue derived from trigeminovascular or cerebral biopsies,the blood DNA was from Migraineurs recruited at International Headache Center of Chinese PLA General Hospital. N = 26 Matched healthy controls with no significant differences in gender and age compared to migraineurs group. N = 25 Patients recruited at International Headache Center of Chinese PLA General Hospital DNA extraction from EDTA blood samples obtained through the cubital vein Analysis of CpG islands of the RAMP1 gene in a 3000 bp region including putative promoter sequences and the first exon Investigate if the methylation pattern of the promoter of RAMP1 gene in peripheral leukocyte is associated with migraine Low methylation trend without statistical significance in migraine vs. control (total average methylation level: 8.41 % ±1.92 % vs. 9.90 % ± 3.88 %, P = 0.197) The ROC the curve and Youden criterion have been used for the determination of the optimum cut- off of methylation level No significant difference in the DNA methylation pattern of RAMP 1 between migraine and control groups Table 1 Main characteristics of the studies included in the analysis. General characteristics of studies included in the analysis. OR = odds ratio; CI = confidence interval; P = P value; HWE = Hardy–Weinberg equilibrium. Polymorphisms of the gene encoding CGRP (Continued) Study Design Disease (N) Control (N) Ethnicity Sample Variant Outcome Genotype 1 Genotype 2 Genotype 3 HWE Results log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance inheritance; D = dominant model of inheritance; R = recessive model of inheritance log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance rs7590387 [RAMP 1 (C > G)] CC: (A) OR (95 %CI) 1.11 (0.75–1.65), P = 0.60; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance CG: (D) OR (95 %CI) 0.84 (0.46–1.54), P = 0.57; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance GG: (R) OR (95 %CI) 1.69 (0.86–3.30), P = 0.12; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance Wan et al., 2015 [48] Exploratory analysis. Limitations reported by the Authors: small sample, the analysis could not be conducted on the specific tissue derived from trigeminovascular or cerebral biopsies,the blood DNA was from multiple cell lineages with likely different DNA methylation Migraineurs recruited at International Headache Center of Chinese PLA General Hospital. N = 26 Matched healthy controls with no significant differences in gender and age compared to migraineurs group. N = 25 Patients recruited at International Headache Center of Chinese PLA General Hospital DNA extraction from EDTA blood samples obtained through the cubital vein Analysis of CpG islands of the RAMP1 gene in a 3000 bp region including putative promoter sequences and the first exon Investigate if the methylation pattern of the promoter of RAMP1 gene in peripheral leukocyte is associated with migraine Low methylation trend without statistical significance in migraine vs. control (total average methylation level: 8.41 % ±1.92 % vs. Polymorphisms of the gene encoding CGRP A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance CC: (R) OR (95 %CI) 0.80 (0.30–2.11), P = 0.65; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance HWE tested. PHWE rs3781719 = 0.52; PHWE rs3754701 = 0.77; PHWE rs7590387 = 0.41. For only MOH patients: (PHWE rs3781719 = 0.18; PHWE rs3754701 = 1; PHWE rs7590387 = 0.69) No significant association for endpoint 1 [risk of inconsistent response to triptans (being consistent responders defined as patients experiencing a ≥2 point reduction after triptan administration in at least 2 out of 3 consecutive attacks)]; significant association of RAMP1rs7590387GG [OR (95 %CI) (R) 0.27 (0.13– 0.57) P = 0.0002)] for endpoint 2 (risk of trasformation of episodic migraine to MOH) rs3754701 [RAMP 1 (T > A)] TT; (A) OR (95 %CI) 0.90 (0.60–1.37), P = 0.63; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = TA; (D) OR (95 %CI) 0.73 (0.41–1.32), P = 0.30; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = log-additive model of AA; (R) OR (95 %CI) 1.19 (0.53–2.67), P = 0.66; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. A = Page 8 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Table 1 Main characteristics of the studies included in the analysis. General characteristics of studies included in the analysis. OR = odds ratio; CI = confidence interval; P = P value; HWE = Hardy–Weinberg equilibrium. (Continued) Study Design Disease (N) Control (N) Ethnicity Sample Variant Outcome Genotype 1 Genotype 2 Genotype 3 HWE Results log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance inheritance; D = dominant model of inheritance; R = recessive model of inheritance log-additive model of inheritance; D = dominant model of inheritance; R = recessive model of inheritance rs7590387 [RAMP 1 (C > G)] CC: (A) OR (95 %CI) 1.11 (0.75–1.65), P = 0.60; logistic regression analysis adjusted for triptan (frovatriptan vs. other triptans), and for polymorphisms, associated with triptan response. Polymorphisms of the gene encoding CGRP 9.90 % ± 3.88 %, P = 0.197) The ROC the curve and Youden criterion have been used for the determination of the optimum cut- off of methylation level No significant difference in the DNA methylation pattern of RAMP 1 between migraine and control groups Moreno- M d Prospective, b i l Diagnosis of h i i i Diagnosis of h i Caucasian h i i Genomic DNA i f rs3781719 (CALC A) Investigate the ff f 3781719 TT: 53.85 % d d TC: 38 46 % CC: 7 69 % All of the i i Significant differences in i k l k f DNA methylation o- domo 019 Prospective, observational, multicentre, study enrolling female participants. Diagnosis of chronic migraine according to ICHD- III edition, beta version. Diagnosis of chronic migraine according to ICHD- III Caucasian ethnicity and Spanish origin Genomic DNA extraction from peripheral blood anticoagulated rs3781719 (CALC A) Investigate the effect of rs3781719 on the response to OnabotulinumtoxinA TT: 53.85 % responders and 27.27 % non responders TC: 38.46 % responders and 63.63 % non responders CC: 7.69 % responders and 9.09 % non responders All of the variants wer in HWE Apart from rs222749 of Significant differences in risk to lack of response to onabotulim toxin A for the SNP rs3781719 of gene Page 9 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Table 1 Main characteristics of the studies included in the analysis. General characteristics of studies included in the analysis. OR = odds ratio; CI = confidence interval; P = P value; HWE = Hardy–Weinberg equilibrium. (Continued) Study Design Disease (N) Control (N) Ethnicity Sample Variant Outcome Genotype 1 Genotype 2 Genotype 3 HWE Results Sample power calculation reported classified as responders to onabotulinum toxinA (defined as defined as a reduction of at least 50 % in the number of monthly migraine days 3 months after the second administration. N = 117 edition, beta version. classified as non responders to onabotulinum toxinA. N = 33 in EDTA-K3 TRPV 1 gene. CALC A: OR (95 %CI) (dominant model) 3,11 (1,33 −7,26), (codominant model) 1,6 (0,85 −3,0), (recessive model) 1,2 (0,31 −4,71) Page 10 of 14 Page 10 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Scuteri et al. The Journal of Headache and Pain determined as chronic migraineurs using the ICHD-III edition, beta version. Polymorphisms of the gene encoding CGRP The demographic and clinical char- acteristics between case and control groups have re- sulted comparable, or subjected to further subgroup analyses, in the studies by Cargnin et al., Lemos et al., Menon et al., and Moreno-Mayordomo et al. Apart from multiple comparisons, adjusted analyses for confounding effects, e.g. for triptan type or for SLC6A4 STin2 VNTR and COMT val158me polymorphisms associated to trip- tan response [46], have been conducted in the studies by Cargnin et al., 2015, Moreno-Mayordomo et al., 2019, Menon et al., 2011 and Wan et al., 2015. Weak linkage disequilibrium, indirect genetic association with the true causal variant [49], has been reported in the study by Lemos et al., 2010. Linkage disequilibrium has been per- formed also in the study by Cargnin et al. and in the study by Sutherland et al. The methods of genotyping have been reported by all the studies. Compromised quality of DNA and successful genotyping have been re- ported and samples removed from analysis where occur- ring, e.g. Lemos et al., Menon et al., and Sutherland et al. On the contrary, no discrepancies in genotyping, that had even been re-conducted for validation in about 10 % of the samples, have been found in the study by Cargnin and coworkers. In the study by Guldiken and collaborators smoking and family history of vascular dis- ease were significantly more frequent in the migraine group, but no analysis adjusted for confounders has been reported. In the study by Moreno-Mayordomo and co- workers two of the considered variables have resulted to present significant differences after correction for mul- tiple comparisons between the groups of responders and non-responders. In the study by Cargnin and collabora- tors, the Authors report that the possibility that some in- dividuals present in the control sample might be affected by MOH cannot be excluded, but that this fraction would be unlikely to be higher than that observed in the general population. The sample power calculation has been reported in the studies by Lemos et al., Menon et al., Sutherland et al., Cargnin et al., Wan et al., and Moreno-Mayordomo et al. Polymorphisms of the gene encoding RAMP 1 y The SNP rs3754701, in the promoter region, and the rs7590387 of the gene encoding RAMP1 have been in- vestigated for the first time by Sutherland and collabora- tors [45], but no significant associations with migraine susceptibility have been identified. The SNP rs3754701 has been tested also with the SNP rs7590387 of RAMP 1 in migraineurs not presenting aura for association with response to triptans and as risk factors for MOH by Cargnin et al., [46]. Using the log-additive, the dominant and the recessive model of inheritance response to trip- tans has not been correlated, but the rs7590387G allele and the rs7590387GG genotype reduce significantly the risk of transformation of episodic migraine into MOH [OR (95 %CI) (R) 0.27 (0.13–0.57) P = 0.0002)] [46]. Inci- dentally, in patients affected by migraine a methylation trend (lower in females) at the promoter region of the gene encoding RAMP 1 without significant differences in the DNA methylation level has been detected by Wan et al., [48]. Polymorphisms of the gene encoding CGRP However, in the study by Lemos et al., the Authors highlight that sample size is a limitation of the study not providing enough power to detect a variant with an OR < 1.5 and that the study had a power of 64 % to detect an association with the in- cluded sample (for a nominal significance level of 0.05). Cargnin et al. report that the study is underpowered to detect small genetic main effects but, its power is suffi- cient for medium-large effect sizes of clinical relevance. Wan et al. state that sample size calculation could not be accurate since it was the first study investigating RAMP1 methylation pattern and that therefore a wider (dominant model) 3,11 (1,33 −7,26), (codominant model) 1,6 (0,85 −3,0), (recessive model) 1,2 (0,31 − 4,71)] in the study by Moreno-Mayordomo and co- workers [47]. Assessment of quality of the studies Interestingly, the relation- ship of this SNP with cerebral infarction has been exam- ined in a Japanese population, suggesting the T-A-C haplotype to represent a genetic marker for cerebral in- farction [56]. Only one study investigating the methyla- tion pattern with regard to CGRP pathway has resulted from the search. The results of this systematic analysis must be interpreted based on the characteristics of the included studies. In fact, it is important to notice that the genetic variations examined are different within the studies and, also when the same SNP has been investi- gated, the outcomes considered differed markedly among these studies. Also, the methods of analysis of the results do not make comparison feasible. Moreover, they have been performed on populations not compar- able for gender and ethnicity, thus not allowing the generalization of the results. Although the study design is the case-control typical to test for direct genetic asso- ciations and the overall quality of the studies meet lot of the HuGe criteria, some concern has been raised: the as- certainment of diagnosis in cases and the matching with controls is not always efficient and clearly reported; the features of migraine considered are different; the adjus- tement for confounders is not always present and not in each study all the parameters assessing the HWE and ef- ficiency of genotyping are reported completely. Together with the described heterogeneity between the studies, the small sample size and the lack of an adequate num- ber of references for power calculation is a fundamental issue strengthening the need for more investigation in this research field. This is more evident for the explora- tory analysis study by Wan et al. Often genetic associ- ation studies show initial significant association, but the latter needs to be confirmed by replication studies. These studies must be adequately powered to detect true associations, also in case of small effect conferred by genotype or allele, in order to exclude the risk of false- multiple cell lineages with likely different DNA methyla- tion and the issue concerned with the impossibility to conduct the analysis on the specific tissue derived from trigeminovascular or cerebral biopsies. All the studies are devoid of reporting bias. Assessment of quality of the studies The quality of the studies included in the present sys- tematic analysis has been assessed following the HuGE systematic reviews and meta-analyses risk-of-bias score for genetic association studies [38] considering the fol- lowing 4 outcomes: (1) Information bias, evaluating the accuracy of diagnosis of migraine, the ascertainment of controls matched to cases and the quality of genotyping; (2) Confounding bias, in which all the confounders e.g. population stratification, different ethnicity/gender, sam- ple power calculation and statistical adjustment for con- founders have been considered; (3) Selective reporting of outcomes, i.e. mentioning only significant associations with SNPs; (4) HWE assessment. The studies by Lemos et al., 2010, Guldiken et al., 2013, Sutherland et al., 2013, Cargnin et al., 2015 and Moreno-Mayordomo et al., 2019 do not present risk of bias since the criteria for diagnosis of cases vs. controls have been clearly stated and have followed the criteria of the IHS and of the ICHD in effect at the time of the study. In the other studies, clinical neurological assessment, also according to international criteria [43], has been reported, originat- ing unclear risk of bias. The ascertainment of sex, age and ethnicity matched controls has been mentioned in the study by Lemos et al., 2010, Menon et al., 2011, Sutherland et al., 2013, Cargnin et al., 2015, Wan et al., 2015. In the study by Moreno-Mayordomo et al., 2019 both responders and non responders have been Page 11 of 14 Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Scuteri et al. The Journal of Headache and Pain after cesarean section. [53]. The rs3781719C allele has been demonstrated to represent a risk factor for this postoperative chronic pain condition [53]. According to our analysis, the second most investigated SNP affecting the CGRP pathway is the rs7590387 of RAMP 1 locus [45, 46], that is another SNV (C > G), also in this case without a demonstrated clinical significance [54]. The latter had been previously investigated in a genome-wide association study finding correlation with migraine [55], but these data had not been published. Although no sig- nificant association with migraine susceptibility in gen- eral and both with and without aura [45] and with the response to triptans in all the three genotypes under study [46], the rs7590387G allele and the rs7590387GG genotype have been found to reduce significantly the risk of transformation MOH [46]. Assessment of quality of the studies The HWE has been assessed in all the studies and ROC curve and Youden criterion have been used for the determination of the optimum cut-off of methylation level in the study by Wan et al., but the PHWE values have been reported only in the studies by Cargnin et al. and Menon et al. Authors’ contributions All h h ib All authors have contributed equally to the work. All authors have read and approved the final manuscript. Abbreviations ACE i i Abbreviations ACE: angiotensin converting enzyme; AV: arterial vessels; BIOMIGA: BIOmarkers of MIGraine; CALCRL: human G protein-coupled recep- tor calcitonin receptor-like receptor; CDEM: chronic disorders with episodic manifestations; CI: confidence interval; CGRP: calcitonin-gene related peptide; COMT: catechol-O-methyltransferase; CSD: cortical spreading depression; Glu: glutamate; HLA: human leukocyte antigens; HuGE: Human Genome Epidemiology; HWE: Hardy–Weinberg equilibrium; mAbs: monoclonal antibodies; MAO: monoamine oxidase; MTHFR: methylenetetrahydrofolate reductase; MTR: methionine synthase; MTRR: methionine synthase reductase; NO: nitric oxide; OR: odds ratio; PICOS: participants/population, interventions, comparisons, outcomes, and study design; PRISMA: Preferred Reporting Items for Systematic reviews and Meta-Analyses; PROSPERO: International prospective register of systematic reviews; RAMP-1: receptor activity- modifying protein; SNP: single-nucleotide polymorphism; SNPs : single- nucleotide polymorphisms; YLD: Years Lived with Disability Discussion and conclusions Moreover, the first comprehensive genetic asso- ciation study of patients with chronic and high- frequency migraine has underscored rs2956 of CALC A gene and rs302680 of RAMP1 to be nominally associated with chronic migraine, although these associations have not resulted significant in the replication stage [57]. Therefore, a role of genetic variants affecting the CGRP pathway and, thus the responsiveness to therapeutics, emerges from this systematic review and it warrants fur- ther investigation. Remarkably, no genetic association studies investigating the possible effect of CGRP SNPs on the responsiveness to the most recent pharmaco- logical approaches, i.e. anti-CGRP(R) mAbs, gepants and ditans, have been found, apart from the ongoing INTE RROGATE trial. The latter mAbs are a fundamental weapon in the arsenal of migraine therapy even more for difficult-to-treat patients who do not find relief from treatment. In fact, the efficacy of mAbs targeting CGRP in refractory patients has provided very encouraging re- sults [58]. Furthermore, these mAbs are well tolerated and titres of neutralizing and not anti-mAbs antibodies that have been reported were low and not affecting sig- nificantly efficacy and safety [58, 59]. Therefore, the ex- istence of clinically relevant genetic variants along the CGRP signaling deserves further investigation since these could account for some percentage of non re- sponders to treatment with mAbs. In addition, several conditions may lead to lack of responsiveness with con- sequent persistent migraine [60]: it is possible that CGRP is not completely inhibited in its action or that, although fully blocked, adrenomedullin can still induce vasodilation through heteromerization of its receptor with CGRP(R). Furthermore, the role of other peptides, e.g. pituitary adenylate cyclase-activating polypeptide (PACAP) or the vasoactive intestinal peptide (VIP) de- serves investigation [60]. An attempt to predict the re- sponders to erenumab has highlighted that a lower baseline mean blood flow velocity in cerebral arteries is associated to increased effectiveness, with cerebral blood flow increase after treatment in good responders [61]. Incidentally, early non responders to galcanezumab can Acknowledgements D S i d f d D.S. is a post-doc funded by S. Anna Institute, Crotone, Italy. D.S. is a post-doc funded by S. Anna Institute, Crotone, Italy. Funding Not applicable. Open Access funding enabled and organized by Projekt DEAL. Discussion and conclusions The Journal of Headache and Pain provide a hypothesis of functional significance of the polymorphisms found in the context of CGRP signalling and migraine, it is mandatory to consider that the data that have resulted close to significance but without reaching it may have been affected by: (1) the small sam- ple size of the cohorts, underpowered to detect small genetic main effects and for further stratification ana- lysis; (2) the confounding influence of environmental factors. However, the results of this systematic review suggest a role of rs7590387GG of RAMP1 in the trans- formation of episodic migraine into MOH. Interestingly, the collected data have highlighted that rs3781719 of gene CALC A influences the response to onabotulinum- toxinA. Moreover, the first comprehensive genetic asso- ciation study of patients with chronic and high- frequency migraine has underscored rs2956 of CALC A gene and rs302680 of RAMP1 to be nominally associated with chronic migraine, although these associations have not resulted significant in the replication stage [57]. Therefore, a role of genetic variants affecting the CGRP pathway and, thus the responsiveness to therapeutics, emerges from this systematic review and it warrants fur- ther investigation. Remarkably, no genetic association studies investigating the possible effect of CGRP SNPs on the responsiveness to the most recent pharmaco- logical approaches, i.e. anti-CGRP(R) mAbs, gepants and ditans, have been found, apart from the ongoing INTE RROGATE trial. The latter mAbs are a fundamental weapon in the arsenal of migraine therapy even more for difficult-to-treat patients who do not find relief from treatment. In fact, the efficacy of mAbs targeting CGRP in refractory patients has provided very encouraging re- sults [58]. Furthermore, these mAbs are well tolerated and titres of neutralizing and not anti-mAbs antibodies that have been reported were low and not affecting sig- nificantly efficacy and safety [58, 59]. Therefore, the ex- istence of clinically relevant genetic variants along the CGRP signaling deserves further investigation since these could account for some percentage of non re- sponders to treatment with mAbs. In addition, several conditions may lead to lack of responsiveness with con- sequent persistent migraine [60]: it is possible that CGRP is not completely inhibited in its action or that, although fully blocked, adrenomedullin can still induce vasodilation through heteromerization of its receptor with CGRP(R). Furthermore, the role of other peptides, e.g. pituitary adenylate cyclase-activating polypeptide (PACAP) or the vasoactive intestinal peptide (VIP) de- serves investigation [60]. Discussion and conclusions An attempt to predict the re- sponders to erenumab has highlighted that a lower baseline mean blood flow velocity in cerebral arteries is associated to increased effectiveness, with cerebral blood fl i f i d d [6 ] turn into responders in the second/third month [62]. Also, recently an association between iron deposits in the periaqueductal gray of patients suffering from chronic migraine and poor response to onabotulinum- toxinA has been observed [63], pointing at the possible involvement of metal deposits in the responsiveness to anti-migraine treatments. Thus, it is fundamental to understand how SNPs and epigenetic modifications may affect the response to anti-migraine treatment, since most drugs directly or indirectly target the latter path- way, in order to predict treatment efficacy and safety. A window is opening on the association between disabling primary headache and preclinical familial Alzheimer’s disease [64]. Moreover, both dementia and migraine are main symptoms of CADASIL (cerebral autosomal dom- inant arteriopathy with subcortical infarcts and leukoen- cephalopathy, OMIM#125,310) [65]. Therefore, the genetic association of these widespread disabling dis- eases needs to be studied. Furthermore, aged popula- tions need to be included in migraine genetic association studies, as well as in clinical trials for migraine treat- ment [66–71]. Hence, adequately powered studies fol- lowing the criteria for rigorous genetic association are needed to provide high quality evidence on the impact of genetic variants affecting CGRP signaling on migraine susceptibility and clinical manifestation and to predict the response to therapy in terms of efficacy and safety. provide a hypothesis of functional significance of the polymorphisms found in the context of CGRP signalling and migraine, it is mandatory to consider that the data that have resulted close to significance but without reaching it may have been affected by: (1) the small sam- ple size of the cohorts, underpowered to detect small genetic main effects and for further stratification ana- lysis; (2) the confounding influence of environmental factors. However, the results of this systematic review suggest a role of rs7590387GG of RAMP1 in the trans- formation of episodic migraine into MOH. Interestingly, the collected data have highlighted that rs3781719 of gene CALC A influences the response to onabotulinum- toxinA. Discussion and conclusions Interest in migraine and in SNPs likely linked to suscep- tibility to its development and clinical features has been growing over the last years. However, little is known about the clinical relevance of these polymorphisms and their effect on the response to anti-migraine treatment. Moreover, the new era of migraine preventative treat- ments has been opened by the anti-CGRP(R) mAbs, but some 40 % non-responders still represent a pharmaco- logical unmet need. This is the first systematic review that intends to identify SNPs affecting different segments of the CGRP pathway and assessing how they can influ- ence migraine from its development, to the presence of aura and to the efficacy and safety of treatment. From an initial screening of the 800 records identified through database searching, only 7 studies met the inclusion cri- teria. This is the first obvious red flag that SNPs con- cerned with the CGRP signaling of clinical relevance are poorly investigated. Within these 7 studies for a total of 2413 patients SNPs and methylation affecting CALC A and RAMP 1 genes have been detected and the most re- curring is the rs3781719 exchange (T > C), a single nu- cleotide variation (SNV) originating a 2KB upstream variant in the CALC A gene promoter, of which clinical significance is not determined [50]. From our analysis no significant association has been found between the SNV rs3781719 and the clinical characteristics of mi- graine manifestation, i.e. increased frequency of mi- graine, presence of aura and gender differences [45]. Not even any correlations with responsiveness to triptans under the log-additive, the dominant and the recessive model of inheritance have been highlighted [46], but it has been associated with an increased risk of lack of re- sponse to onabotulinumtoxinA [51]. These results have been obtained in population of different gender and eth- nicity, being Australian [45], Italian [46] and females of Spanish origin [51]. However, from the study by Guldi- ken and coworkers we have learnt that the rs3781719 T- 692 C is not associated to migraine attacks frequency and severity and to the occurrence of aura in Turkish fe- males [52]. Incidentally, the effects of CALC A rs3781719 (-692T > C) have been studied in Chinese Han women with chronic postsurgical pain 6 months Scuteri et al. The Journal of Headache and Pain (2021) 22:87 Page 12 of 14 Page 12 of 14 Scuteri et al. 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J Headache Pain 8(6):334–339 Author details 1Pharmacotechnology Documentation and Transfer Unit, Preclinical and Translational Pharmacology, Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy. 2Regional Center for Serious Brain Injuries, S. Anna Institute, Crotone, Italy. 3Department of Health Sciences, University “Magna Graecia” of Catanzaro, 88100 Catanzaro, Italy. 4German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. 22. Simmaco M, Borro M, Missori S, Martelletti P (2009) Pharmacogenomics in migraine: catching biomarkers for a predictable disease control [corrected]. Expert Rev Neurother 9(9):1267–1269 23. Capi M, Gentile G, Lionetto L, Salerno G, Cipolla F, Curto M et al (2018) Pharmacogenetic considerations for migraine therapies. Expert Opin Drug Metab Toxicol 14(11):1161–1167 Received: 23 May 2021 Accepted: 12 July 2021 Received: 23 May 2021 Accepted: 12 July 2021 24. 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Reproducible Quantification of the Microstructure of Complex Quenched and Quenched and Tempered Steels using Modern Methods of Machine Learning
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metals Article Reproducible Quantification of the Microstructure of Complex Quenched and Quenched and Tempered Steels Using Modern Methods of Machine Learning Björn-Ivo Bachmann 1,2 , Martin Müller 1,2 , Dominik Britz 2 , Thorsten Staudt 3 and Frank Mücklich 1,2, * 1 2 3 * Citation: Bachmann, B.-I.; Müller, M.; Britz, D.; Staudt, T.; Mücklich, F. Reproducible Quantification of the Microstructure of Complex Quenched and Quenched and Tempered Steels Using Modern Department of Materials Science, Saarland University, 66123 Saarbrücken, Germany; bjoernivo.bachmann@uni-saarland.de (B.-I.B.); martin.mueller1@uni-saarland.de (M.M.) Materials Engineering Saarland (MECS), 66123 Saarbrücken, Germany; d.britz@mec-s.de Aktien-Gesellschaft der Dillinger Hüttenwerke, 66763 Dillingen, Germany Correspondence: frank.muecklich@uni-saarland.de; Tel.: +49-681-302-70500 Abstract: Current conventional methods of evaluating microstructures are characterized by a high degree of subjectivity and a lack of reproducibility. Modern machine learning (ML) approaches have already shown great potential in overcoming these challenges. Once trained with representative data in combination with objective ground truth, the ML model is able to perform a task properly in a reproducible and automated manner. However, in highly complex use cases, it is often not possible to create a definite ground truth. This study addresses this problem using the underlying showcase of microstructures of highly complex quenched and quenched and tempered (Q/QT) steels. A patch-wise classification approach combined with a sliding window technique provides a solution for segmenting entire microphotographs where pixel-wise segmentation is not applicable since it is hardly feasible to create reproducible training masks. Using correlative microscopy, consisting of light optical microscope (LOM) and scanning electron microscope (SEM) micrographs, as well as corresponding data from electron backscatter diffraction (EBSD), a training dataset of reference states that covers a wide range of microstructures was acquired in order to train accurate and robust ML models in order to classify LOM or SEM images. Despite the enormous complexity associated with the steels treated here, classification accuracies of 88.8% in the case of LOM images and 93.7% for high-resolution SEM images were achieved. These high accuracies are close to super-human performance, especially in consideration of the reproducibility of the automated ML approaches compared to conventional methods based on subjective evaluations through experts. Methods of Machine Learning. Metals 2023, 13, 1395. https:// doi.org/10.3390/met13081395 Keywords: microstructure classification; microstructure segmentation; machine learning; quenched steel; martensite; bainite Academic Editor: João Manuel R. S. Tavares Received: 7 July 2023 1. Introduction Revised: 25 July 2023 Due to the excellent combination of properties, the ability to selectively adjust the desired properties at relatively low costs, and the high sustainability in its value chain through high recyclability, steel is justifiably one of the most widely used materials of our time. The complexity of microstructures in modern high-strength steels is increasing enormously because of ever-higher demands on the material and constant optimization of the manufacturing processes as well as ever-tighter tolerances in quality control due to customer demands. Thus, modern steels have a large number of different microstructural constituents, not all of which can be easily distinguished from one another. Reliable analysis and characterization of the complex steel microstructure are essential to establish, on the one hand, a well-founded development of new materials accompanied by substantial quality control, as well as later process–property correlations to better understand the influences of the process parameters on the resulting microstructure and ultimately, on the properties of the material. The status quo of conventional microstructure analysis is still Accepted: 27 July 2023 Published: 3 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Metals 2023, 13, 1395. https://doi.org/10.3390/met13081395 https://www.mdpi.com/journal/metals Metals 2023, 13, 1395 2 of 27 characterized by a high degree of subjectivity, as well as the experience of the respective metallographer, and is clearly limited by the resolution of the methodology used. Thus, the microstructures are usually evaluated only qualitatively, or only rough estimates of the fractions of the microstructural constituents are given. The experts’ assessments are also characterized by poor reproducibility and a lack of objectivity. This also depends strongly on the type and quality of the underlying etching method used to observe the steels. The use of modern computer vision approaches holds enormous potential for serial application in microstructure analysis, especially in the quantitative evaluation of those, which still is subject to a high degree of subjectivity and a lack of reproducibility. Previous publications demonstrate the valuable advantages of machine learning (ML) in microstructural analysis, particularly the increase in efficiency through automation, as well as the associated reproducibility combined with objectivity, insofar as the ground truth is well-funded. Stuckner et al. [1] could transfer common deep learning (DL) approaches to successfully segment microstructures based on a large microscopy dataset in general. De Cost et al. [2] were able to distinguish different microstructural image data classes through an image classification approach according to general microstructural classes, such as brass, different types of cast iron, and hypoeutectoid steel, among others. More specifically, in the case of steels, Azimi et al. [3] could implement a sophisticated approach in order to segment more complex microstructures: they succeeded in performing a pixel-wise segmentation approach using a fully connected neural network (FCNN) in dual-phase steels with the aid of correlative microscopy using a light optical microscope (LOM) and scanning electron microscope (SEM) images. The final model was able to successfully and robustly segment the matrix as well as the second phase based on microscopic images and identify the second phase object according to the distinguished classes martensite, tempered martensite, bainite, and pearlite. Müller et al. [4] extended this classification approach and further differentiated between very complex bainite subclasses. There, morphological features, as well as textural features, were used in a traditional ML approach. In the case of even more complex segmentation problems, UNets, which originally were developed in order to segment medical images [5], could show their superior performance on microstructural images. Through a sophisticated approach, correlative electron backscatter diffraction (EBSD) measurements could help to create objective ground truths in the form of annotated masks to train UNets for semantic segmentation. Here, Durmaz et al. [6] succeeded in training the complex features of lath-shaped bainite to a DL model capable of separating it from polygonal ferrite. Work on ML-based classification approaches for quenched steels is still limited to date. Tsuitsui et al. [7] used specially heat-treated low-carbon steels to classify entire types of microstructures using SEM images with the aid of texture-based information in the form of Haralick features. A similar approach was followed by Zhu et al. [8], who compared classification based on textural features using conventional ML with DL approaches in the form of a convolutional neural network (CNN) for feature extraction. Bachmann et al. [9] succeeded in using EBSD reconstructions to automatically create masks for an efficient and reproducible segmentation of prior austenite grain boundaries (PAGB) based on Nital-etched microstructural images of quenched steels using only optical microscopy. The authors have no knowledge of other approaches that aim at a direct multiclass segmentation of entire microstructural images, neither on the basis of LOM nor SEM images of quenched or quenched and tempered steels. This would be of great added value for quality control and microstructure-based process development of these complex steels. The steels investigated in this work are quenched, as well as quenched and tempered steels with low carbon contents. This type of steel is characterized by a particularly high degree of complexity since the constituents of the steel often differ morphologically only in very fine features. The most common phases found in quenched and tempered steels are martensite, tempered martensite, lower bainite, and partially upper bainite, illustrated in Figure 1. Metals 2023, 13, x FOR PEER REVIEW Metals 2023, 13, 1395 3 of 28 3 of 27 are martensite, tempered martensite, lower bainite, and partially upper bainite, illustrated in Figure 1. Martensite is formed during cooling from the high-temperature phase austenite at very high coolingisrates. Theduring cooling rate required martensitic transformation depends Martensite formed cooling from thefor high-temperature phase austenite at very high cooling Thecomposition cooling rate required for Martensite martensiticis transformation mainly mainly on the rates. chemical of the steel. identifiable bydepends its plate/lathon structure. the chemical of the steel. identifiable bymartensitic its plate/lath-like like Duecomposition to the diffusionless and Martensite displacing is character of the transstructure. the Due to thehas diffusionless and displacing character of the martensitic transforformation, carbon no time to diffuse or to precipitate in the form of carbides but is mation,dissolved the carbon has no diffuse or to precipitate in the distortion form of carbides but is forcibly within thetime solidtosolution, leading to a tetragonal of the cubic forcibly dissolved within the solid solution, leading to a tetragonal distortion of the cubic lattice [10]. The individual martensite laths can be identified with the help of the clearly lattice [10]. topographical The individualdifferences martensiteafter lathscontrasting. can be identified with the help of the clearly pronounced pronounced contrasting. Temperedtopographical martensite isdifferences formed byafter tempering processes within the martensite. This Tempered martensite is formed within the martensite. This tempered state can be created either bybyantempering annealingprocesses process adjacent to the cooling step tempered state can be created either by an annealing process adjacent to the cooling step or by self-tempering effects due to residual heat within the material during the coolingor by self-tempering effects due to residual heat within the material during the cooling process process itself [11]. During the tempering, the trapped carbon precipitates from the tetragitself [11]. During the tempering, the trapped carbon precipitates from the tetragonally onally distorted lattice by temporarily allowing diffusion in the form of carbides, which distorted lattice by distinguishing temporarily allowing diffusion in the form of carbides, which represents represents its main criterion from conventional martensite. its main distinguishing criterion from conventional martensite. Bainite formation exhibits both a diffusion-controlled and a displacive character [12]. Bainite formation exhibits both a diffusion-controlled andofa martensite displacive character Thus, it also forms at cooling rates intermediate between those and those[12]. of Thus, it also forms at cooling rates intermediate between those of martensite those of fully diffusion-controlled pearlite. Lower bainite is constituted of lath-shaped and featureless fully diffusion-controlled pearlite.within Lowerthe bainite is laths. constituted of lath-shaped featureless ferrite with cementite precipitated ferrite The upper bainite is composed ferrite with cementite precipitated within the ferrite laths. The upper bainite is composed of similar lath-like ferrite with continuous cementite precipitates at the boundaries of the of similar lath-like ferrite with continuous cementite precipitates at the boundaries of the laths. Hereby, the cooling rate of lower bainite is higher than the cooling rate at which laths. Hereby, the cooling rate of lower bainite is higher than the cooling rate at which upper bainite forms. The resulting more restricted diffusion is thus responsible for the upper bainite forms. The resulting more restricted diffusion is thus responsible for the different appearances of cementite precipitation within the different bainitic phases. For different appearances of cementite precipitation within the different bainitic phases. For the sake of simplicity and clarity, no further subdivision, as seen in [4], is used in this study the sake of simplicity and clarity, no further subdivision, as seen in [4], is used in this study investigating complex Q/QT steels. investigating complex Q/QT steels. Figure Figure1.1.Schematic SchematicIllustration Illustrationofofthe themorphology morphologyofofthe thecommon commonmicrostructural microstructuralconstituents constituents (martensite and tempered martensite—modified from [13], and upper and (martensite and tempered martensite—modified from [13], and upper andlower lowerbainite—modified bainite—modified from steels. from[14] [14]ininQ/QT Q/QT steels. Despite Despitethe thedifferent differentmicrostructure microstructuredevelopment developmentmechanisms, mechanisms,allallthese thesedifferent different phases can occur simultaneously within a microstructure in industrial plates. phases can occur simultaneously within a microstructure in industrial plates.The Themain main reasons reasonsfor forthis thisare arethe thelimited limitedthermal thermalconductivity conductivityassociated associatedwith withhigh highmaterial materialthickthicknesses nessesand andchemical chemicalirregularities irregularitiessuch suchasassegregation segregationleading leadingtotoinhomogeneous inhomogeneouscritical critical cooling coolingrates ratesalong alongthe theplate platethickness. thickness.For Forthe thereproducible reproduciblecharacterization characterizationofofthese these highly highlycomplex complexmicrostructures, microstructures,a aprecise preciseidentification identificationofofthe thefine finedifferences differencesisisrequired required totoidentify identifythe thepresent presentphases. phases. The Thebiggest biggestobstacle obstacleininapplying applyingcomputer computervision visionapproaches approachestotothe thefield fieldofofmaterials materials science scienceisisestablishing establishinga arepresentative representativeand andobjective objectiveground groundtruth. truth.InInevery everyone oneofofthe the aforementionedworks, works,ititwas waspossible possibletotocreate createappropriate appropriatetraining trainingmasks maskswith withthe thehelp help aforementioned correspondingdomain domainexpertise, expertise,asaswell wellasascorrelative correlativemicroscopy, microscopy,ififnecessary. necessary.This This ofofcorresponding allowedDL DLmodels modelstotobebetrained trainedininorder ordertotobebeable abletotoapply applythe thelearned learnedknowledge knowledgetoto allowed unseenimages imagesand andsegment segmentthem thementirely. entirely. unseen Unfortunately, this is not readily possible with the complex microstructures of Q/QT steels. Even based on the high-resolution SEM images, there are many regions where a clear and doubtless assignment to a class is not possible. The identification of the boundaries between the different structural components is particularly complex, as they Metals 2023, 13, 1395 4 of 27 frequently merge into one another. This is due to the formation process of the microstructure components. Diffusion in quenched steels is strongly limited, and even small differences in local chemical composition have a significant influence on the microstructure development. Furthermore, the characteristic morphological features of the different phases are mostly, if at all, only visible in high-resolution images. These include the spatial distribution and the shape of the carbide precipitates. Only based on LOM images in most cases, even experienced experts cannot clearly identify these characteristics. To make the analysis of such complex microstructures more reproducible and efficient, this work aims to combine modern methods of microstructure analysis with promising deep learning approaches from the field of computer vision. 2. Materials and Methods 2.1. Material Due to the high level of complexity combined with a variety of different manifestations of the individual microstructural constituents in QT steels, sample selection is critical to the success of the interdisciplinary approach. In order to cover a sufficient number of features that occur in industrial QT steels, a total of 22 specimens were fully investigated. These include 10 industrial samples from heavy plates taken after being thermo-mechanically treated and 12 dilatometry samples. All samples have a carbon content between 0.16 and 0.22%. The dilatometry samples were cooled down continuously at different cooling rates (8–278 K/s) after being fully austenitized at 1273 K for 10 min. In addition, the sample with the highest cooling rate, entirely consisting of martensite, was annealed at 773 K for 60 min to form reference sections of tempered martensite. Furthermore, other reference samples of the same chemical composition were isothermally cooled to generate additional bainitic reference states. After austenitizing, these were cooled at 50 K/s to the respective holding temperature (698 K, 748 K, 798 K) and held there for a certain time (300 s, or 500 s at 798 K) and then cooled again at 50 K/s to room temperature. All dilatometry samples used in the scope of this study are taken from [4]. The industrial samples were taken from five different heavy plates with thicknesses of 15 mm, 20 mm, 30 mm, and 180 mm. The latter was divided into five different samples in order to map the entire plate thickness. The heavy plates went through different process routes, including direct quenching and conventional quenching, and partially, subsequent annealing. In the case of direct quenching, the quenching process immediately takes place after the thermomechanical rolling. For conventional quenching, the heavy plate gets time to cool down after rolling before it is heated up again to be quenched [15]. Due to the high material strength of the 180 mm heavy plate and the hereby limited heat transfer, selfannealing effects after the quenching influenced the material’s microstructure significantly. Overall, the hereby used materials mainly consist of martensite (M), tempered martensite (MST), and lower (LB), as well as upper bainite (UB). Figure 2 shows respective LOM and SEM images of representative and homogeneous areas of some of the specimens used for this study, illustrating distinct features of the different microstructural constituents. The different colorations in the micrographs are attributed to different phases, originating from different dissolved carbon concentrations, as well as to an orientation influence [16,17]. The high resolution of the SEM images reveals the highly complex features of each phase. Only with the help of those fine details a clear identification of the different microstructural sections can be made. Figure 2a illustrates the significant coloring of a martensitic microstructure in combination with the strongly pronounced topography of the disordered needle structure. MST in (b) shows a decrease in coloration due to the tempering process. Nevertheless, the coloration is much stronger than with LB or UB. In the SEM image, the topography and the disordered carbide precipitates can be identified. In the case of the LB (c) and the UB (d), the ordered lath structure is noticeable. The main focus here is on the differences in the carbide precipitates. In LB, shown in (c), the carbon is precipitated in a preferential orientation within the laths in the Metals 2023, 13, 1395 stronglypronounced pronouncedtopography topographyofofthe thedisordered disorderedneedle needlestructure. structure.MST MSTinin(b) (b)shows showsa a strongly decreaseinincoloration colorationdue duetotothe thetempering temperingprocess. process.Nevertheless, Nevertheless,the thecoloration colorationisismuch much decrease strongerthan thanwith withLB LBororUB. UB.InInthe theSEM SEMimage, image,the thetopography topographyand andthe thedisordered disorderedcarcarstronger bideprecipitates precipitatescan canbebeidentified. identified.InInthe thecase caseofofthe theLB LB(c)(c)and andthe theUB UB(d), (d),the theordered ordered 5 of 27 bide lathstructure structureisisnoticeable. noticeable.The Themain mainfocus focushere hereisisononthe thedifferences differencesininthe thecarbide carbidepreprelath cipitates. In LB, shown in (c), the carbon is precipitated in a preferential orientation within cipitates. In LB, shown in (c), the carbon is precipitated in a preferential orientation within thelaths laths theform formofof finecarbides. carbides. thecase case ofUB, UB, shown (d),coherent coherent carbides the ininthe InInshown the shown inin(d), carbides form of fine carbides. Infine the case of UB, inof (d), coherent carbides form between the form between the bainitic laths. form between the bainitic laths. bainitic laths. Figure2.2.2.Representative Representativemicrographs micrographs from LOM (upper row) andcorresponding corresponding SEMimages images Figure micrographs from LOM (upper row) Figure Representative from LOM (upper row) andand corresponding SEM SEM images (lower (lower row) showing the described microstructural constituents: (a)—martensite, (b)—tempered (lower row) showing the described microstructural constituents: (a)—martensite, (b)—tempered row) showing the described microstructural constituents: (a)—martensite, (b)—tempered martensite, martensite, (c)—lowerbainite, bainite, (d)—upperbainite. bainite. martensite, (c)—lower(c)—lower bainite, (d)—upper(d)—upper bainite. Figure3,3, 3,on onthe theother otherhand, hand,shows showsareas areaswhere whereaaaclear clearidentification identificationofof ofthe thepresent present Figure Figure on the other hand, shows areas where clear identification the present phases and, especially, an identification of the borders between them is no longer possible phases phasesand, and,especially, especially,an anidentification identificationofofthe theborders bordersbetween betweenthem themisisno nolonger longerpossible possible withoutfurther furtherado ado(red (redarrows). arrows).Thus, Thus,various varioussections sectionsshow showcharacteristic characteristicfeatures featuresofof of without without further ado (red arrows). Thus, various sections show characteristic features differentmicrostructural microstructuralconstituents constituentsatatthe the same time. Furthermore, the characteristic ardifferent same time. Furthermore, the characteristic ardifferent microstructural constituents same time. Furthermore, the characteristic areas easare areextremely extremely diffuse andinterwoven interwoven thatno noclear clear boundaries can drawn. Aneas diffuse sosothat boundaries bebedrawn. Anare extremely diffuse andand interwoven so that no clear boundaries cancan be drawn. Another otherproblem problem lies themulti-layered multi-layered contrasts thedifferent different microstructural constituother lies inin the contrasts ofofthe microstructural constituproblem lies in the multi-layered contrasts of the different microstructural constituents, ents,which which areintensified intensified onthe the one hand theunderlying underlying contrasting technique, which are intensified on theon one hand by the underlying contrasting technique, which is ents, are one hand bybythe contrasting technique, which well known belimited limited interms termsofofreproducibility, reproducibility, andon onthe the other handbyby well known toknown be limited terms ofinreproducibility, and on the other hand by the respective which isiswell totobein and other hand therespective respective imaging settingsofofthe themicroscopes. microscopes. imaging settings of thesettings microscopes. the imaging Figure 3. Representative micrographs from LOM (upper row) and corresponding SEM images (lower row) showing more complex microstructures of the materials investigated containing mixtures of different phases (labeled arrows) as well as no clear borders between them and respective prior austenite grain boundaries (PAGB—marked with black arrows). Red arrows mark regions where a clear identification is not possible. Metals 2023, 13, 1395 6 of 27 2.2. Correlative Microscopy In order to guarantee a reliable application of modern DL approaches in the field of material science, high-quality data are essential. To establish an objective and wellfounded ground truth, it is often not sufficient to limit oneself to a single methodology used for such complex steels, as demonstrated in Figures 2 and 3. Therefore, all samples were characterized using LOM, SEM, as well as EBSD. In correlative microscopy, these methods were combined to benefit from the advantages of the different approaches and to eliminate their respective disadvantages and thereby overcome their limits. In this way, the complementary information necessary for a holistic characterization can be collected on different length scales and from different contrasting mechanisms. However, the goal behind the correlative microscopy approach is to apply the detailed information obtained from more advanced methods to the simplest methodology possible. All specimens were first ground with 80–1200 grit SiC abrasive paper and then subjected to diamond polishing using 6, 3, and 1 µm suspension. Subsequently, the sample preparation was completed by a colloidal OPS polish to achieve the best possible surface quality for the subsequent EBSD measurement. The respective region of interest (ROI) with a size of 400 × 400 µm, at which the correlative microscopy is being conducted, are marked using hardness indentations. After each EBSD measurement, which was made on a Zeiss Merlin (Zeiss, Oberkochen, Germany) with an EDAX detector (EDAX, Pleasanton, CA, USA) under an accelerating voltage of 25 kV and a beam current of 10 nA at a working distance of 15 mm with a step size of 0.35 µm, the sample was briefly polished again using OP-S to remove the contamination layer before further optical examination. The EBSD data were post-processed with the help of the EDAX OIM software (Version 7, TSL Solutions, Nishihashimoto, Japan). For this purpose, a standard routine with a filter operation for poorly indexed measurement points was applied. Subsequently, the samples were contrasted for 25 s using a 2.5% alcoholic Nital solution, and the micrographs of the respective ROIs were captured in LOM and SEM. As LOM, an Olympus LEXT OLS 4100 laser scanning microscope (Olympus, Shinjuku, Japan) was used, and the images were taken at a 1000× magnification, resulting in a pixel size of 126.6 nm. The SEM images were acquired using a ZEISS Supra SEM (Zeiss, Oberkochen, Germany) using a secondary electron contrast, using an acceleration voltage of 5 kV and a working distance of 5 mm, at a magnification of 850× with a respective image size of 2048 × 1536 px corresponding to a pixel size of 47.5 nm. The brightness and contrast settings were adjusted so that the gray-level histogram was approximately normally distributed. In order to capture an entire ROI, several single images need to be taken with a respective overlap and, subsequently, stitched together. Therefore, Microsoft Image Composite Editor was used. In order to superimpose the different image data congruently, image registration is necessary due to the different contrast generation mechanisms as well as unequal perspectives on the respective sample location. For the registration operation, the ImageJ [18] Plugin bUnwarpJ [19], as proposed in [16,20], was used. In contrast to their presented procedure, however, the individual features within the different images had to be selected manually. No corresponding features could be found using common automated feature extraction algorithms such as SIFT (Scale-Invariant Feature Transform), due to the high complexity, as well as the fine visual features of the microstructural images of the investigated steels. First, the high-resolution SEM image was registered on the corresponding image quality (IQ) map of the EBSD measurement. Afterward, the LOM micrograph was registered on the already registered SEM image. Another obstacle here is the different resolutions of the individual methods: either the lower-resolution method must be scaled up and thus interpolated, or important details of the higher-resolution methods may be lost when scaling down. Since the microscopy images are later used in DL algorithms, it is reasonable to adjust, for the sake of simplicity, Metals 2023, 13, 1395 image quality (IQ) map of the EBSD measurement. Afterward, the LOM micrograph was registered on the already registered SEM image. Another obstacle here is the different resolutions of the individual methods: either the lower-resolution method must be scaled up and thus interpolated, or important details of the higher-resolution methods may be lost when scaling down. Since the microscopy 7 of 27 images are later used in DL algorithms, it is reasonable to adjust, for the sake of simplicity, the image dimensions to powers of 2. Here, it is recommended to deviate as little as possible from the native resolutions of the microscopes. the image dimensions to powers of 2. Here, it is recommended to deviate as little as possible Hence, the final DL approaches take LOM as well as SEM images as input; the EBSD from the native resolutions of the microscopes. mappings will be resized to the native resolution of LOM/SEM in order to properly create Hence, the final DL approaches take LOM as well as SEM images as input; the EBSD the ground truth annotations using all complementary information. This results in image mappings will be resized to the native resolution of LOM/SEM in order to properly create dimensions of 4096 × 4096 px for LOM and 8192 × 8192 px for SEM for respective imaging the ground truth annotations using all complementary information. This results in image of the mentioned sample sections of 400 × 400 µm. The EBSD mappings with a native dimensions of 4096 × 4096 px for LOM and 8192 × 8192 px for SEM for respective imaging resolution of 1320 × 1320 px, measured in a hexagonal grid using the mentioned step size of the mentioned sample sections of 400 × 400 µm. The EBSD mappings with a native of 0.35 µm, were resized accordingly. resolution of 1320 × 1320 px, measured in a hexagonal grid using the mentioned step size Thus, the correlative datasets of the respective samples were aligned and congruent of 0.35 µm, were resized accordingly. to beThus, used directly as complementary information for the later used in the correlative datasets of the respective samples wereannotations aligned andbeing congruent to the deep learning methodology. be used directly as complementary information for the later annotations being used in the 4 shows a section of a correlative data set consisting of LOM, SEM, and EBSD deepFigure learning methodology. data Figure containing all phases to be There, the corresponding added value of 4 shows a section of adistinguished. correlative data set consisting of LOM, SEM, and EBSD the information for the identification of the occurring phases datacomplementary containing all phases to be distinguished. There, the corresponding addedbecomes value of clear. With the help of the high resolution of the SEM, fine carbide precipitates can be the complementary information for the identification of the occurring phases becomes clearly identified, and thus LB and MST can be reliably detected. In addition, a distinction clear. With the help of the high resolution of the SEM, fine carbide precipitates can be between LB and UB possible based thebelocalization and orientation of athe fine carclearly identified, andisthus LB and MSToncan reliably detected. In addition, distinction bides, which would not always be the case based purely on light microscopy images. Furbetween LB and UB is possible based on the localization and orientation of the fine carbides, thermore, contrasting artifacts, as well ambiguous can beFurthermore, considered which would not always be the case basedaspurely on lightmorphologies, microscopy images. with the help of crystallographic EBSD information. Though, theconsidered most helpful information contrasting artifacts, as well as ambiguous morphologies, can be with the help of from EBSD data EBSD for identifying the Though, differentthe microstructural constituentsfrom is provided by crystallographic information. most helpful information EBSD data the misorientation. for identifying the different microstructural constituents is provided by the misorientation. Figure 4. Excerpt Excerpt of of aa correlative correlative dataset dataset consisting consisting of LOM (a), IQ Map (b), SEM SEM (c), kernel kernel average ◦ misorientation (KAM) (d), and thresholded misorientation borders (red—2°, green—5°, misorientation (KAM) (d), and thresholded misorientation borders (red—2 , green—5◦ , blue—15°) blue—15◦ ) (e) overlayed with SEM, respectively. This region contains representative areas of all four considered microstructural constituents (labeled arrows). The magnifications show the slight differences between Martensite (green) and tempered Martensite (red) with the finely precipitated carbides (red arrows). Metals 2023, 13, 1395 (e) overlayed with SEM, respectively. This region contains representative areas of all four considered microstructural constituents (labeled arrows). The magnifications show the slight differences between Martensite (green) and tempered Martensite (red) with the finely precipitated carbides (red arrows). 8 of 27 2.3. Used Quantification Approach: Patchwise Classification as Alternative to Semantic Segmentation 2.3. Used Quantification Approach: Patchwise Classification as Alternative to Semantic segmentation is the common DL approach for segmenting more complex Semantic Segmentation problems that cannot be solved conventional approaches, such as more threshold segSemantic segmentation is theusing common DL approach for segmenting complex mentation. It is a pixelwise classification: each pixel of the image is assigned a class, and problems that cannot be solved using conventional approaches, such as threshold segmenthereby, the entire image is segmented it assigned is possible to carry segtation. It is a pixelwise classification: each[21]. pixelAdditionally, of the image is a class, and out thereby, mentation with more than two classes in the same segmentation step, which offers great the entire image is segmented [21]. Additionally, it is possible to carry out segmentation added value. Intwo order to train a model for semantic segmentation, masks must be created with more than classes in the same segmentation step, which offers great added value. in which every pixel can clearly be assigned to a specific class. In order to train a model for semantic segmentation, masks must be created in which every segmentation problems, pixel In cancontrast clearly to bemost assigned to a specific class. for the microstructures of Q/QT steels, it is notInstraightforward tosegmentation create unambiguous masks train a DL segmentation approach. contrast to most problems, for to the microstructures of Q/QT steels, of dual-phasetoand complex-phase steels, thetocomplementary informationapof itInisthe notcase straightforward create unambiguous masks train a DL segmentation the correlative images is sufficient to complex-phase clearly identify steels, the respective microstructural conproach. In the case of dual-phase and the complementary informastituents. There, the images respective phases to can be clearly frommicrostructural each other by tion of the correlative is sufficient clearly identifyseparated the respective grain/phase boundaries [3,6]. Morphology and corresponding misorientation information constituents. There, the respective phases can be clearly separated from each other by allow us to boundaries confidently [3,6]. differentiate one phase from another. Due to the complex forgrain/phase Morphology and corresponding misorientation information mation Q/QT steelsone andphase the resulting interwoven of the microallow usmechanisms to confidentlyindifferentiate from another. Due to structure the complex formation structural constituents, it is not to reproducibly the of boundaries of the difmechanisms in Q/QT steels andpossible the resulting interwovendefine structure the microstructural ferent phaseitregions. In addition, various areas ofthe theboundaries microstructure cannot be constituents, is not possible to reproducibly define of theoften different phase clearly assigned to a various corresponding Even crystallographic EBSD as well regions. In addition, areas ofclass. the microstructure often cannot beinformation, clearly assigned to aascorresponding Even crystallographic EBSD information, as well asannotation corresponding correspondingclass. high-resolution imaging techniques, often do not allow with high-resolution imaging techniques, do not allow annotation required confirequired confidence according to the often common classification schemes,with which is illustrated dence according to the common classification schemes, which is illustrated in Figure 5. in Figure 5. Figure 5. 5. Excerpt from two different samples (in a(in row) containing LOMLOM (a,d) Figure Excerptof ofcorrelative correlativedatasets datasets from two different samples a row) containing as well as SEM (b,e) micrographs and the corresponding IQ overlayed with inverse pole figure (IPF) (a,d) as well as SEM (b,e) micrographs and the corresponding IQ overlayed with inverse pole figure (c,f). Many different microconstituents can be identified (MST—purple, LB—green, UB—blue), but (IPF) (c,f). Many different microconstituents can be identified (MST—purple, LB—green, UB—blue), also regions that cannot properly be identified (red—dashed). Furthermore, the underlying problem but also regions that cannot properly be identified (red—dashed). Furthermore, the underlying of the definition of clear borders becomes clear. problem of the definition of clear borders becomes clear. A reasonable approach would be to annotate only unambiguous regions and then assign the ambiguous regions to a common mixed class. An obvious disadvantage of this would be the great diversity of the resulting mixed class. This would accordingly mix up the characteristic visual features of the individual classes. Accordingly, it can be assumed that Metals 2023, 13, 1395 would decrease. Therefore, an alternative approach to segment the different microstructural constituents in Q/QT steels as reliably and unambiguously as possible was chosen in this work. In contrast to the pixel-by-pixel approach used in semantic segmentation, this approach reduces the microstructural images to individual patches. These patches are 9clasof 27 sified individually, and the result is considered representative of the particular microstructure section, as illustrated in Figure 6. In order to be able to characterize an entire microstructure, the individual microstructural images are scanned and segmented by a CNN the significance of the decisive features for determining the clearly defined classes would patch by patch using a sliding window approach [22]. Thereby, the complex problem of decrease. Therefore, an alternative approach to segment the different microstructural segmentation is reduced to a simple classification. This saves a decent amount of time constituents in Q/QT steels as reliably and unambiguously as possible was chosen in during annotation or makes annotation possible in the first place in complex cases. this work. The respective patch size is predefined by the CNN architecture’s input size. HowIn contrast to the pixel-by-pixel approach used in semantic segmentation, this apever, in order to achieve a higher “segmentation resolution” and to better map the fine proach reduces the microstructural images to individual patches. These patches are classitransitions between the areas of the individual microstructural components, the step size fied individually, and the result is considered representative of the particular microstructure can be adjusted in the in scanning process. The smaller the step size, the the resolution section, as illustrated Figure 6. In order to be able to characterize anhigher entire microstructure, of the resultingmicrostructural classification map. As aare result, the possible resolution the result the individual images scanned and segmented by aofCNN patchdepends by patch on the original resolution of the entire recording, as well as the step size with the correusing a sliding window approach [22]. Thereby, the complex problem of segmentation is sponding size,classification. and is, thus, dependent the total amount of input images, limited reduced toinput a simple This saves aondecent amount of time during annotation or by computing resources. makes annotation possible in the first place in complex cases. Figure Figure 6. 6. Illustration Illustrationof ofthe theproposed proposedapproach approachof ofsegmentation segmentationof ofan anentire entireinput inputimage image(1024 (1024px) px) using usingthe thepatch-wise patch-wiseclassification classificationapproach approach using usingaasliding slidingwindow windowtechnique technique(orange (orangesquare) square)with with respective respectivestep-sizes step-sizes (128 (128 px, px, 64 64 px, px, 32 32 px) px) resulting resulting in in different different resolutions resolutions for for segmentation. segmentation. In this approach,patch a classification model isby trained with architecture’s patches of reference statesHowthat The respective size is predefined the CNN input size. are representative of the astructural constituents that are present. respect to fine the ever, in order to achieve higher “segmentation resolution” and toWith better map the transitions between thefor areas of the the individual thereference step size patches to be selected training models,microstructural only relativelycomponents, unambiguous can beare adjusted in the scanning process. theThe stepidea size,behind the higher resolution states selected. Inconclusive patchesThe aresmaller left out. this the is to classify of theobjectively resulting classification map. As result, the possible resolution of the of result depends these and reproducibly by athe ML model. Due to the versatility the individon phases the original resolution of steels, the entire recording, asmust well cover as thethe step size with the speccorreual occurring in Q/QT the training data widest possible sponding input size, andfeatures is, thus,and dependent ontheir the total images, trum of microstructural represent highamount varianceofasinput accurately aslimited possiby computing resources.between the amount of data and the confidence with which the ble. There is a trade-off Inlabeled. this approach, model trained with patches of reference data is Hence, a 22classification correlative data setsisfrom the above-mentioned samples states were that are representative of course the structural thattoare present. With respect to the elaborately created in the of this constituents work and used extract sufficient high-quality patches to be aselected for training the models, only relatively unambiguous reference states data to build final training dataset. are selected. Inconclusive patches are left out. The idea behind this is to classify these objectively and reproducibly by the ML model. Due to the versatility of the individual phases occurring in Q/QT steels, the training data must cover the widest possible spectrum of microstructural features and represent their high variance as accurately as possible. There is a trade-off between the amount of data and the confidence with which the data is labeled. Hence, 22 correlative data sets from the above-mentioned samples were elaborately created in the course of this work and used to extract sufficient high-quality data to build a final training dataset. The probabilistic character of the CNN classification approach makes it possible to take the uncertainty of the ambiguous regions into account when evaluating entire microstructural images. Usually, classification CNNs give the corresponding probabilities of the available classes as output, from which a final prediction can be derived. A confidence component can be built into this approach by using appropriate thresholds with respect to the class probabilities. Thus, individual patches can be declared as uncertain with no clear Metals 2023, 13, 1395 10 of 27 class affiliation, which is an analogy to the microstructural regions that are ambiguous for experts during the characterization of the respective microstructure. This means that even ambiguous structure sections containing a combination of different phases can be identified and considered in the evaluation routine by summarizing the uncertain predictions in a separate class. 2.4. Annotations and Final Data Set Based on the microscopy images, a comparison of the respective structural components can be made using the morphological information after appropriate contrast. In the case of LOM, further information is obtained by looking at the coloration. The major advantage of SEM methodology is the ability to resolve substructures, such as carbides and finer lath boundaries, which delivers essential information about class affiliation. Considering the related step size of 0.35 µm, the resolution of the EBSD is behind those of LOM and SEM. Nevertheless, the crystallographic and misorientation information provides crucial added value in contributing to an objective labeling process. Thus, upper and lower bainite can be distinguished on the basis of the quantity and type of misorientations or boundaries that occur [14]. Upper bainite contains a higher fraction of low misorientations (<20◦ ), as well as a low proportion of misorientations (>40◦ ). In the case of the lower bainite, this situation is reversed. Bainitic objects also exhibit more global misorientations that create intra-structural gradients visible in the inverse pole figure (IPF). Furthermore, the packet size can provide information about the distinction between martensite and the different bainite types [23]. In addition, the IQ can be used to compare local dislocation densities qualitatively. Thus, this information can provide further evidence for an assignment to the ground truth [24,25]. Due to the displacive forming mechanism, martensite exhibits the highest misorientations, as well as the highest density of dislocations. The individual martensite plates are very well visible and separable from each other in the IPF as well as in the IQ map [26]. Their lath boundaries are strongly contrasted and characterized by high misorientations (>50◦ ). It was observed that the contrast of the lath boundaries is weaker in tempered martensite within the IQ map. In general, the contours become more blurred. The amount of high misorientations decreases slightly, whereas the amount of smaller misorientations increases during the tempering process. This is shown in Figure 7b by the number of green interfaces. This is the same material as in Figure 7a, except that it has been subjected to a subsequent annealing process. These phenomena can be attributed to carbon diffusion during the tempering process. The key to this approach of quantitative structural evaluation via patch-wise classification lies in the creation of representative and well-founded data sets. A crucial parameter for the creation of the datasets is the choice of the patch size. To use the native resolutions of the microscopes to avoid resizing and thus falsification of the morphological features by interpolation, the size scales of the microstructural constituents within the specimens examined were used as a guide. As a result, it was decided to create two separate datasets, each optimized with respect to the different microscope types, LOM and SEM. To achieve the highest possible classification resolution in the evaluation routine described above, it makes sense to keep the size of the squared patches to be classified as small as possible. The size of the characteristic morphological features of the different structural components is another limiting factor. A patch should therefore be as small as possible, but it must contain enough information to be able to assign a well-founded and objective label for the ground truth. As a result, a patch size of 128 px was chosen for the SEM-optimized dataset, which corresponds to a size of 6.25 µm. Due to the lower resolution of the LOM, a patch size of 128 px was selected, which in this case corresponds to an actual length of 12.5 µm. In the labeling process of each dataset, the corresponding microscopy images of SEM and LOM, respectively, were first inspected. Each section, which could be a candidate to be added to the training dataset, was validated with the help of the corresponding complementary information from correlative microscopy. Metals 2023, 2023, 13, 13, 1395 x FOR PEER REVIEW 11 of 27 28 ◦ , green—5 ◦ , blue—15 ◦ ), and IQ 7. LOM LOM micrograph, micrograph, SEM SEM ++ overlayed overlayed boundaries boundaries (red—2 (red—2°, green—5°, blue—15°), Figure 7. map of of aa fully fully martensitic martensitic dilatometry dilatometry sample sample (a) (a) and and the the identical identical sample sample after after annealing annealing (b). (b). The The map tempering process can be presumed to reduce major misorientations. Instead, the density of lower tempering process can be presumed to reduce major misorientations. Instead, the density of lower misorientations (red and green) is apparently higher. Furthermore, the boundaries of the individual misorientations (red and green) is apparently higher. Furthermore, the boundaries of the individual battens within the structure appear less pronounced. battens within the structure appear less pronounced. The different key to this approach of quantitative structural evaluation via patch-wise The representations were overlaid in an image processing program toclassifiextract cation lies in the of representative well-founded data A crucial paramethe patches. Forcreation this purpose, LOM, SEMand images, as well as thesets. EBSD information in ter for theof creation of and the datasets is the choice of the patch size. To use the native resoluthe form IPF, IQ, kernel average misorientation (KAM) maps and, mostly, the tions of the microscopes to avoid resizing andinthus of the morphological fearepresentation of the different misorientations the falsification form of boundaries with the threshold tures by interpolation, the size scales of the microstructural constituents within the specivalues 2, 5, and 15 degrees, were used. To mark the labeled sections, a square brush tool mensused examined were used guide. As four a result, it was decided tofor create two separate was to create masks ofas thea respective classes, respectively, the LOM and the datasets, each optimized with respect to the different types, LOMextract and SEM. SEM optimized dataset. These binary masks were thenmicroscope used to automatically the To achieve the highest possible classification resolution in the evaluation routine decorresponding labeled patches from LOM and SEM images, respectively. scribed above, it makes sense to keep the size of the squared patches to be classified as When creating the SEM data set, superimposed representations of the misorientations small as possible. The size of the characteristic morphological features of the different combined with the SEM images were used in order to be able to reproducible assign more structural components another factor. A patch should therefore be as small of the unclear areas to is one of the limiting classes, which may have appeared ambiguous due as to possible,contrasting but it mustartifacts. contain enough information to bee.g., able to assignthe a well-founded possible The crucial information, regarding orientation ofand the objective label for thewhich ground As a result, patch size of 128 px was chosen is for the precipitated carbides, is truth. to be regarded as aadecisive distinguishing criterion, most SEM-optimized dataset, which corresponds size of 6.25 µm. Due to the lower resoluevident in the SEM images themselves, as it to is athe highest-resolution method. Therefore, tion of theitLOM, a patch size time-consuming of 128 px was selected, which in this case data corresponds an although proved to be very to create a well-founded set from to SEM actual length 12.5 µm. In labeling process ofthe each dataset, theconsidered, corresponding patches due toofthe volume of the information as well as criteria to be it canmibe evaluated with great confidence as objective and were representative. croscopy images of SEM and LOM, respectively, first inspected. Each section, which 8 shows ato selection of characteristic thevalidated individual classes within couldFigure be a candidate be added to the trainingfeatures dataset,ofwas with the help of the corresponding different complementary information sources. Thus, LB, shown in (a) and (b), is complementary information from correlative microscopy. characterized by moderate misorientations, as well the orientation size of The different representations were overlaid in anasimage processing and program to the excarbides within the laths. The IPF overlay shows similar orientations of the adjacent laths. tract the patches. For this purpose, LOM, SEM images, as well as the EBSD information in UB, as shown in IQ, (c) and is characterized by elongated(KAM) carbide precipitation between the the form of IPF, and(d), kernel average misorientation maps and, mostly, the replaths, whichof have homogeneous orientationinbythe comparison, sometimeswith withthe pronounced resentation the adifferent misorientations form of boundaries threshold gradients laths and least the remaining structural values 2, 5,within and 15the degrees, werethe used. To misorientation. mark the labeledFor sections, a square brush tool components M, shown (f), andfour MST, shown in (g) andfor (h),the the disordered was used to create masksinof(e) theand respective classes, respectively, LOM and the orientation of thedataset. individual laths can masks be confirmed by looking the IPF. Here, MST can SEM optimized These binary were then used to at automatically extract the corresponding labeled patches from LOM and SEM images, respectively. Metals 2023, 13, 1395 Figure 8 shows a selection of characteristic features of the individual classes within the different complementary information sources. Thus, LB, shown in (a) and (b), is characterized by moderate misorientations, as well as the orientation and size of the carbides within the laths. The IPF overlay shows similar orientations of the adjacent laths. UB, as shown in (c) and (d), is characterized by elongated carbide precipitation between the laths, 12 graof 27 which have a homogeneous orientation by comparison, sometimes with pronounced dients within the laths and the least misorientation. For the remaining structural components M, shown in (e) and (f), and MST, shown in (g) and (h), the disordered orientation of individual laths can bedisordered confirmed by looking at the IPF.visible Here, MST be identified be the identified by the fine and carbide precipitates in thecan high-resolution by theimage. fine and disordered carbide precipitates visible in the high-resolution SEM image. SEM 8.Overview Overview selected representative patches ofSEM-optimized the SEM-optimized dataset (6.25 µm) Figure 8. of of selected representative patches of the dataset (6.25 µm) showing LB (a,b), (c,d), (e,f), M and(e,f), MSTand (g,h). In addition, corresponding correlative information showing LB UB (a,b), UBM(c,d), MST (g,h). Inthe addition, the corresponding correlative is shown as overlays + misorientation boundaries (red—2°, green—5°, SEM◦+), information is shown(SEM as overlays (SEM + misorientation boundaries (red—2◦ ,blue—15°), green—5◦ , and blue—15 IPF, see Figure 5 for color-coding), which was consulted for labeling. and SEM + IPF, see Figure 5 for color-coding), which was consulted for labeling. The creation of the LOM data set proved to be much more complex. In order to be able to able to to assign thethe patches to ato class, the input image must must have to train trainaamodel modelthat thatis is able assign patches a class, the input image sufficient characteristic visual features. For some patches that can assigned to a have sufficient characteristic visual features. For some patches thatbe canclearly be clearly assigned class based on the image, a clear classification based on the images waswas not to a class based on SEM the SEM image, a clear classification based on LOM the LOM images possible. The resolution of theofLOM was simply not sufficient to represent the finethe micronot possible. The resolution the LOM was simply not sufficient to represent fine structural features appropriately. For this the corresponding input input size ofsize the of LOM microstructural features appropriately. Forreason, this reason, the corresponding the LOM dataset was adjusted upward, as mentioned above, capturemore morecontext contextand and more dataset was adjusted upward, as mentioned above, to to capture global characteristics in the dataset accordingly. When selecting the LOM patches, care had to be taken to ensure that the decisive features could be identified solely based on the LOM image. The corresponding correlative information from SEM and EBSD should therefore only be consulted as additional information. Due to the increased input size, the number of possible representative areas was reduced. The reason for this is that the consideration of larger sample areas, accompanied by larger patch size, results in fewer homogeneous areas that can be predominantly assigned to one microstructural constituent. This results, on the one hand, in a smaller amount of training data, which is not insignificant for DL applications, and, on the other hand, in patches that also include more visual features of further microstructural components in comparison to the SEM patches. Figure 9 shows a selection of labeled patches based on the LOM dataset. Using the same characteristics outlined in the case of Figure 8, the potential reference ranges were validated using the complementary information from the correlative datasets. Metals 2023, 13, 1395 mogeneous areas that can be predominantly assigned to one microstructural constituent. This results, on the one hand, in a smaller amount of training data, which is not insignificant for DL applications, and, on the other hand, in patches that also include more visual features of further microstructural components in comparison to the SEM patches. Figure 9 shows a selection of labeled patches based on the LOM dataset. Using the of 27 same characteristics outlined in the case of Figure 8, the potential reference ranges13were validated using the complementary information from the correlative datasets. Figure 9. 9. Overview of selected representative patches of the LOM-optimized dataset (12.5 µm) Figure showing LB and MST (g,h). In addition, the corresponding correlative inforshowing LB (a,b), (a,b),UB UB(c,d), (c,d),MM(e,f), (e,f), and MST (g,h). In addition, the corresponding correlative ◦ , green—5 ◦, mation is shown (SEM(SEM and Overlay: SEM +SEM misorientation boundaries (red—2°, green—5°, blue— information is shown and Overlay: + misorientation boundaries (red—2 15°)), which was consulted for labeling. blue—15◦ )), which was consulted for labeling. However, the the explained explained compromise compromise regarding regarding the the adjusted adjusted patch patch size size of of the the LOM LOM However, dataset leads leads to to the the fact fact that that many, many, especially especially bainitic bainitic areas, areas, are are too too small small to to fill fill an an entire entire dataset patch (Figure (Figure 8d)). 8d)). As a result, features of the adjacent adjacent phases are also also visible visible on on these these patch patches, which is ofof data required to capture the the vapatches, is unavoidable unavoidableconsidering consideringthe theamount amount data required to capture riety of the phases occurring in real Q/QT steels. Thereby, care was taken to ensure that variety of the phases occurring in real Q/QT steels. Thereby, care was taken to ensure the characteristic areasareas occupy most most of theofpatch area and located in the in center. Howthat the characteristic occupy the patch areaare and are located the center. ever, this could prove to betoadvantageous for the routine explained since the However, this could prove be advantageous for evaluation the evaluation routine explained since patches often contain more than oneone phase when thethe sliding window approach is is used. the patches often contain more than phase when sliding window approach used. An intensive intensive review review of of the the correlative correlative data sets sets in in a cross-scale cross-scale and and holistic approach approach An revealed ground truth when no no complerevealed fundamental fundamentalproblems problemsininassigning assigningwell-founded well-founded ground truth when commentary information waswas available andand only thethe LOM images were used forfor thethe labeling plementary information available only LOM images were used labelprocesses. In the not only are the micrographs accepted as such, but they ing processes. In process, the process, not only arepresent the present micrographs accepted as such, but are critically scrutinized throughout the entire process of data acquisition. Thus, additional information, such as differences in chemical composition and the manufacturing processes of the samples, as well as contrasting and methodological influences, are also considered. Here, correlative microscopy enhances the interpretation of this information. Figure 10 illustrates patches of the LOM-optimized dataset with a size of 12.5 µm, for which the exclusive use of LOM images for the labeling process could be quite misleading. Here, based on the visual characteristics, it can quickly lead to false labels regarding the assignment of ground truth. Thus, Figure 10a appears to be lower bainite based on the LOM patch. This is indicated by the visible preferred orientation of the bainite plates, as well as the brownish coloration, which is caused by the finely precipitated carbides. However, if we now look at the highresolution SEM image, it becomes apparent that the majority of the carbides have not precipitated within the laths but that they have formed between the very fine bainitic laths. Thus, this is extremely fine upper bainite according to the common classification schemes. Metals 2023, 13, 1395 ditional information, such as differences in chemical composition and the manufacturing processes of the samples, as well as contrasting and methodological influences, are also considered. Here, correlative microscopy enhances the interpretation of this information. Figure 10 illustrates patches of the LOM-optimized dataset with a size of 12.5 µm, for which the exclusive use of LOM images for the labeling process could be quite misleading. 14 of 27 Here, based on the visual characteristics, it can quickly lead to false labels regarding the assignment of ground truth. Figure 10. Showcases Showcases where where aa clear clear classification classification based based purely purely on on the the LOM LOM recordings recordings (upper (upper row) row) might lead to false labels. labels. The The necessary necessary information information for for correct correct classification classification can only be taken taken from from the lom. The the high-resolution high-resolution SEM SEM image image (lower (lower row). row). In In (a), (a), LB LB can can be be assumed assumed based based on on the the lom. The SEM SEM first resolves the carbide sufficiently, whereupon one can assume UB. In (b), the finely precipitated first resolves the carbide sufficiently, whereupon one can assume UB. In (b), the finely precipitated carbides of the MST are only visible on the basis of the SEM. Thanks to the brown coloration, M can carbides of the MST are only visible on the basis of the SEM. Thanks to the brown coloration, M can be assumed for (c). However, closer examination under the SEM reveals a morphology of the carbe assumed for (c). However, closer examination under the SEM reveals a morphology of the carbides bides that can be assigned to UB. Due to the lack of topographic information from the LOM, no clear that can becan assigned to UB. Due LB to the topographic information from the LOM, clear distinction be made between andlack MST.ofThe SEM image is again supportive in this no respect. distinction can be made between LB and MST. The SEM image is again supportive in this respect. In In (d), confusion between MST and LB can easily occur. This is due to missing topography infor(d), confusion between MSTOnly and LB can basis easilyofoccur. Thisthe is due to missing topography mation based on the LOM. on the the SEM martensitic needles can be information recognized. based on the LOM. Only on the basis of the SEM the martensitic needles can be recognized. Thus, Figure 10a appears to be lower bainite based on the LOM patch. This is indiat the LOM micrograph of theofpatch Figure plates, 10b, oneaswould possibly choose catedLooking by the visible preferred orientation the bainite well as the brownish martensite for classification expert. This is supported by ifthe coloration, which is caused as byan theexperienced finely precipitated carbides. However, wedisordered, now look lathlike morphology and the distinct coloration of the features. However, with aid of at the high-resolution SEM image, it becomes apparent that the majority of thethe carbides the corresponding SEM image, the extremely fine, disordered precipitation of the cementite have not precipitated within the laths but that they have formed between the very fine particleslaths. becomes clear, in combination with the previously morphology, is a bainitic Thus, thiswhich is extremely fine upper bainite according named to the common classificharacteristic of tempered martensite. cation schemes. Based onatthe dark of brown coloration, misinterpreting lath Looking thepronounced LOM micrograph the patch Figure as 10b,well oneas would possibly choose boundaries, the LOM patch shown in Figure 10c indicates a martensitic affiliation. Like martensite for classification as an experienced expert. This is supported by the disordered, Figure 10a, however, the coloration can be attributed to the fineness of the cementite lathlike morphology and the distinct coloration of the features. However, with the aid of lamellae, which is even more pronounced in this case. Again, it can be assumed that the corresponding SEM image, the extremely fine, disordered precipitation of the cementaccording to the characteristics, it deals with very fine upper bainite. ite particles becomes clear, which in combination with the previously named morphology, Due to the dark spots in combination with a light brown coloration, both correspondis a characteristic of tempered martensite. ing to a fine dispersion of small cementite precipitations, one expects an assignment to Based on the pronounced dark brown coloration, as well as misinterpreting lath lower bainite or tempered martensite for Figure 10d. With the help of the SEM, the disboundaries, the LOM patch shown in Figure 10c indicates a martensitic affiliation. Like tinct topography becomes visible, which provides higher confidence in an assignment to Figure 10a, however, the coloration can be attributed to the fineness of the cementite lathe latter. mellae, which is even more pronounced in this case. Again, it can be assumed that accordThe holistic approach via correlative microscopy, which also takes into account the ing to the characteristics, it deals with very fine upper bainite. significant influence of contrasting as part of specimen preparation with regard to imaging, can identify further misleading factors. Figure 11 again illustrates the problematic reproducibility of electrochemical etching processes. After contrasting under apparently identical conditions Figure 11b,c, the correlative LOM images reveal an obvious difference. In Figure 11c, especially the precipitated carbides within the microstructure, which was identified as tempered martensite, are clearly more pronounced, which simplifies an assessment as such. Based on Figure 11b, one could also assume that it is martensite. Once again, only the high-resolution SEM image provides clarity regarding an unambiguous assignment. Metals 2023, 13, 1395 ing, can identify further misleading factors. Figure 11 again illustrates the problematic reproducibility of electrochemical etching processes. After contrasting under apparently identical conditions Figure 11b,c, the correlative LOM images reveal an obvious difference. In Figure 11c, especially the precipitated carbides within the microstructure, which was identified as tempered martensite, are clearly more pronounced, which simplifies an assessment as such. Based on Figure 11b, one could also assume that it is martensite. Once again, only the high-resolution SEM image provides clarity regarding an unambiguous assignment. 15 of 27 Figure of of thethe influence of contrasting methods using the same electrochemical etching etching Figure11. 11.Example Example influence of contrasting methods using the same electrochemical procedure (a) as corresponding correlative SEM micrograph to (b). Though etching under similar procedure (a) as corresponding correlative SEM micrograph to (b). Though etching under similar conditions at different times, the micrograph (c) shows a significantly stronger etching effect, resultconditions different times, the micrograph (c) shows a significantly stronger etching effect, resulting ing in moreatdistinct visual features. This can lead to a shift in the significance of the individual in more which distinct visual features. This can to a shift in the significance of the individual features, features, has a significant influence on lead the corresponding classification. which has a significant influence on the corresponding classification. An orientation dependence of the Nital etching was observed across all specimens. Crystal of dependence <100> show a significantly effect, as shown Anorientations orientation of the Nitalreduced etchingetching was observed acrossinallFigspecimens. ure 12 [17]. As a result,ofthe areas show in the LOM appear bright, similaretching to areaseffect, associated Crystal orientations <100> a significantly reduced as shown in with upper bainite. SEM appear micrograph reveals the presence Figure 12 [17]. As Though, a result,the thehigh-resolution areas in the LOM bright, similar to areas of associated martensitic topography, which due to the reduced etchability, is not as pronounced as with upper bainite. Though, the high-resolution SEM micrograph reveals the presence of those regions with different crystal orientations. Therefore, it is not visible in the LOM martensitic topography, which due to the reduced etchability, is not as pronounced as those image. Additionally, selective contrasting of the lath boundaries of the martensite reinregions with different crystal orientations. Therefore, it is not visible in the LOM image. forces the false impression so that confusion with upper bainite can quickly arise (red Metals 2023, 13, x FOR PEER REVIEW 16 of 28reinforces the Additionally, selective contrasting of the lath boundaries of the martensite marking). false impression so that confusion with upper bainite can quickly arise (red marking). Figure 12. micrographs of the sample regionregion (a)—LOM, (b)—LOM + IPF, (c)— Figure 12.Correlative Correlative micrographs of same the same sample (a)—LOM, (b)—LOM + IPF, (c)—SEM, SEM, (d)—SEM + IPF). The orientation dependence of the Nital etchant becomes clear: Regions with (d)—SEM + IPF). The orientation dependence of the Nital etchant becomes clear: Regions with <100> orientation topography (reddish in IPF) show a significantly weaker etching effect with a cor<100> orientation topography (reddish IPF) show a significantly weaker etching effect with a respondingly softer topography (only visible inin SEM). correspondingly softer topography (only visible in SEM). Considering the problems demonstrated in this section, it can be concluded again that the most challenging problem behind the application of modern ML approaches in the field of materials science is finding an absolute ground truth. Considering the available complementary information, as well as the occurring misleading factors, two final datasets were created based on the 22 correlative datasets optimized for SEM and LOM images, respectively. Unfortunately, these can only be compared indirectly due to different Metals 2023, 13, 1395 16 of 27 Considering the problems demonstrated in this section, it can be concluded again that the most challenging problem behind the application of modern ML approaches in the field of materials science is finding an absolute ground truth. Considering the available complementary information, as well as the occurring misleading factors, two final datasets were created based on the 22 correlative datasets optimized for SEM and LOM images, respectively. Unfortunately, these can only be compared indirectly due to different patch sizes and different underlying slices. In conclusion, it can be stated that despite the reduction to the most homogeneous patches possible, it is not possible to guarantee the correctness of all labels. Nevertheless, a lot of time and consideration of all complementary information was used to work out the underlying ground truth. This represents, at the current state of research, the best approach to a classification in terms of the structural constituents that occur in combination with the samples investigated and taking into account the widely recognized identification criteria of the common phases occurring in modern steels. In order to train the model to a certain degree of variance and to make it as robust as possible, more critical patches were also included in the data set. These partially deviate from the reference states of the respective microstructural constituents to be able to characterize real Q/QT steels as successfully as possible. Ultimately, an SEM-optimized training data set of 6680 individual patches (14% LB—41% M—25% MST—20% UB), as well as a LOM-optimized training data set of 2246 individual patches (17% LB—40% M—27% MST—15% UB), has successfully been created to train respective DL Classification models as good as possible. 2.5. Deep Learning Methodology From DL’s perspective, this results in a multiclass classification problem with the four introduced classes. To include additional objectivity, three independent models based on different architectures have been trained. For each of the three independent models, a pre-trained CNN is used based on the principle of transfer learning [27]. This is performed by taking advantage of the fact that each model is already capable of reliably recognizing important visual features and extracting corresponding low- and high-level features in order to allow accurate classification of the respective structures. Several publications could confirm that transfer learning, using pretrained weights, even using data from different domains, is beneficial to the training process and the final model’s performance in material science [27–29], especially if, compared to usual DL applications, only little amount of data is accessible. As model backbones, Xception [30], ResNet50 [31], and DenseNet201 [32] seemed to yield the best results in the scope of the pre-tests. Each of the architectures was used without the respective dense layers. After the convolution layers as feature extraction, a GlobalMaxPooling operation was implemented to reduce the features to a 1D feature vector [33], which is fed into a fully connected network. The latter consists of a dense layer with 64 neurons that are connected to a dense layer with four neurons, corresponding to the respective number of classes. The final prediction layer uses the SoftMax activation function to be able to interpret its entries as classification probability. To reduce the overfitting, an additional dropout layer has been added after the feature extraction part and the dense layer with 64 neurons. For both SEM and LOM dataset, 128 pixels has been selected as input size in order to use the native resolution of the respective microscope, corresponding to the earlier mentioned sample section of 6.25 µm tiles for SEM and 12.5 µm tiles for LOM, respectively. As a preparation step, the default preprocessing function of the individual CNN architectures has been used, zero-centering each color channel with respect to the ImageNet dataset, which was used to pre-train the weights. Afterward, each dataset was normalized to a maximum value of 1 to be processible by the CNNs. The image labels were one-hot-encoded prior to the training process. The training dataset was divided into a training and a test set (75–25%). To tackle the problem of class imbalance, class weights according to the total number of images with respect to each class within the training dataset were included [34]. Metals 2023, 13, 1395 17 of 27 As a loss function, the categorical cross-entropy was used in combination with classification accuracy as a validation metric. In terms of data augmentation, common operations such as zoom (70–130%), rotations (0–360◦ ), random contrast and brightness adjustments (0–10%), as well as vertical and horizontal flips, were selected. The thereby emerging empty areas are filled using the “reflect” argument. Each model was trained for 25 epochs using the Adam optimizer at a learning rate of 0.0001 and subsequently, for 10 epochs at a learning rate of 0.00001 as fine-tuning, using the Keras API of the Tensorflow implementation [35] in Google Colab Pro with access to an NVIDIA Tesla T4 GPU. 2.6. Post-Processing: Combining Predictions of Different Models Since different CNN architectures learn, extract, and process visual features in different ways, it seems reasonable to combine the predictions of several different models. This allows for increased objectivity, as well as higher robustness. For this work, three independent optimized models were trained with the same data sets. In the scope of a combined evaluation, each of these models independently classify the individual patches of the microstructure image to be analyzed. The corresponding predictions then contribute accordingly to the final classification. These are summarized and equally influence the final prediction within the framework of the majority voting approach used. In the majority voting approach, the final prediction emerges based on the majority occurrence of the predictions after consideration of the respective threshold value. Accordingly, the final prediction must correspond to at least two of the three predictions resulting from the models used. In case either three different predictions are made by the separate models, or in case the majority of the predictions are to be assigned to the additional class of uncertainty after filtering by the confidence threshold, this will thus be the prognosis as the final prediction. A subsequent median filter with a kernel size corresponding to the corresponding step size leads to the removal of undesired artifacts, as well as a smoothing of the boundaries of the respective phase regions. 3. Results and Discussion 3.1. Classification Model Results After training the respective architectures with each dataset, LOM, and SEM, the following performance could be achieved on the unseen test sets. As mentioned in the methodology part, model A is based on Xception, model B uses a DenseNet backbone, whereas model C consists of a ResNet. The classification results on the test-sets of the respective models are summarised in Table 1. Overall, the three models could achieve an average accuracy of 88.8% for the LOM and 93.7% for the SEM dataset, respectively. These are exceptional results considering the high complexity due to the fine differences between the microstructural patches. With a standard deviation of 0.7% between the different architectures and 0.6% in the case of the SEM dataset, the performance between the models is quite homogeneous on both datasets. Using the SEM patches delivers significantly higher performances. For example, the mean precision in the classification of martensite reached a value of 98.3%. Thus, martensite was most reliably identified as such among all the classes due to its distinct morphology, which differs more from the other classes. Surprisingly, in the SEM dataset, the objects belonging to the UB class achieved the lowest relative accuracy. During the annotation process, usually, the UB class was the easiest to assign since it has very distinctive characteristics due to the morphology of the elongated carbide precipitations between the bainitic laths. In the LOM dataset, however, the UB patches were the second best identified as such. There, the martensite class also achieves the highest precision. Thus, in the case of the SEM images, each model was able to detect the subtle differences between LB and MST. From this, it can be concluded that the models in relation to SEM images are successfully Metals 2023, 13, 1395 18 of 27 able to identify and process the differences in topography, as well as the fine discrimination criteria regarding the precipitation of the carbides in microscopic images of Q/QT steels. Once again, the extraordinary ability of modern DL approaches to independently learn decisive criteria based on visual features and to apply them in a reproducible manner became evident. Table 1. Summary of the confidence matrix combined with respective metrics, such as precision, recall, and F1 score and overall accuracy of the three different architectures (Model A, B, and C) for being trained on the SEM and LOM datasets. Model A—SEM Dataset Accuracy: Pred. LB Model A—LOM Dataset 93.4% Accuracy: Label Label Label Label Class LB M MST UB Precision 201 0 5 15 92% Pred. LB 88.1% Label Label Label Label Class LB M MST UB Precision 86 0 4 4 70% 97% Pred. M 1 645 16 14 98% Pred. M 4 224 6 4 Pred. MST 10 10 401 20 93% Pred. MST 31 6 112 5 92% Pred. UB 6 3 11 312 86% Pred. UB 1 2 0 73 85% Class recall 91% 95% 91% 94% Class recall 91% 94% 73% 96% Class F1 score 92% 97% 92% 90% Class F1 score 80% 95% 81% 90% Model B—SEM Dataset Accuracy: Model B—LOM Dataset 93.5% Accuracy: 89.8% Label Label Label Label Class Label Label Label Label Class LB M MST UB Precision LB M MST UB Precision Pred. LB 210 0 3 8 87% Pred. LB 84 0 9 1 76% Pred. M 3 640 21 12 99% Pred. M 3 223 7 5 98% Pred. MST 14 5 407 15 92% Pred. MST 23 4 125 2 88% Pred. UB 90% Pred. UB 90% 14 3 11 304 1 1 1 73 Class recall 95% 95% 92% 92% Class recall 89% 94% 81% 96% Class F1 score 91% 97% 92% 91% Class F1 score 82% 96% 84% 93% Model C—SEM Dataset Accuracy: Model C—LOM Dataset 93.1% Accuracy: Label Label Label Label Class 88.6% Label Label Label Label Class LB M MST UB Precision LB M MST UB Precision Pred. LB 209 0 3 9 89% Pred. LB 83 0 10 1 75% Pred. M 1 638 26 11 98% Pred. M 3 221 10 4 97% Pred. MST 15 8 399 19 91% Pred. MST 24 4 121 5 86% Pred. UB 9 4 10 309 89% Pred. UB 0 3 0 73 88% Class recall 95% 94% 90% 93% Class recall 88% 93% 79% 96% Class F1 score 92% 96% 91% 91% Class F1 score 81% 95% 82% 92% When looking at the LOM data, however, the comparatively low precision of averaged 73.7% in the classification of the LB class is noticeable. This also reflects the problems and resulting concerns during the annotation process of the LOM images. Based purely on LOM images, it was often not possible to distinguish LB from MST, so the corresponding SEM images had to be consulted. Representing this fact, most of the misclassified patches of the LB class are assigned to the class of the MST. Metals 2023, 13, 1395 19 of 27 Despite the remarkable classification accuracies, it is of great importance to investigate the models’ misclassifications in detail. Only in this way can the possibilities and limits of the models really be assessed. When considering the misclassified patches of the SEM dataset, it must be admitted that almost none of the misclassified excerpts can undoubtedly be assigned to the corresponding label. Often, these examples were assigned to the respective ground truth class on the basis of complementary information and a more global view, with the aim of being able to represent as much variance as possible. Most of them leave some room for interpretation and a residual degree of subjectivity. Patch (a), shown in Figure 13, was incorrectly classified as UB from two of the three models. It clearly shows oriented lath-like structures with slightly pronounced carbides along the axis (red arrows). Due to the majority of particles being precipitated within the laths, it was labeled as LB (black arrows). However, this lack of clarity is expressed in a low prediction probability of less than 60% for each model. The counterpart to 20 this is Metals 2023, 13, x FOR PEER REVIEW of 28 provided by (b). Here, due to the fact that the majority of the carbides run coherently along the laths, a classification to class UB was chosen (black arrows). Nevertheless, carbide precipitates also occur occasionally within the laths (red arrows), which would confirm the prediction of of the the model. model. In first two two examples, examples, patch patch (c) (c) was was assigned assigned to to prediction In contrast contrast to to the the first the wrong class with high confidence (>90% probability). Due to the pronounced parallel the wrong class with high confidence (>90% probability). Due to the pronounced parallel and comparatively homogeneous boundaries of the and comparatively homogeneous edges edges throughout throughout the the patch, patch, these these boundaries of the martensitic lathswere weremisinterpreted misinterpretedasas those of the arrows). However, addimartensitic laths those of the UB UB (red(red arrows). However, additional tional information largerfor range for ainput given input size) could to a corglobal global information (e.g., a (e.g., largerarange a given size) could lead to a lead correction by rection by the model by identifying it at martensite. the model by identifying it at martensite. Figure 13. Showing Showing misclassified misclassified patches based on the SEM dataset with a corresponding corresponding patch size of 6.25 µm. of 6.25 µm. (a) (a) was was labeled labeled as as LB LB and and classified classified as as UB. UB. (b) (b) was was classified classified as as LB LB but but has has UB UB as as label. label. (c) was labeled as martensite but classified as UB, and (d) has MST as label, but was classified as (c) was labeled as martensite but classified as UB, and (d) has MST as label, but was classified as LB. LB. In MST patch patch (d), (d),which whichwas waslabeled labeledasassuch suchbased-on based-on topography, significance topography, thethe significance of of aligned laths in combination with carbides precipitated within (black arthethe aligned laths in combination with the the carbides precipitated within themthem (black arrows) rows) apparently predominated, so the model classified apparently predominated, so the model classified this as this LB. as LB. In summary, using the SEM dataset, no misclassified patches could be identified, for which the model’s prediction could be considered totally inconclusive. Thus, no decisive argument can be found to question the general validity and reliability of the models in ofaagiven givensimilarity similarity specimens being considered within this study this view of toto thethe specimens being considered within this study at thisatpoint. point.For the LOM dataset, the number of correctly classified patches in the LB, M, and UB classes onlydataset, marginally. Here, the different Fordiffers the LOM the number ofgreatest correctlydifference classifiedbetween patches the in the LB, M,architecand UB tures is differs expressed the MST class. majority of the overall misclassified patches, classes onlyinmarginally. Here,For thethe greatest difference between the different archithe insufficient resolution of the LOM was found to be the cause. For the corresponding tectures is expressed in the MST class. For the majority of the overall misclassified patches, patches, the respective ground truth waswas selected underlying the insufficient resolution of the LOM foundbased to beon thethe cause. For thecomplementary corresponding information. Minimal misinterpretations to incorrect decisions by the model, patches, the respective ground truth wasalready selectedlead based on the underlying complemenwhich is, however,Minimal very comprehensible on closer examination. tary information. misinterpretations already lead to incorrect decisions by the Forwhich the classification thecomprehensible LOM images, patch (a) from Figure 14 was predicted as model, is, however,of very on closer examination. UB and LB, whereas it was labeled as MST. These decisions are both understandable For the classification of the LOM images, patch (a) from Figure 14 was predicteddue as to the slightly brownish coloration in combination with the precipitated carbides, as well UB and LB, whereas it was labeled as MST. These decisions are both understandable due as the dueslightly to possible contrasting lath boundaries of the avoidable bainite. However, due to to brownish coloration in combination with the precipitated carbides, as well thedue needle-like topography, which only enhanced the SEMbainite. image, However, the decision was as to possible contrasting lath is boundaries of theon avoidable due to made in favor of MST during the labeling process. Though, since all three models predicted the needle-like topography, which is only enhanced on the SEM image, the decision was made in favor of MST during the labeling process. Though, since all three models predicted different classes, the interpretability will be represented as an unclear label in the following post-processing routine during the quantification of an entire microstructural image. Metals 2023, 13, 1395 20 of 27 Metals 2023, 13, x FOR PEER REVIEW 21 of 28 different classes, the interpretability will be represented as an unclear label in the following post-processing routine during the quantification of an entire microstructural image. Figure 14. 14. Showing with a correspondFigure Showing misclassified misclassifiedpatches patchesbased basedon onthe theLOM LOMdataset dataset(upper (upperrow) row) with a corresponding ing patch size of 12.5µm and correlative SEM patch (lower row) for clarification. (a) was labeledas asMST patch size of 12.5µm and correlative SEM patch (lower row) for clarification. (a) was labeled MST and classified as UB and LB. (b) was classified as LB from two models but had MST as label. and classified as UB and LB. (b) was classified as LB from two models but had MST as label. (c) was (c) was labeled as M but classified as UB from all three models, and (d) has M as label but was labeled asas MUB. but The classified as UBof from allpartially three models, andwith (d) has as label but was classified as classified SEM image (d) is overlayed theM corresponding IPF (consult UB. The SEM image of (d) is partially overlayed with the corresponding IPF (consult Figure 5 for Figure 5 for color-coding) map in order to identify the orientation dependence of the Nital etchant. color-coding) map in order to identify the orientation dependence of the Nital etchant. A similar case occurs in patch (b). Here, the as M incorrectly classified patch (c) with A similar case occurs inthe patch (b). Here, the as M classified ground truth UB represents problem that already hasincorrectly been shown in Figurepatch 10c in(c) anwith ground truth UB represents the problem that already has been shown in Figure 10c in even more difficult way. There, the bainitic laths are extremely fine, which allows misinan even more difficult way. There, the bainitic laths are extremely fine, which allows terpretation towards the martensitic class. Additionally, in contrast to the previous exammisinterpretation towards the martensitic in contrast to the previous ple from Figure 10c, the bainitic features do class. not fillAdditionally, the entire patch but are additionally example from 10c, the bainitic features notone fillof the entire but areexamples additionally surrounded byFigure martensitic regions. Hence, thisdo was the mostpatch ambiguous within the test surrounded bydataset. martensitic regions. Hence, this was one of the most ambiguous examples Inthe patch an inverse misclassification compared to (c) can be seen. There, the white within test(d), dataset. shimmering the sufficiently contrasted lath boundaries on be theseen. upper There, center, the In patchareas (d), between an inverse misclassification compared to (c) can as wellshimmering as the whiteareas region on the lower right, leadcontrasted through the otherwise brownish white between the sufficiently lath boundaries on thecolupper orationasofwell the as microstructure to a on falsified impression. Through looking at the IPF, the center, the white region the lower right, lead through the otherwise brownish assignmentofofthe those areas to the <100> can be found. Thus, the orientation coloration microstructure to a orientation falsified impression. Through looking at theconIPF, the trast observed during the labeling process is also involved in a misjudgment of the CNN. assignment of those areas to the <100> orientation can be found. Thus, the orientation However,observed the fine and disordered needleprocess structure theinvolved martensiteinplates can be identicontrast during the labeling isof also a misjudgment of the fied by SEM, which is why this class was chosen in the labeling process. CNN. However, the fine and disordered needle structure of the martensite plates can be In summary, the classification of LOM performed better than expected identified by SEM, which is why this classpatches was chosen in themuch labeling process. at theInbeginning. Compared to the SEM images, the biggest bottleneck of using the LOM summary, the classification of LOM patches performed much better than expected images is the resolution, which is accompanied by a lack of crucial detailed information. at the beginning. Compared to the SEM images, the biggest bottleneck of using the LOM Nevertheless, very high accuracies were achieved, and the wrong decisions of CNN could images is the resolution, which is accompanied by a lack of crucial detailed information. be comprehended in most cases and, most likely, would be confirmed by human experts Nevertheless, very high accuracies were achieved, and the wrong decisions of CNN could in a similar manner. be comprehended in most cases and, most likely, would be confirmed by human experts in All in all, based on the results presented here, it can be concluded that the trained a similar manner. models are very well able to perform a good differentiation between the individual classes All in all, based on the results presented here, it can be concluded that the trained modbased on both LOM and SEM images. The surprisingly high accuracies, despite the unels are very well able to perform a good differentiation between the individual classes based derlying complexity, illustrate the adaptability of the modern DL approaches. Despite the on both LOM and SEM images. The surprisingly high accuracies, despite the underlying inevitable room for interpretation in a classification based on the given classification complexity, the adaptability theis modern DL approaches. Despite the inevitable schemes andillustrate the residual subjectivity of that unavoidable in these approaches to microroom for interpretation a classification based onhave the given classification schemes and the structure evaluation, theinclassification approaches the ability to successfully recogresidual subjectivity that is unavoidable in these approaches to microstructure evaluation, nize and process the fine discriminatory criteria. It can be assumed that the performance the classification approaches have the to ability toan successfully recognize and process the fine of the models trained here is very close that of experienced expert, given the richness discriminatory criteria. It can be assumed that the performance of the models trained here Metals 2023, 13, 1395 Metals 2023, 13, x FOR PEER REVIEW 21 of 27 22 of 28 is very close to that of an experienced expert, given the richness of the complementary of the complementary information used to find an and objective ground truthability and itstooverinformation used to find an objective ground truth its overwhelming apply whelming ability to apply this knowledge. Nevertheless, this knowledge is limited exthis knowledge. Nevertheless, this knowledge is limited to exactly these types oftoQ/QT actly these types of Q/QT steels with which the model was trained. The great added value steels with which the model was trained. The great added value here, however, is the here, however, is the existing reproducibility compared to conventional expert opinions. existing reproducibility compared to conventional expert opinions. 3.2. 3.2. Patch-Wise Patch-Wise Classification ClassificationUsing UsingaaSliding SlidingWindow WindowTechnique TechniqueasasSegmentation SegmentationApproach Approach In the following section, the trained models are used to quantify entire microstrucIn the following section, the trained models are used to quantify entire microstructural tural images. The sliding window approach forpurpose this purpose allows segmentation images. The sliding window approach used used for this allows segmentation using using classification models that need a fixed patch size of 128 px, respectively, an input. classification models that need a fixed patch size of 128 px, respectively, as anasinput. Here, with each other, Here, the the results resultsof ofLOM LOMrecordings recordingsand andSEM SEMimages imagesare arecompared compared with each other, and and the the similarities similarities and and differences differencesare arediscussed. discussed.Thereby Therebythe theaim aimisistotoclarify clarifytotowhat what extent extent aa quantification quantificationbased basedon onthe theLOM LOMimages imagesisissufficient sufficientasasthe thesimplest simplestand andfastest fastest method forfor aa method and and to to what what extent extentmore morecomplex complexhigh-resolution high-resolutionSEM SEMimages imagesare areneeded needed representative evaluation. representative evaluation. In 15a–c, the the different different segmentation segmentation results results of of the thetrained trainedmodels modelscan canbe beexamexIn Figure Figure 15a–c, amined separately. It is noticeable that the majority of the color-coded predictions (LB— ined separately. It is noticeable that the majority of the color-coded predictions (LB—green, green, M—yellow, MST—purple, UB—blue, uncertain—red) are very comparable M—yellow, MST—purple, UB—blue, uncertain—red) are very comparable betweenbethe tween the individual models. Nevertheless, there are isolated areasthe in which themodels different individual models. Nevertheless, there are isolated areas in which different also models also produce different described in the section, methodology section, the is produce different estimates. Asestimates. described As in the methodology the final decision final decision is made by a majority vote. This allows an increase in objectivity and a remade by a majority vote. This allows an increase in objectivity and a reduction of errors, duction of errors, similar to consultation between experts. similar to consultation between experts. Figure result using thethe proposed patch-wise classifiFigure 15. 15. Showing Showingan anoverview overviewofofthe thesegmentation segmentation result using proposed patch-wise classication approach. (a–c) show the respective results using each model, A, B, and C, based on an fication approach. (a–c) show the respective results using each model, A, B, and C, basedSEM on an micrograph independently. (d) shows the combined result using the MaxVoting approach. (e) shows SEM micrograph independently. (d) shows the combined result using the MaxVoting approach. the combined result after applying a median filter with a kernel size corresponding to the selected (e) shows the combined result after applying a median filter with a kernel size corresponding to the step size of the sliding window technique to smooth the borders and eliminate artifacts. The colors selected steptosize the sliding window technique to smoothpurple—MST, the borders and eliminate artifacts. The correspond the of following classes: green—LB, yellow—M, blue—UB, and red—uncolors correspond to result the following green—LB, yellow—M, purple—MST, blue—UB, and certain. (f) shows the as shownclasses: in (e) overlayed with the respective input SEM image. red—uncertain. (f) shows the result as shown in (e) overlayed with the respective input SEM image. In order to eliminate individual artifacts and to smooth the boundaries of the deorderranges, to eliminate individual artifacts andsize to smooth the boundaries of the detected tectedInphase a median filter with a kernel corresponding to the respective step phase median filterofwith a kernel size corresponding to the respective step size is size is ranges, used as athe final step post-processing. used as the final step of post-processing. Metals 2023, 13, 1395 22 of 27 Consequently, an evaluation of the majority vote requires a classification by each of the models used and then unification of the predictions. The quantification of a 200 × 200 µm test area with a high-resolution SEM image with a step size of 16 px (approx. 0.8 µm) required 3 min in the Colab environment used for this purpose. The duration is thus dependent on the dimension of the section to be quantified, as well as the step size, which corresponds to the resolution of the evaluation, and can thus be adjusted accordingly. The confidence threshold for decision-making in this case was deliberately chosen to be extremely high at 75%. This is usually the majority, i.e., 50%. In this case, however, this illustrates the problems discussed in the segmentation of complex Q/QT steels. Similar to the labeling process by experts, the transition zones between two phase areas have been classified as ambiguous (red). This was the basic idea behind the patch-wise approach: to be able to precisely map this uncertainty without having to add a separate class within the training data, which then contains characteristic visual features of various other classes. Looking at Figure 16, at first glance, a large part of the segmented areas between LOM and SEM image matches. The discrepancies become clearer in the enlarged section (red). There, the UB fraction (blue) in the LOM is overestimated. This is due to the strong expression of the laths in the LOM. A closer look at the SEM confirms the prediction of the model based on the SEM data regarding classification in favor of the MST class. Furthermore, there are occasional confusions between UB (blue) and LB (green) within the LOM. As explained in the section above, this is due to the insufficient resolution of the Metals 2023, 13, x FOR PEER REVIEW 24 of 28 precipitated carbides between and within the ferritic laths, again representing the greatest limitation of an evaluation based on LOM as a simpler methodology. Figure 16.Segmentation Segmentation results as colored for the sample shown in Figures Figure 16. results as colored overlayoverlay for the sample shown in Figures 4 and 15 due to4 and 15 due to in occurring phases based based on correlative LOM (a) and SEM including magnified its wide widevariety variety in occurring phases on correlative LOM (a)(b), and SEM (b), including magnified regions. The colors correspond to the following classes: green—LB, yellow—M, purple—MST, regions. The colors correspond to the following classes: green—LB, yellow—M, purple—MST, blue—UB, and red—uncertain. blue—UB, and red—uncertain. In summary, the functionality and applicability of the proposed approach could be demonstrated. The relatively high variance within the elaborate datasets, consisting of 22 samples, allowed us to train a robust model that allows a successful application of the learned criteria and characteristic visual features. Thus, LOM and SEM microstructural images that included the diverse microstructural constituents were successfully seg- Metals 2023, 13, 1395 23 of 27 It would be desirable to reduce the proportion of misclassifications and to increase the lack of unambiguity by an increased uncertainty expressed by a larger proportion of the corresponding uncertain class. This can be achieved by adjusting the confidence threshold upward. Thus, in general, the proportion of the additional class representing the model uncertainty can be interpreted as a measure of the complexity of the analyzed microstructure. This martensitic (yellow) steel with areas of upper bainite (blue) illustrates the robustness of the underlying models (see Figure 17). The quantification based on the LOM Metals 2023, 13, x FOR PEER REVIEW 25 of 28 image corresponds, apart from minimal differences, to that of the SEM image. Thus, it can be concluded that there are applications in which the evaluation based on the easierto-generate LOM image is undoubtedly sufficient for reliable characterization. Although additionally represent an even variance to chemical composition, prothe corresponding UB areas arehigher relatively smallwith and respect the differences in the visual features cess well as acquisition conditions, including more contrasting of theconditions, UB, in this as case to that of the M, are only minor, the model wasvariable able to identify all methods. corresponding areas from the LOM image. Figure17. 17. Correlative Correlative LOM LOM (a) (a) and and SEM SEM (b) (b)images images of ofan anunseen unseensample samplethat thatwas was not notincluded included in in Figure the training datasets, including the respective overlay of the segmentation results of the combined the training datasets, including the respective overlay of the segmentation results of the combined approachatata aconfidence confidence threshold of 75% after using the smoothing operation. The colors correapproach threshold of 75% after using the smoothing operation. The colors correspond spond to the following classes: yellow—M, blue—UB, and red—uncertain. to the following classes: yellow—M, blue—UB, and red—uncertain. In contrast, Figure 18 again illustrates the limitations of evaluating only LOM images. Based on the LOM image, most of this section is assigned to the LB phase. Based on the SEM image, the majority can be assigned to the UB phase. Both decisions are comprehensible based on the respective images. However, only the SEM image reveals the specific morphology of the precipitated carbides. Although these are also finely distributed and Metals 2023, 13, 1395 24 of 27 not excessively coherent, a closer look reveals that they were precipitated between the extremely fine laths along their boundaries, which confirms the assessment as UB. Thus, it can be concluded once again that the evaluation of both recording methods can also be carried out on unseen steels, but that the significance of the LOM recording depends on the complexity and fineness of the present structure. If the characteristic features are too fine, it is not possible to evaluate the LOM recording alone to yield the desired result. Nevertheless, even in the LOM, the isolated areas belonging to the MST class (purple) could be correctly Metals 2023, 13, x FOR PEER REVIEWidentified, which reduces the aforementioned concerns about limited distinguishability 26 of 28 between LB and MST. In this case, the topography of the MST is sufficiently contrasted so that it can be reliably distinguished from the LB based on the LOM micrograph. Figure18. 18.Correlative CorrelativeLOM LOM(a) (a)and andSEM SEM(b) (b)images imagesof ofanother anotherunseen unseensample samplethat thatwas wasnot notincluded included Figure in the training datasets, including the respective overlay of the segmentation results of the combined in the training datasets, including the respective overlay of the segmentation results of the combined approach confidencethreshold thresholdofof 60% after using smoothing operation. colors correapproach at aa confidence 60% after using thethe smoothing operation. The The colors correspond spond to the following classes: green—LB, yellow—M, purple—MST, blue—UB, and red—uncerto the following classes: green—LB, yellow—M, purple—MST, blue—UB, and red—uncertain. tain. In summary, the functionality and applicability of the proposed approach could be 4. Conclusions demonstrated. The relatively high variance within the elaborate datasets, consisting of 22 samples, allowed usas toquenched train a robust thatsteels allows a successful of the Quenched as well and model tempered are known for application their wide range learned criteriaand andhigh characteristic visual features. Thus, LOMcan andoften SEMmainly microstructural of applications complexity of microstructures which be qualiimages that included thehigher-resolution diverse microstructural constituents were successfully segmented tatively assessed using microscopy methods due to their fine microand quantified. An example was used to show the extent to which it is sufficient to autocarry structural features. For the segmentation of these types of steel microstructures, an out quantification using the simpler LOM methodology and where its limitations are. Thus, mated, objective, and reproducible machine learning (ML) approach is proposed. The four high-resolution constituents SEM imagesmartensite, are necessary for a meaningful of the fine and microstructural tempered martensite,evaluation lower bainite, and upper more complex microstructural constituents. However, this also applies to a conventional bainite are considered for the segmentation, which can all be present simultaneously in a evaluation byof a metallographer. great added value insegmentation, the automationa of this evaluation single region interest. InsteadThe of the typical semantic patch-wise clasroutine by the DL approach presented here, therefore, also lies in reproducibility. Assuming sification is used, which significantly simplifies the annotations required for the ML apreproducible contrasting andwindow constantapproach recording conditions forsuitable comparable steels, the proach. An advanced sliding combined with post-processing quantifications by this approach are preferable to the subjective evaluations by nevertheless enables a finely resolved segmentation based on the classifications. different experts. In order to further improve the robustness and reliability of this approach, it is The foundation for a successful implementation is a correlative microscopy aprecommended to generate additional well-founded training data, which may additionally proach, combining light optical microscope (LOM), scanning electron microscope (SEM), represent an even higher variance with respect to chemical composition, process conditions, and electron backscatter diffraction (EBSD). Additional information from EBSD was esas well as acquisition conditions, including more variable contrasting methods. sential for a well-funded and objective assignment of the ground truth. From this correlative characterization, datasets with a large microstructural diversity were generated for a 4. Conclusions LOM and an SEM classification aiming to evaluate the limits of the proposed model’s roQuenched as well as quenched and tempered steels are known for their wide range of bustness. applications and high complexity of microstructures which can often mainly be qualitatively The ML models achieved an accuracy of 88.8% for the LOM patches and 93.7% for assessed using higher-resolution microscopy methods due to their fine microstructural feathe SEM patches. Building on this, the segmentation of new unseen images was demontures. For the segmentation of these types of steel microstructures, an automated, objective, strated. The possibilities and limitations of segmentation, also with regard to potential and reproducible machine learning (ML) approach is proposed. The four microstructural uncertainties in the assignment of the ground truth, were discussed in detail. In addition, the authors specifically investigated which performance is still achievable using only lowresolution LOM images and for which use cases higher resolution SEM images are preferable. The presented approach offers a universally valid alternative to a quantitative evaluation of highly-complex microstructures, where an unambiguous generation of pixel- Metals 2023, 13, 1395 25 of 27 constituents martensite, tempered martensite, lower bainite, and upper bainite are considered for the segmentation, which can all be present simultaneously in a single region of interest. Instead of the typical semantic segmentation, a patch-wise classification is used, which significantly simplifies the annotations required for the ML approach. An advanced sliding window approach combined with suitable post-processing nevertheless enables a finely resolved segmentation based on the classifications. The foundation for a successful implementation is a correlative microscopy approach, combining light optical microscope (LOM), scanning electron microscope (SEM), and electron backscatter diffraction (EBSD). Additional information from EBSD was essential for a well-funded and objective assignment of the ground truth. From this correlative characterization, datasets with a large microstructural diversity were generated for a LOM and an SEM classification aiming to evaluate the limits of the proposed model’s robustness. The ML models achieved an accuracy of 88.8% for the LOM patches and 93.7% for the SEM patches. Building on this, the segmentation of new unseen images was demonstrated. The possibilities and limitations of segmentation, also with regard to potential uncertainties in the assignment of the ground truth, were discussed in detail. In addition, the authors specifically investigated which performance is still achievable using only low-resolution LOM images and for which use cases higher resolution SEM images are preferable. The presented approach offers a universally valid alternative to a quantitative evaluation of highly-complex microstructures, where an unambiguous generation of pixel-wise training masks is not possible in a reproducible way, similar to this showcase of Q/QT steels. This ML-based quantification is noted for its automation, objectivity, and reproducibility, and enables microstructural analyses of previously unfeasible quality and detail and, thus, can form the basis for future process–microstructure–property correlations as well as for improving industrial quality control. Author Contributions: Conceptualization, B.-I.B., M.M., and D.B.; methodology, B.-I.B., M.M.; software, B.-I.B.; validation, B.-I.B., M.M.; formal analysis, B.-I.B., M.M.; investigation, B.-I.B.; resources, B.-I.B.; data curation, B.-I.B.; writing—original draft preparation, B.-I.B., M.M.; writing—review and editing, B.-I.B., M.M., D.B., and T.S.; visualization, B.-I.B.; supervision, F.M., D.B.; project administration, T.S., D.B., and F.M.; funding acquisition, M.M., D.B., F.M., and T.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the EFRE Funds of the European Commission. Data Availability Statement: The dataset presented in this article is not readily available yet because it is still part of an ongoing study. Requests to access the datasets should be directed to bjoernivo.bachmann@uni-saarland.de. Acknowledgments: The authors thank steel manufacturer Aktien-Gesellschaft der Dillinger Hüttenwerke for the strategic collaboration in which this research project was elaborated and for providing the sample material. The EFRE Funds of the European Commission and the State Chancellery of Saarland for support of activities within the ZuMat project are acknowledged. We acknowledge support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation). Conflicts of Interest: TS was employed by the company Aktien-Gesellschaft der Dillinger Hüttenwerke. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References 1. 2. 3. 4. Stuckner, J.; Harder, B.; Smith, T.M. Microstructure Segmentation with Deep Learning Encoders Pre-Trained on a Large Microscopy Dataset. Npj Comput. Mater. 2022, 8, 200. 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Phosphorescent soft salt for ratiometric and lifetime imaging of intracellular pH variations
Chemical science
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aInstitute of Molecular Functional Materials, Department of Chemistry and Partner State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Waterloo Road, Hong Kong, P. R. China. E-mail: rwywong@hkbu.edu.hk; Fax: +852 34117348; Tel: +852 34117074 bKey Laboratory for Organic Electronics & Information Displays (KLOEID), Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications, Nanjing 210023, P. R. China. E-mail: iamqzhao@njupt.edu.cn; Fax: +86 25 85866396; Tel: +86 25 85866396 cInstitute of Polymer Optoelectronic Materials and Devices, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, P. R. China † Electronic supplementary information (ESI) available: UV-visible spectrum, photoluminescence spectrum, 1H NMR spectra, MS spectra and cell imaging experiment. See DOI: 10.1039/c5sc04624f Phosphorescent soft salt for ratiometric and lifetime imaging of intracellular pH variations† In contrast to traditional short-lived fluorescent probes, long-lived phosphorescent probes based on transition-metal complexes can effectively eliminate unwanted background interference by using time- resolved luminescence imaging techniques, such as photoluminescence lifetime imaging microscopy. Hence, phosphorescent probes have become one of the most attractive candidates for investigating biological events in living systems. However, most of them are based on single emission intensity changes, which might be affected by a variety of intracellular environmental factors. Ratiometric measurement allows simultaneous recording of two separated wavelengths instead of measuring mere intensity changes and thus offers built-in correction for environmental effects. Herein, for the first time, a soft salt based phosphorescent probe has been developed for ratiometric and lifetime imaging of intracellular pH variations in real time. Specifically, a pH sensitive cationic complex (C1) and a pH insensitive anionic complex (A1) are directly connected through electrostatic interaction to form a soft salt based probe (S1), which exhibits a ratiometric phosphorescent response to pH with two well- resolved emission peaks separated by about 150 nm (from 475 to 625 nm). This novel probe was then successfully applied for ratiometric and lifetime imaging of intracellular pH variations. Moreover, quantitative measurements of intracellular pH fluctuations caused by oxidative stress have been performed for S1 based on the pH-dependent calibration curve. Received 1st December 2015 Accepted 4th February 2016 DOI: 10.1039/c5sc04624f www.rsc.org/chemicalscience the lifetime difference between the probe and interference signal.2–5 In addition, lifetime as the detected signal is inde- pendent of excitation laser intensity, target molecule concen- tration and photobleaching, and is very benecial for imaging applications. Phosphorescent transition-metal complexes (PTMCs), typically exhibiting long emission lifetime, large Stokes shiand high photostability,6–11 are ideal candidates for biological applications, especially for lifetime imaging appli- cations,12–21 although examples of PTMCs for monitoring intracellular biomolecules by PLIM are quite rare.12–19 Chemical Science EDGE ARTICLE View Article Online View Journal | View Issue Chemical Science EDGE ARTICLE View Article Online View Journal | View Issue This journal is © The Royal Society of Chemistry 2016 † Electronic supplementary information (ESI) available: UV-visible spectrum, photoluminescence spectrum, 1H NMR spectra, MS spectra and cell imaging experiment. See DOI: 10.1039/c5sc04624f EDGE ARTICLE Cite this: Chem. Sci., 2016, 7, 3338 Received 1st December 2015 Accepted 4th February 2016 DOI: 10.1039/c5sc04624f www.rsc.org/chemicalscience cle. Published on 04 February 2016. Downloaded on 6/19/2018 12:38:11 PM. s article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Chem. Sci., 2016, 7, 3338 Phosphorescent soft salt for ratiometric and lifetime imaging of intracellular pH variations† Yun Ma,a Hua Liang,b Yi Zeng,a Huiran Yang,b Cheuk-Lam Ho,a Wenjuan Xu,b Qiang Zhao,*b Wei Huangb and Wai-Yeung Wong*ac aInstitute of Molecular Functional Materials, Department of Chemistry and Partner State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Waterloo Road, Hong Kong, P. R. China. E-mail: rwywong@hkbu.edu.hk; Fax: +852 34117348; Tel: +852 34117074 Open Access Article. Published on 04 February 2016. Downloaded on 6/19/2018 12:38:11 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Design concept of a ratiometric pH probe and chemical structures of complexes A1, C1 and S1. (C1) was prepared by reuxing bis(cyclometalated) iridium(III) dichloro-bridged dimer in the presence of an excess of qpy ligand.55 The anionic iridium(III) complex [Ir(dfppy)2(CN)2]- Bu4N+ (A1) was synthesized from [Ir(dfppy)2Cl]2 and tetrabu- tylammonium cyanide (10 equiv.) in dichloromethane at 50 C for 4 h. By mixing two oppositely charged iridium(III) complexes A1 and C1 (1 : 1 molar ratio) in a mixture of CH3CN–H2O (1 : 1, v/v) at room temperature, the sosalt (S1) was obtained through the metathesis reaction. The above complexes were character- ized by 1H and 13C NMR spectroscopy, MALDI-TOF spectrom- etry and elemental analysis. Here we present the rst example of a sosalt based ratio- metric probe for imaging and measuring pH variations in living cells. Intracellular pH is a crucial parameter associated with cellular behaviors and pathological conditions, such as cell proliferation, apoptosis,44 drug resistance,45 enzymatic activity,46 and ion transport.47 Abnormal cellular pH value is an indicator of inappropriate cellular functions, which are associ- ated with many common diseases, for example, stroke,48 cancer,49 and Alzheimer's disease.50 It is thus vital to monitor pH alterations in biological cells and tissues to understand physiological and pathological processes.51,52 Introduction Fluorescence bioimaging based on uorescent probes provides a powerful approach for visualizing morphological details in biological systems with subcellular resolution.1 However, most traditional uorescent probes oen suffer from interference due to autouorescence and scattered light, which increases background noise and reduces the signal-to-noise ratio (SNR). Recently, an emerging technique, namely, photoluminescence lifetime imaging microscopy (PLIM), has offered an effective way of eliminating unwanted background interference based on Despite the advantages of PTMCs-based probes, most previously reported probes were based on single emission intensity changes. The diversities in cell morphology within different districts might inuence the quality and quantity of emission signals, which can result in substantial misinterpre- tations when dynamic changes of intracellular biomolecules are investigated. Therefore, accurate and quantitative measure- ments of the actual concentrations of intracellular biomole- cules or the relative changes of concentrations in living cells are difficult. Ratiometric measurement is normally used to address this issue. It can permit simultaneous recording of the relative changes of two separated wavelengths instead of measuring single emission intensity changes and thus offers built-in correction for environmental effects, leading to a more favor- able system for imaging living cells and tissues.22–25 However, This journal is © The Royal Society of Chemistry 2016 3338 | Chem. Sci., 2016, 7, 3338–3346 View Article Online Edge Article Chemical Science Fig. 1 Design concept of a ratiometric pH probe and chemical structures of complexes A1, C1 and S1. most ratiometric probes developed recently for imaging of biological molecules are based on organic dyes or nano- particles,26–31 only a few ratiometric PTMCs probes have been reported.32–34 It is still a challenge to design PTMCs-based ratiometric probes due to their complex excited-state properties.35–38 Luminescent ion pairs, which consist of two photoactive coordination complexes with opposite charges, are called “so salts” due to the sonature of the ions.39–43 Recently, Thompson and co-workers have studied the photophysical properties of sosalts in detail and successfully applied them in organic light-emitting diodes.39 However, extensive studies on sosalts have not received much attention yet. Considering that two emission wavelengths from a sosalt can be easily separated by chemical modication of cyclometalated ligands of the two ionic complexes, sosalts will be a good and versatile platform for the design of phosphorescent ratiometric probes. To date, however, the applications of sosalts in chemical sensing and biological systems are still unexploited areas. Photophysical properties The photophysical data of A1, C1 and S1 are summarized in Table S1 (ESI†). Spectroscopic results of A1 and C1 in acetoni- trile are shown in Fig. 2a. The emission peaks of anionic complex A1 at around 451 and 475 nm display vibronic Fig. 1 schematically describes the design concept. We selected the cationic complex C1 with pendant pyridyl moie- ties as a pH-sensitive phosphor and the anionic complex A1 as a pH-insensitive phosphor. These two luminophores are con- nected by electrostatic interaction to form the sosalt complex S1. Complex S1 is expected to give two emission bands, namely pH-insensitive blue and pH-sensitive red phosphorescence emissions. Thus, the ratio of the phospho- rescence intensities can respond to different pH values (2.03– 7.94). Moreover, S1 exhibits two well-resolved emission peaks separated by about 150 nm (from 475 to 625 nm), which avoids mutual interference of two emission bands and allows for high-resolution and sensitive ratiometric response of pH variations. Hence, complex S1 could act as an ideal ratiometric probe to monitor pH variations in biological cells. Further- more, to utilize the long phosphorescence lifetime of complex S1, PLIM experiments were carried out to monitor intracellular pH alterations. Fig. 2 (a) Normalized absorption and photoluminescence spectra of A1 and C1 in acetonitrile solution. (b) Photoluminescence spectra of S1 at different concentrations in acetonitrile solution. (c) Photo- luminescence spectra of anionic complex A1 (105 M) in acetonitrile solution with various amounts of cationic complex C1 (0–1.0  105 M). (d) Stern–Volmer plot of the quenching study between C1 and A1 ([Q] is the concentration of quencher). Ratiometric response to pH variations Fig. 3 (a) Changes in the phosphorescence emission spectra of A1 (2.0  105 M) in the pH range of 2.03–7.94 in CH3CN/buffer (1 : 9, v/ v). (b) Changes in the phosphorescence emission spectra of C1 (2.0  105 M) in the pH range of 2.03–7.94 in CH3CN/buffer (1 : 9, v/v). (c) Changes in the phosphorescence emission spectra of S1 (2.0  105 M) in the pH range of 2.03–7.94 in CH3CN/buffer (1 : 9, v/v). (d) Plot of I625 nm/I451 nm versus pH values. I625 nm and I451 nm indicate the phosphorescence intensity at 625 nm and at 451 nm, respectively. The phosphorescence emission spectra of A1, C1 and S1 were examined in acetonitrile/buffer (1 : 9, v/v) at various pH values (2.03–7.94). There are no obvious spectral variations for A1 solutions of different pH values (Fig. 3a). For C1, the emission intensity at 625 nm decreases dramatically with the decrease in pH value, which can serve as an on–offsingle intensity based pH probe (Fig. 3b). Protonation makes the pyridine ring in the ancillary ligand of C1 a stronger electron acceptor, which can cause quenching of the phosphorescence. To realize the ratio- metric probe, a sosalt S1 constituted of A1 and C1 by elec- trostatic interaction has been developed. The phosphorescence spectral changes of S1 at different pH values are displayed in Fig. 3c. Increasing the pH value results in a higher phospho- rescence intensity of C1 at 625 nm (I625 nm), while the emission intensity of A1 at 451 nm (I451 nm) remains unchanged. Such a change in phosphorescence emission color from blue to red with increasing pH value can be easily observed by the naked eye (Fig. 3c). The relative ratio of phosphorescence intensities (I625 nm/I451 nm) increased by 16-fold (from 0.18 to 2.86) over the pH range of 2.03–7.94 (Fig. 3d), which covers most physiological pH values. In addition, the phosphorescence response of S1 to pH value displays an excellent reversibility (Fig. S3†). Fig. 4 (a) Living HepG-2 cells co-stained with 10 mM A1 and C1 for 1 h at 37 C, and (b) living HepG-2 cells incubated with S1 under the same conditions. Fig. 4 (a) Living HepG-2 cells co-stained with 10 mM A1 and C1 for 1 h at 37 C, and (b) living HepG-2 cells incubated with S1 under the same conditions. Synthetic procedures Fig. 2 (a) Normalized absorption and photoluminescence spectra of Fig. 2 (a) Normalized absorption and photoluminescence spectra of A1 and C1 in acetonitrile solution. (b) Photoluminescence spectra of S1 at different concentrations in acetonitrile solution. (c) Photo- luminescence spectra of anionic complex A1 (105 M) in acetonitrile solution with various amounts of cationic complex C1 (0–1.0  105 M). (d) Stern–Volmer plot of the quenching study between C1 and A1 ([Q] is the concentration of quencher). Fig. 2 (a) Normalized absorption and photoluminescence spectra of A1 and C1 in acetonitrile solution. (b) Photoluminescence spectra of S1 at different concentrations in acetonitrile solution. (c) Photo- luminescence spectra of anionic complex A1 (105 M) in acetonitrile solution with various amounts of cationic complex C1 (0–1.0  105 M). (d) Stern–Volmer plot of the quenching study between C1 and A1 ([Q] is the concentration of quencher). The cyclometalated iridium(III) chloro-bridged dimer [Ir(C^N)2Cl]2 (C^N ¼ 2-(2,4-diuorophenyl)pyridine (dfppy) or 2- phenylpyridine (ppy)) and the 2,20:4,400:40,4000-quaterpyridyl (qpy) ligand were synthesized according to the literature methods.53,54 The cationic iridium(III) complex [Ir(ppy)2qpy]+Cl This journal is © The Royal Society of Chemistry 2016 Chem. Sci., 2016, 7, 3338–3346 | 3339 Edge Article View Article Online Edge Article View Article Online View Article Online Chemical Science Fig. 3 (a) Changes in the phosphorescence emission spectra of A1 (2.0  105 M) in the pH range of 2.03–7.94 in CH3CN/buffer (1 : 9, v/ v). (b) Changes in the phosphorescence emission spectra of C1 (2.0  105 M) in the pH range of 2.03–7.94 in CH3CN/buffer (1 : 9, v/v). (c) Changes in the phosphorescence emission spectra of S1 (2.0  105 M) in the pH range of 2.03–7.94 in CH3CN/buffer (1 : 9, v/v). (d) Plot of I625 nm/I451 nm versus pH values. I625 nm and I451 nm indicate the phosphorescence intensity at 625 nm and at 451 nm, respectively. The quenching study was carried out to investigate the energy transfer between the two ionic components in S1. As shown in Fig. 2c, with the addition of an increasing amount of cationic complex C1 into an acetonitrile solution of the anionic complex A1 (105 M), the red emission increased gradually at the expense of the blue emission from A1. This quenching effect can be attributed to the intermolecular triplet–triplet energy transfer.58–60 The results indicate that the energy transfer/ quenching process is very efficient between the two ionic complexes. Ratiometric response to pH variations We investigated the interference to the pH measurement by biological molecules, and the phosphorescence spectral responses of S1 in the presence of oxidative-stress-associated redox chemicals (such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and H2O2) and essential metal ions (such as K+, Na+, Zn2+, Cu2+, Ca2+, Mn2+, Mg2+, Fe2+ and Fe3+) were measured. There are no apparent spectroscopic changes detected (Fig. S4†), which indicates that S1 could act as a phosphorescent probe for the detection of intracellular pH alterations without any interference. In addition, the stability of S1 in acetonitrile/buffer (1 : 9, v/v) at 37 C was investigated. Fig. S5† shows that the relative ratio of phosphorescence intensities (I625 nm/I451 nm) barely changed at 37 C even aer 2 h, indicating the good stability of S1. progressions, which is a result of the triplet ligand-centered (3LC) transition on the cyclometalated ligands.56 The cationic complex C1 shows a broad and featureless spectrum with the emission maximum at 625 nm. Therefore, C1 is expected to emit from a metal-to-ligand charge-transfer (MLCT) state.57 The photoluminescence (PL) spectrum of S1 exhibits concentration dependence (Fig. 2b). The intensity ratio of emission peaks of A1 and C1 varies signicantly depending on the solution concentration, suggesting that the degree of energy transfer between the two complexes is different. At 380 nm excitation, the emission is mainly from the anionic complex A1 at a rela- tively low concentration of 106 M, which might be due to the fact that the quantum efficiency of A1 is much higher than that of C1. The blue emission decreases as the concentration of solution increases, with the cationic complex C1 acting as a quencher of A1, and the emission of A1 is barely observed at a concentration of 103 M or above. Synthetic procedures The quenching rate constant (Kq) can be derived by dividing the slope of the tted straight line by I0 (the lumines- cence intensity with no quencher present) (Fig. 2d). The calcu- lation yielded a Kq value of 5.49  1010 M1 s1. Open Access Article. Published on 04 February 2016. Downloaded on 6/19/2018 12:38:11 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cytotoxicity The cytotoxicity towards HepG-2 cells was evaluated by the standard MTT (MTT ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) assay. The results are illustrated in This journal is © The Royal Society of Chemistry 2016 3340 | Chem. Sci., 2016, 7, 3338–3346 Chemical Science View Article Online Edge Article Fig. 5 (a) Phosphorescence images of S1 in HepG-2 cells clamped at pH 3.98, 5.02, 6.08, 7.01 and 8.01, respectively. The excitation wavelength was 405 nm and the images of the first row (blue channel) and second row (red channel) were collected in the ranges of 430–480 nm and 600– 700 nm, respectively. Overlay images (third row) and ratio images obtained from the red and blue channels (fourth row). (b) Phosphorescence emission spectra of the HepG-2 cells at pH 3.98 and 8.01. Open Access Article. Published on 04 February 2016. Downloaded on 6/19/2018 12:38:11 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence Fig. 5 (a) Phosphorescence images of S1 in HepG-2 cells clamped at pH 3.98, 5.02, 6.08, 7.01 and 8.01, respectively. The excitation wavelength was 405 nm and the images of the first row (blue channel) and second row (red channel) were collected in the ranges of 430–480 nm and 600– 700 nm, respectively. Overlay images (third row) and ratio images obtained from the red and blue channels (fourth row). (b) Phosphorescence emission spectra of the HepG-2 cells at pH 3.98 and 8.01. Table 1 Emission lifetimes of S1 at different pH values pH 3.99 pH 5.11 pH 6.07 pH 6.95 pH 7.94 s451 nm (ns) 705  1.6 697  1.4 739  1.2 725  1.1 711  0.9 s625 nm (ns) 73  3.2 131  1.5 169  1.7 184  1.6 328  1.7 Fig. 6 Phosphorescence lifetime images of S1 in living HepG-2 cells at different pH values. HepG-2 cells were incubated for 1 h at 37 C. Table 1 Emission lifetimes of S1 at different pH values luminescence was observed in HepG-2 cells (Fig. S7†). The overlay of confocal luminescence and bright-eld images demonstrated that luminescence was evident in the cytoplasm region. To determine the kinetics of complex internalization, time-lapse imaging was carried out to monitor the progression of A1, C1 and S1 in HepG-2 cells via a Live Cell Workstation. Confocal images were obtained aer 15 min, 30 min, 45 min and 1 h, respectively. As shown in Fig. Cytotoxicity Table 2 Emission lifetimes of S1 at different pH values via TCSPC- PLIM pH 3.98 pH 5.02 pH 6.08 pH 7.01 pH 8.01 RPMI (ns) 261  1.4 238  1.3 211  1.7 176  1.4 141  2.8 Cells (ns) 312  3.1 271  2.3 247  2.2 209  1.9 178  3.2 Table 2 Emission lifetimes of S1 at different pH values via TCSPC- PLIM Fig. S6.† Aer treatment of living HepG-2 cells with different concentrations of S1 for 24 h, the cellular viabilities were esti- mated to be approximately 85% at 200 mM, apparently indi- cating good biocompatibility and low cytotoxicity of S1. Fig. S6.† Aer treatment of living HepG-2 cells with different concentrations of S1 for 24 h, the cellular viabilities were esti- mated to be approximately 85% at 200 mM, apparently indi- cating good biocompatibility and low cytotoxicity of S1. Next, these complexes were used for monitoring intracellular pH changes. The HepG-2 cells were cultured with 10 mL niger- icin (10 ng mL1) for 10 min to homogenize the intracellular pH value rst. Remarkable intracellular luminescence enhance- ment was observed with the increase in pH value for C1 (Fig. S10 and S11†). In contrast, no obvious luminescence intensity change can be detected in HepG-2 cells for A1 (Fig. S10 and S11†). We subsequently exploited the ratiometric probe S1 to examine the change in the cellular pH value in living cells. As This journal is © The Royal Society of Chemistry 2016 Cytotoxicity S8,† only very weak luminescence was observed in the cells in the rst 45 min for A1, indicating a slow cellular uptake rate of A1. In contrast, for C1, notable luminescence was detected in the cells within a short period of time (15 min). ig. 6 Phosphorescence lifetime images of S1 in living HepG-2 cells at ifferent pH values. HepG-2 cells were incubated for 1 h at 37 C. These ndings suggest that the cellular uptake rates are different for A1 and C1, which might be due to the different ionic natures of the complexes. However, when S1 was treated with HepG-2 cells, both the blue and red luminescence were observed in the cells at 15 min (Fig. S9†), implying that the cellular uptake rate for each counterpart of the sosalt is similar. In addition, the overlapping rate was calculated, which showed that the two counterparts of the sosalt remained intact in the cells. From Fig. 4, we can see that the overlapping rate between the blue and red channels was calculated to be 70.3% when the living cells were co-stained with A1 and C1. However, aer the treatment of living cells with S1, the over- lapping rate of the two channels was 94.6%. These observations suggest that the cationic and anionic parts of S1 remain intact rather than fall apart in the cells. Fig. 6 Phosphorescence lifetime images of S1 in living HepG-2 cells at different pH values. HepG-2 cells were incubated for 1 h at 37 C. Table 2 Emission lifetimes of S1 at different pH values via TCSPC- PLIM pH 3.98 pH 5.02 pH 6.08 pH 7.01 pH 8.01 RPMI (ns) 261  1.4 238  1.3 211  1.7 176  1.4 141  2.8 Cells (ns) 312  3.1 271  2.3 247  2.2 209  1.9 178  3.2 Fig. S6.† Aer treatment of living HepG-2 cells with different concentrations of S1 for 24 h, the cellular viabilities were esti- mated to be approximately 85% at 200 mM, apparently indi- cating good biocompatibility and low cytotoxicity of S1. Chem. Sci., 2016, 7, 3338–3346 | 3341 Open Access Article. Published on 04 February 2016. Downloaded on 6/19/2018 12:38:11 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 7 (a) Intracellular pH calibration curve of S1 in HepG-2 cells. (b) Ratiometric images and (c) phosphoresce ntact cells, H2O2 (100 mM) treated, NEM (100 mM) treated and NAC (100 mM) treated cells were incubated for Fig. 7 (a) Intracellular pH calibration curve of S1 in HepG-2 cells. (b) Ratiometric images and (c) phosphorescence lifetime images of S1 (10 mM). Intact cells, H2O2 (100 mM) treated, NEM (100 mM) treated and NAC (100 mM) treated cells were incubated for 1 h at 37 C. shown in Fig. 5a, the luminescence from the red channel (600– 700 nm) in cells increases with increasing pH value, whereas that from the blue channel (430–480 nm) hardly alters. These imaging results have further been demonstrated by phospho- rescence emission spectra of HepG-2 cells at pH 3.98 and 8.01 (Fig. 5b), which show signicant variation in the red channel but small change in the blue channel. The obtained intracel- lular phosphorescence emission spectra are also similar to those measured in solution. Thus, the variations in the ratio of blue to red intensity reveal the ability of S1 to measure a pH- dependent signal linearly over the pH range of 4–8 (R2 ¼ 0.9854; Fig. 7a). In addition, it is important to understand how S1 behaves at different concentrations inside the cells because S1 shows concentration-sensitive emission. Therefore, living cells were incubated with 1 mM S1 to detect the intracellular pH variations. As shown in Fig. S12,† the obtained imaging results are similar to those measured at the concentration of 10 mM. can see that the variation trend of emission lifetime of S1 is similar in the solution and cells. However, it is found that the emission lifetimes in the solution are shorter than those in the cells. This might be because the oxygen content inside cancer cells is lower than that in air-equilibrated solution. The PLIM experiment collected photons randomly from both blue and red emissions, and only the red emission was affected by the change in pH value. Therefore, more photons from the blue emission are collected as the pH decreases due to the reduction in the red emission intensity. And the fact that s625 nm is decreasing as the pH decreases (Table 1) indicates that the increase in average photoluminescence lifetime from pH 8 (savg ¼ 178  3.2 ns) to pH 4 (savg ¼ 312  3.1 ns) is reasonable. Open Access Article. Published on 04 February 2016. Downloaded on 6/19/2018 12:38:11 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In addition, savg of S1 (1 mM) is consistent with the results obtained at the concentration of 10 mM (Table 2, Fig. S18 and S19†). Thus, the result has demonstrated that S1 has the ability to detect the intracellular pH alterations by the photoluminescence lifetime, which highlights the capability of removing the background uorescence. Lifetime imaging To utilize the long phosphorescence lifetime of complex S1, a PLIM experiment was carried out for living HepG-2 cells. We expect that PLIM can separate the long-lived phosphorescence signal from other contributions to the total photoluminescence. The emission lifetimes of S1 were rst measured in CH3CN/ buffer (1 : 9, v/v) of different pH values (Table 1). The lifetime from 625 nm is increased with increasing pH value (from 73 to 328 ns), but that from 451 nm stays unchanged (around 700.0 ns), which is similar to the variation trend of the emission intensity. Then, the PLIM experiment was performed aer the living HepG-2 cells were incubated with 10 mM S1 at 37 C for 1 h. As shown in Fig. 6, the average photoluminescence lifetime (savg) of S1 experienced an obvious increase with the decrease in pH value. The savg of S1 was determined to be 178  3.2 ns by PLIM when the intracellular pH value was 8.01. As the intra- cellular pH value decreased from 8.01 to 3.98, the savg of S1 was measured to be 209  1.9 ns, 247  2.2 ns, 271  2.3 ns and 312  3.1 ns, respectively (Fig. S14†). The s averages of S1 from cell growth media at varying pH values were collected by confocal TCSPC-PLIM (TCSPC ¼ time-correlated single photon counting) to study how well the s average from cells matched with the solution data at varying pH value (Fig. S16†). From Fig. S17† we This journal is © The Royal Society of Chemistry 2016 Ratiometric imaging Practical application of complexes C1, A1 and S1 in lumines- cence imaging of living HepG-2 cells was investigated using confocal luminescence microscopy. Aer incubation with 10 mM of C1 (A1 or S1) for 1 h at 37 C, notable intracellular This journal is © The Royal Society of Chemistry 2016 Chem. Sci., 2016, 7, 3338–3346 | 3341 View Article Online Chemical Science Edge Article Fig. 7 (a) Intracellular pH calibration curve of S1 in HepG-2 cells. (b) Ratiometric images and (c) phosphorescence lifetime images of S1 (10 mM). Intact cells, H2O2 (100 mM) treated, NEM (100 mM) treated and NAC (100 mM) treated cells were incubated for 1 h at 37 C. Quantitative measurement of intracellular pH uctuations The ratio channel, obtained based on the above two distin- guishable emission channels, shows a characteristic pH- dependent signal, demonstrating the ability of S1 to examine a pH-dependent signal linearly over the pH range of 3.98 to 8.01 (Fig. 7a). According to this calibration curve, the averaged intracellular pH value of intact HepG-2 cells was measured to be 6.80  0.20 (Fig. 7). Furthermore, the effects of different redox substances on intracellular pH uctuations were investigated based on the calibration curve. As shown in Fig. 7a and b, the pH value for H2O2 treated cells was determined to be 7.20  0.15, which indicates that H2O2 makes the HepG-2 cells more basic. This observation is in good agreement with the previous report that oxidative stress (such as H2O2) can cause inactiva- tion of lysosomal V-ATPase, consequently resulting in increasing the pH value of lysosomes.61 Then, NEM (N-ethyl- maleimide, a GSH inhibitor) or NAC (N-acetylcysteine, a GSH precursor) was applied to HepG-2 cells to control the intracel- lular GSH level. The intracellular pH value was measured to be 7.10  0.12 aer decreasing the concentration of GSH by NEM (Fig. 7). Possible reasons for the basication of cells are as This journal is © The Royal Society of Chemistry 2016 3342 | Chem. Sci., 2016, 7, 3338–3346 Chemical Science View Article Online View Article Online Edge Article Chemical Science follows: (i) an oxidative cellular environment caused by the decrease of GSH induces lysosomal inactivation; (ii) the func- tion of the Na+/H+ antiporter may be affected by the decrease of GSH level.62 Interestingly, the generation of GSH by NAC decreases the intracellular pH to 4.80  0.16 (Fig. 7). We believe that a reductive cellular environment caused by the high concentration of GSH induces the activation of lysosomal V- ATPase, which is a possible explanation for this acidication.63 Ratiometric images directly reveal the intracellular pH changes caused by the oxidative stress (Fig. 7b). To take advantage of ratiometric measurement, accurate and quantitative determi- nations of the actual intracellular pH value and its relative changes can be well achieved. Moreover, when the lifetime serves as a signal, these intracellular pH variations could also be detected by PLIM. Fig. 7c shows the phosphorescence lifetime images of intact cells, H2O2 treated, NEM treated and NAC treated cells, and their savg were determined to be 217 ns, 206 ns, 201 ns and 263 ns, respectively. Conclusions A mixture of iridium(III) bis(2-phenylpyridine) dichloro-bridged dimer (34.5 mg, 0.032 mmol) and 2,20:4,400:40,4000-quaterpyridyl (25 mg, 0.08 mmol) was dissolved in a mixture of dichloro- methane and methanol (2 : 1, v/v) and the mixture was reuxed for 16 h. The solution was concentrated and washed with hexane to afford the crude product. Aerwards, the product was recrystallized by vapor diffusion of diethyl ether into acetoni- trile. Yield 65%. 1H NMR (400 MHz, acetonitrile-d3): d (ppm) 8.97 (s, 2H), 8.82 (d, J ¼ 8 Hz, 4H), 8.10 (d, J ¼ 4 Hz, 4H), 7.87– 8.81 (m, 10H), 7.70 (d, J ¼ 4 Hz, 2H), 7.09 (dd, J ¼ 16 Hz, 4H), 6.98 (t, J ¼ 16 Hz, 2H), 6.33 (d, J ¼ 8 Hz, 2H). 13C NMR (100 MHz, acetone-d6), d (ppm): 168.67, 157.71, 152.21, 151.90, 151.23, 150.28, 149.41, 144.96, 143.86, 139.74, 132.50, 131.40, 127.37, 125.95, 124.61, 123.98, 123.58, 122.48, 120.96. MS (MALDI-TOF) [m/z]: 811.8 [M  Cl]+. Elemental analysis (calcd, found for C40H48F4IrN5): C (55.41, 55.74), H (5.58, 5.93), N (8.08, 8.24). In conclusion, we have developed a novel sosalt based phos- phorescent probe. This type of probe consists of two oppositely charged ionic complexes with two distinguishable emission colors, which makes it a perfect candidate as a ratiometric probe. The emission color of S1 changes from blue to red with increasing pH value. S1 is cell-permeable and exhibits low cytotoxicity, and it has been successfully applied for ratiometric pH imaging with the use of confocal microscopy, demon- strating its great potential for intracellular environmental monitoring. Furthermore, phosphorescence lifetime imaging experiments can detect intracellular pH variations by photo- luminescence lifetime measurements, which allowed for elim- inating background uorescence and selecting long-lived phosphorescence images. Quantitative measurement of intra- cellular pH uctuations caused by oxidative stress has been successfully carried out for S1 based on the pH-dependent calibration curve. To our knowledge, this work represents the rst example of a sosalt based probe for chemical sensing and biological applications. We expect that this work can provide valuable information for the future rational design of phos- phorescent ratiometric and lifetime probes. Synthesis of 2,20:4,400:40,4000-quaterpyridyl (qpy) 4,40-Bipyridine (2 g) and 10% palladium on carbon (400 mg) in DMF were heated at 180 C for 48 h under an inert atmosphere of nitrogen. Aer being cooled to room temperature, the mixture was ltered and DMF was removed under reduced pressure. The residual solid was recrystallized from acetone to give white crystals. Yield 19%. 1H NMR (400 MHz, CDCl3): d (ppm) 8.81 (d, J ¼ 8 Hz, 2H), 8.76 (d, J ¼ 8 Hz, 6H), 7.67 (dd, J ¼ 4 Hz, 4H), 7.59 (dd, J ¼ 8 Hz, 2H). MS (MALDI-TOF) [m/z]: 310.4 [M+]. Synthesis of A1 Iridium(III) bis(2-(2,4-diuorophenyl)pyridine) dichloro-bridged dimer (146 mg, 0.12 mmol) was combined with tetrabuty- lammonium cyanide (360 mg, 1.2 mmol) in dichloromethane at 50 C for 4 h. Aer removing dichloromethane under reduced pressure, the product was puried by aluminum oxide (chro- matography) with dichloromethane and methanol (10 : 1, v/v) as the eluent. Yield 78%. 1H NMR (400 MHz, DMSO-d6): d (ppm) 9.53 (d, J ¼ 8 Hz, 2H), 8.20 (d, J ¼ 8 Hz, 2H), 8.02 (t, J ¼ 16 Hz, 2H), 7.44 (t, J ¼ 12 Hz, 2H), 6.64–6.59 (m, 12H), 5.52 (d, J ¼ 8 Hz, 2H), 3.33–3.17 (m, 8H), 1.61–1.53 (m, 8H), 1.34–1.25 (m, 8H), 0.92 (t, J ¼ 16 Hz, 12H). 13C NMR (100 MHz, DMSO-d6): d (ppm) 170.2, 163.42, 154.97, 145.83, 139.76, 137.44, 132.17, 129.64, 124.72, 123.36, 121.62, 120.11, 59.49, 31.15, 23.52, 19.67, 13.95. MS (MALDI-TOF) [m/z]: 625.1 [M  Bu4N]. Elemental analysis (calcd, found for C42H30ClIrN6): C (59.60, 59.95), H (3.57, 3.89), N (9.93, 10.11). This journal is © The Royal Society of Chemistry 2016 Quantitative measurement of intracellular pH uctuations This result highlights that the detection of intracellular pH alterations by the lifetime signal can avoid the interferences from autouorescence, scat- tered light as well as other technical artifacts. were recorded with a HP UV-8453 spectrophotometer. Photo- luminescence spectra were measured with an Edinburgh Instrument FLS920 combined uorescence lifetime and steady state spectrophotometer that was equipped with a red-sensitive single-photon counting photomultiplier in Peltier Cooled Housing. The quantum efficiencies of complexes were measured in solutions at room temperature with an aerated aqueous solution of [Ru(bpy)3]Cl2 as an external standard (F ¼ 0.028). Acknowledgements We thank National Basic Research Program of China (973 Project 2013CB834702), Hong Kong Research Grants Council (HKBU12304715), Areas of Excellence Scheme of HKSAR (AoE/P- 03/08), National Natural Science Foundation of China (21171098 and 21201104), Natural Science Foundation of Jiangsu Province of China (BK20130038), Hong Kong Baptist University (FRG1/14-15/084) and Hong Kong Scholars Program (XJ2012053) for nancial support. The work was also supported by Partner State Key Laboratory of Environmental and Biolog- ical Analysis (SKLP-14-15-P011) and Strategic Development Fund of HKBU. Viability (%) ¼ [(mean absorbance value of treatment group)/ (mean absorbance value of control)]  100. Cytotoxicity assay The cytotoxicity of the complexes toward the HepG-2 cells was measured by the methyl thiazolyl tetrazolium assay. Before incubation at 37 C under 5% CO2 atmosphere for 24 h, HepG-2 cells in log phase were seeded into a 96-well cell-culture plate at 1  104 per well. The S1 solution (100 mL per well) at concen- trations of 200, 100, 50 and 25 mM was added to the wells of the treatment group, and MTT containing 0.2% DMSO (100 mL per well) to the negative control group. The cells were incubated at 37 C under 5% CO2 atmosphere for 24 h. 20 mL MTT solution (5 mg mL1) was added to each well of the 96-well assay plate, and the solution was incubated for another 3 h under the same conditions. A Tecan Innite M200 monochromator based multifunction microplate reader was used for measuring the OD570 (absorbance value) of each well referenced at 690 nm. The following formula was used to calculate the viability of cell growth: Cell culture The HepG-2 cell line was supplied by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The HepG-2 cells were grown in RPMI 1640 (Roswell Park Memorial Insti- tute's Medium) supplemented with 10% FBS (Fetal Bovine Serum) at 37 C and 5% CO2. Cells (5  108 per L) were plated on 18 mm glass coverslips and allowed to adhere for 24 h. Intracellular pH calibration The HepG-2 cells were incubated at 37 C for 10 min in high K+ buffer (30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES) with various pH values (3.98–8.01) in the presence of 10 mL nigericin (10 ng mL1). HepG-2 cells were incubated with C1 (or A1 and S1) for 1 h at 37 C. The phosphorescence and lifetime images were then measured, and the pH calibration curve was constructed with an Olympus FV1000 confocal microscope. Materials and general experiments Commercially available chemical reagents were used without further purication. All solvents were puried before use. The solvents were carefully dried and distilled from appropriate drying agents prior to use. 1H and 13C NMR spectra were recorded with a Bruker Ultrashield 400 MHz FT-NMR spec- trometer. Mass spectra were obtained with a Bruker Autoex matrix assisted laser desorption/ionization time of ight mass spectrometer (MALDI-TOF MS). UV-visible absorption spectra This journal is © The Royal Society of Chemistry 2016 Chem. Sci., 2016, 7, 3338–3346 | 3343 Edge Article View Article Online Edge Article View Article Online Chemical Science for 1 h at 37 C. Then, the experiments were carried out on an Olympus FV1000 laser scanning confocal microscope and a 60 oil-immersion objective lens. A semiconductor laser served as the excitation source of the HepG-2 cells incubated with C1 (or A1 and S1) at 405 nm. The overlay images were generated by the Fluoview Viewer soware. The ratio images were analyzed with Kodak Molecular Imaging Soware, and the ratio of region of interest (ROI) was calculated pixel-by-pixel. All data were expressed as mean  standard deviation. PLIM imaging The PLIM image setup is integrated with an Olympus IX81 laser scanning confocal microscope. The uorescence signal was detected by the system of the confocal microscope and correl- ative calculation of the data was carried out by professional soware which was provided by Pico Quant Company. The light from the pulse diode laser head (Pico Quant, PDL 800-D) with excitation wavelength of 405 nm and frequency of 1 MHz (>1 ms) was focused onto the sample with a 40/NA 0.95 objective lens for single-photon excitation. The PLIM data were processed in SymPhoTime 64 pro soware (Pico Quant Company), and exported in ASCII (line proles) or BMP (images) format. PLIM data obtained from 256  256 regions of interest were t using double exponential tailt, binning factor 1 in SymPhoTime 64 pro soware. Fit curves in each pixel, excluding dark regions, yielded a lifetime distribution over the whole image, with a lifetime being displayed on the x-axis and the abundance of each lifetime on the y-axis. The average lifetime (50% of the total integral is reached) and half-width (difference between lifetimes at which half-maximal abundance is reached) were calculated from the distribution curve. Synthesis of S1 [Ir(dfppy)2(CN)2]Bu4N+ (25 mg, 0.035 mmol) and [Ir(ppy)2- qpy]+Cl (25 mg, 0.04 mmol) were added to CH3CN (10 mL). The reaction mixture was stirred for 2 h at room temperature and then extracted with CH2Cl2. The solution was washed with water several times to remove the counterions and concentrated by rotary evaporation. The resulting solid was washed with diethyl ether to afford S1 as a red solid. Yield 85%. 1H NMR (400 MHz, acetonitrile-d3): d (ppm) 9.51 (d, J ¼ 4 Hz, 2H), 8.93 (s, 2H), 8.71 (d, J ¼ 4 Hz, 4H), 8.15 (d, J ¼ 4 Hz, 2H), 8.02 (d, J ¼ 8 Hz, 4H), 7.81–7.74 (m, 12H), 7.62 (d, J ¼ 4 Hz, 2H), 7.17 (t, J ¼ 12 Hz, 2H), 7.02–6.95 (m, 4H), 6.89 (t, J ¼ 12 Hz, 2H), 6.33 (t, J ¼ 16 Hz, 2H), 6.25 (d, J ¼ 8 Hz, 2H), 5.55 (d, J ¼ 8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): d (ppm) 167.22, 164.30, 156.61, 156.36, 154.18, 150.94, 149.68, 147.96, 146.30, 144.88, 144.30, 142.94, 139.37, 138.04, 131.52, 130.80, 128.59, 128.27, 126.89, 125.64, 124.51, 123.86, 123.54, 123.02, 122.82, 122.52, 122.30, 121.88, 120.60, 118.50, 112.30, 97.03, 55.38. MS (MALDI-TOF) [m/z]: 811.8 [M+], 625.1 [M]. 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https://openalex.org/W1957540036
https://revistapsicologia.uchile.cl/index.php/RDP/article/download/36149/37832, https://www.redalyc.org/pdf/264/26435341007.pdf
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Modelado de la intención emprendedora con redes bayesianas
Revista de psicología
2,015
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8,654
2014, 23(2), 71-87 ISSN impreso: 0716-8039 ISSN en línea: 0719-0581 www.revistapsicologia.uchile.cl Revista de Psicología UNIVERSIDAD DE CHILE Modelado de la intención emprendedora con redes bayesianas Modelling entrepreneurial intention with Bayesian networks Jorge López y Ana María Ruiz-Ruano Universidad Católica de Murcia, Murcia, España Resumen El emprendimiento es considerado hoy en día como un elemento clave en las sociedades postindustriales. Desde un punto de vista psicológico, el emprendimiento puede ser entendido como un proceso actitudinal condicionado por la intención emprendedora. En este trabajo se construyeron, testearon y utilizaron tres modelos de red bayesiana destinados a clarificar las relaciones establecidas entre variables tradicionalmente asociadas al emprendimiento y la intención emprendedora. Una muestra de 254 estudiantes universitarios (73 hombres y 179 mujeres), con edades comprendidas entre los 18 y los 50 años (M = 22.47, DT = 4.11), rellenó una encuesta destinada a registrar parámetros asociados con la actitud y la intención emprendedora. Con sus respuestas se construyeron tres modelos de red bayesiana que fueron validados y utilizados para realizar predicciones sobre las variables implicadas en el análisis. Los resultados muestran que la deseabilidad percibida, la viabilidad percibida, el locus de control y la influencia familiar son las variables que más influencian la intención emprendedora en emprendedores potenciales. La influencia de estas variables sobre la intención emprendedora fue analizada considerando diferentes escenarios y las relaciones detectadas fueron discutidas con relación a planteamientos teórico-prácticos en el contexto de la potenciación de actitudes emprendedoras. Abstract Entrepreneurship is nowadays considered as a key element in postindustrial societies. From a psychological point of view, entrepreneurship can be understood as an attitudinal process conditioned by entrepreneurial intention. We built, tested and used three Bayesian models designed to clarify the relationships between variables commonly associated with entrepreneurship and entrepreneurial intention. A sample of 254 undergraduate university students (73 male and 179 female), ages ranging from 18 to 50 (M = 22.47, SD = 4.11), filled in a questionnaire that registered parameters associated with entrepreneurial attitude and intention. Survey answers were used to build three Bayesian networks models which were validated and used to make predictions about the variables involved in the model. Our results show that perceived desirability, perceived feasibility, and the normative social influence derived from the family and the locus of control reliably predict entrepreneurial intention. The influence of these variables over the entrepreneurial intention was analyzed considering different scenarios, and the discovered relationships between variables where discussed from practical and theoretical points of view in the context of promoting entrepreneurial attitudes. Palabras clave: emprendimiento, educación superior, intención, actitudes, redes bayesianas. Keywords: entrepreneurship, higher education, intention, attitudes, Bayesian nets. Contacto: J. López. Facultad de Ciencias de la Salud. Departamento de Ciencias de la Salud. Universidad Católica de Murcia. Campus de los Jerónimos s/n, 30107, Guadalupe, Murcia, España. jpuga@ucam.edu Cómo citar este artículo: López, J. y Ruiz-Ruano, A. M. (2014). Modelado de la intención emprendedora con redes bayesianas. Revista de Psicología, 23(2), 71-87. http://dx.doi.org/10.5354/0719-0581.2014.36149 López y Ruiz-Ruano Introducción El rol de las personas emprendedoras está ganando protagonismo en las sociedades postindustriales como elemento que favorece el desarrollo social y económico tanto a nivel local como global. Como señaló Shapero (1981) hace más de tres décadas, el modelo económico basado en la explotación de recursos humanos poco cualificados por parte de grandes multinacionales es poco deseable en comparación con economías locales que se autorenuevan y que se sostienen por pequeñas empresas. Este segundo modelo, que tiende a ser más sostenible en el tiempo, es el caldo de cultivo ideal para que las personas emprendedoras pongan en marcha su creatividad para dinamizar el desarrollo social y económico de sus regiones. Por ello, el emprendimiento es considerado como la fuente que “proporciona a las comunidades la diversidad y el dinamismo que no sólo favorece el desarrollo continuo, sino que también proporciona un entorno en el que la libertad personal y los derechos individuales pueden florecer” (Shapero, 1985, p. 5). Sin embargo, pese a la relevancia que se atribuye al emprendimiento, este sigue siendo un fenómeno social difícil de estudiar y conceptualizar. La analogía introducida por Rogoff y Lee (1996) es muy ilustrativa en este sentido, ya que equiparan el estudio de las partículas subatómicas, por parte de la física, al estudio de las personas emprendedoras desde la óptica de las ciencias sociales. Según los autores, de igual modo que la física observa el comportamiento de las partículas subatómicas pero no puede explicarlo, las ciencias sociales se percatan de los efectos de las personas emprendedoras pero no pueden explicar su comportamiento. En cierto modo, la creación de empresas es algo caótico, desordenado, turbulento y sensible a las condiciones iniciales que propicia cierta ausencia de replicabilidad del fenómeno (Smilor y Feeser, 1991). Por ello, pese a que se han desarrollado modelos orientados a optimizar las probabilidades de desarrollo de nuevas aventuras empresariales (e.g., Bhave, 1994), únicamente un bajo porcentaje de las empresas de nueva creación sobreviven a los cinco primeros años de vida (Fuller-Love, 2006). 72 Revista de Psicología 2014, 23(2), 71-87 Desde una perspectiva psicológica, el modelo de los rasgos ha sido el paradigma predominante hasta mediados de los años ochenta del pasado siglo (e.g., Fuller-Love, 2006; McKenzie, Ugbah y Smothers, 2007; Thompson, 2004). El modelo de los rasgos asume que las personas que crean empresas se diferencian de las demás por un conjunto de rasgos de personalidad relativamente estables que sirven para catalizar la creación de un nuevo negocio. Así, aspectos como una alta motivación de logro (Boyatzis, 1982; Genescá y Capelleras, 2004; McClelland, 1955, 1961; Veciana, 1989), un perfil de locus de control interno (García, Cano y Gea, 2005; Sánchez, 2003; Stanworth, Stanworth, Granger y Blyth, 1989; Welsch y Young, 1982), el optimismo o la tolerancia al riesgo (Cantillon, 1755/2010; Cooper, Woo y Dunkelberg, 1988; Hmieleski y Baron, 2009; Liang y Dunn, 2008, 2010; Lovallo y Kahneman, 2003; Welsch y Young, 1982) son algunos rasgos que han sido asociados a las personas que crean empresas. Sin embargo, esta perspectiva ha recibido numerosas críticas como paradigma explicativo y predictivo de la creación de empresas (Bird, 1988; Gartner, 1985, 1988). Por ejemplo, Robinson, Stimpson, Huefner y Hunt (1991) apuntaron cuatro grandes bloques de críticas que atacaban la perspectiva de los rasgos como modelo explicativo de los procesos individuales que daban lugar a la creación de empresas. En primer lugar, Robinson et al. (1991) aluden a un problema metodológico, dado que los instrumentos de medida que se venían utilizando en la psicología general se tomaron para el estudio del emprendimiento sin las adaptaciones pertinentes. Por otro lado, la interpretación de las medidas adoleció de los criterios básicos de validez exigibles a cualquier medida de rasgos psicológicos. En tercer lugar, los autores consideran que las teorías de la personalidad surgidas en el seno de la psicología hubiesen merecido la consiguiente adaptación al contexto de la investigación sobre la creación de empresas. Por último, Robinson et al. (1991) consideran que el modelo de los rasgos tendría que haber tenido en consideración las teorías interactivas de la personalidad que se comenzaron a forjar durante el apogeo del citado modelo. Intención emprendedora y redes bayesianas Un paradigma complementario y, en cierto modo, competidor del modelo de los rasgos para comprender o estudiar el fenómeno emprendedor es el basado en las actitudes. Desde esta otra perspectiva, se defiende que la creación de empresas está mediatizada por un proceso intencional y deliberado que conduce al emprendedor potencial a crear un negocio en interacción constante con el entorno social que le circunda. Por tanto, para este modelo la creación de empresas es fruto tanto de las predisposiciones estables de respuesta en la persona potencialmente emprendedora, como de la interacción que se produce entre la persona con las condiciones sociales y culturales en las que se ve envuelta (cf., Bird, 1988; Krueger y Brazeal, 1994; Krueger y Carsrud, 1993; Krueger, Reilly y Carsrud, 2000; Liñán, Battistelli y Moriano, 2008; Moriano, Gómez, Laguna, y Roznowsky, 2008). Licht y Siegel (2006) reconocen que el trabajo de Shapero y Sokol (1982) podría considerarse como un punto arquimédico que marcó un antes y un después con relación al estudio de los condicionantes sociales que influyen en la creación de empresas. Para Shapero y Sokol (1982) el mayor condicionante social que influye en la creación de empresas son los valores que una sociedad fomenta o promueve con relación al emprendimiento. Así, en la medida que una cultura o sociedad concreta presenta la creación de empresas como algo viable y deseable, la probabilidad de que una persona potencialmente emprendedora cree una empresa, aumenta. Sin embargo, en esta propuesta está implícita la idea de que cualquier persona no se convierte en emprendedor ineludiblemente cuando considera viable y deseable el desarrollo de una aventura empresarial. Más bien, para estos autores, la persona se convierte en emprendedora como consecuencia de una experiencia negativa caracterizada por una sensación subjetiva de rechazo por parte de la sociedad (Shapero, 1975). En este sentido, la persona que crea una empresa encajaría en el perfil de un individuo que ha sido “marginado” por la sociedad (e.g., Stanworth et al., 1989; Veciana, 1989) pero que, a la vez, considera el emprendimiento como algo deseable y viable. La propuesta sobre la influencia de la deseabilidad y la viabilidad en la intención emprendedora ha sido testada empíricamente en repetidas ocasiones (e.g., Fitzsimmons y Douglas, 2011; Krueger y Brazeal, 1994; Krueger y Carsrud, 1993; Moriano et al., 2008). Su estudio ha sido realizado bajo la óptica de la Teoría de la Acción Planeada (Ajzen y Fishbein, 2005) entendida como una evolución de la Teoría de la Acción Razonada (Ajzen y Fishbein, 1980; Fishbein y Ajzen, 1975), considerando que la creación de un nuevo negocio es fruto de un proceso intencional mediado por las interpretaciones subjetivas que la persona realiza sobre ciertos elementos sociales. Un metaanálisis reciente que estudia la integración entre el modelo de la intención emprendedora y la Teoría de la Acción Razonada puede encontrarse en Schlaegel y Koening (2014). Sin embargo, los trabajos destinados a estudiar el fenómeno emprendedor con redes bayesianas o metodología bayesiana son más limitados (e.g., Block, Hoogerheide y Thurik, 2012; López y García, 2012; López, Ramírez y Casado, 2012; Sohn y Lee, 2013) y, por ello, uno de los objetivos de este estudio es mostrar la utilidad de este tipo de técnica estadística para modelar la intención emprendedora. Las redes bayesianas son herramientas de modelado estadístico surgidas en el ámbito de la inteligencia artificial y se caracterizan por representar un aspecto concreto de la realidad, tanto desde un punto de vista cualitativo como cuantitativo (López y García, 2011a). Técnicamente, las redes bayesianas pertenecen a una familia de modelos gráficos destinados a modelar un problema de estudio de un modo altamente estructurado (Cowell, Dawid, Lauritzen y Spiegelhalter, 1999; Martínez y Rodríguez, 2003). Como se ha indicado con anterioridad, se considera que las dos claves principales que explican la intención emprendedora son la deseabilidad y la viabilidad (Fitzsimmons y Douglas, 2011; Krueger y Brazeal, 1994; Krueger y Carsrud, 1993; Licht y Siegel, 2006; Moriano et al., 2008; Shapero, 1982) y, por consiguiente, predecimos que en un modelo de red bayesiana estas dos variables también serán las que mayor varianza expliquen. Adicionalmente, se compararán tres modelos diferentes de red bayesiana Revista de Psicología 2014, 23(2), 71-87 73 López y Ruiz-Ruano con el objetivo de identificar la intensidad de las relaciones que se establecen entre las variables. En concreto, se testará un modelo estructural basado en la teoría actual sobre emprendimiento, un modelo de clasificador ingenuo bayesiano que sería análogo a la regresión logística y un modelo TAN (Tree Augmented NaïveBayes) que se diferencia del clasificador ingenuo en que tolera la presencia de relaciones entre las variables diagnósticas o de clasificación (Greiner, Su, Shen y Zhou, 2005; Greiner y Zhou, 2002; Shen, Su, Greiner, Musilek y Cheng, 2003). Por consiguiente, este estudio tendrá una doble utilidad: una finalidad teórico-práctica y otra orientada al aspecto metodológico. En cuanto al aspecto teórico-práctico nuestro trabajo servirá para arrojar luz sobre las relaciones del modelo actitudinal que explica la intención de crear una empresa en emprendedores potenciales. En este sentido, este trabajo podría ser de utilidad frente a la orientación que pudiera ofrecerse a los emprendedores potenciales que aspiran a crear su propio negocio (Fitzsimmons y Douglas, 2011). Por ejemplo, los emprendedores potenciales podrían ser informados sobre las variables que más influyen en su intención emprendedora y advertirles sobre aquellos aspectos que pueden afectar negativamente en el futuro desarrollo de su negocio. Una variable que podría considerarse perniciosa frente a la creación de empresas es el exceso de optimismo (e.g., Cooper et al., 1988; Hmieleski y Baron, 2009; Lovallo y Kahneman, 2003). Así, un modelo de red bayesiana que considerase la influencia negativa de esa variable sobre la intención emprendedora podría ser de utilidad para orientar a los futuros emprendedores. En nuestro trabajo, las variables utilizadas que se relacionan con cierta forma de optimismo (diferentes formatos de riesgo y obstáculos percibidos) podrían servir a tal efecto. Por su parte, este trabajo también presentará las posibilidades que ofrecen las redes bayesianas para modelar un fenómeno psicosocial como el emprendimiento con el consiguiente beneficio que esto podría aportar a la comunidad científica. Así, por ejemplo, el uso de este tipo de herramienta permitirá la representación del modelo estadístico tanto gráfica como 74 Revista de Psicología 2014, 23(2), 71-87 cuantitativamente (Edwards, 1998; Heckerman, 1995). Aunque los modelos gráficos que incorporan información cuantitativa han recibido considerable atención en el área de las ciencias sociales por medio de la utilización de modelos de ecuaciones estructurales; también es cierto que, como indican Anderson y Vastag (2004), las redes bayesianas tienen ciertas ventajas frente a este tipo de modelos. La posibilidad de combinar conocimiento previo con datos observados del problema modelado (Nadkarni y Shenoy, 2004), la no asunción de supuestos distribucionales en los datos (Anderson y Vastag, 2004) y el buen funcionamiento de estas técnicas con muestras pequeñas (López y García, 2011b) son ventajas que se aplican al caso particular que estamos abordando. Participantes Método La muestra estuvo compuesta por 254 estudiantes universitarios. El 28.97% fueron hombres (73) y el 71.03% mujeres (179), cuyas edades oscilaron entre los 18 y 50 años (M = 22.47; DT = 4.11). El 47.22% de los participantes se encontraban cursando estudios de Psicología (119), el 25.79% Licenciatura en Administración y Dirección de Empresas (LADE) (65), el 13.89% empresariales (35) y el 13.1% Magisterio (33). Del total de participantes, 76 se encontraban entre el primer y segundo curso de la carrera (30.16%), 66 cursaban tercero (26.19%), 80 se encontraban cursando cuarto (31.75%) y 30 se encontraban en el último curso de la carrera (11.90%). Instrumentos Para recoger los datos se diseñó una encuesta electrónica con la plataforma LimeSurvey (LimeSurvey Project Team, 2013). La encuesta estuvo integrada por cuatro secciones. En la primera sección se recogieron los datos sociodemográficos necesarios para caracterizar la muestra de participantes. En la sección segunda se presentaron las escalas de locus de control y riesgo percibido. La tercera sección contuvo preguntas relacionadas con los modelos actitudinales de emprendimiento y, por último, la cuarta sección se utilizó para que los participantes informasen voluntariamente de sus datos de Intención emprendedora y redes bayesianas contacto para participar en fases posteriores de la investigación. Para medir el locus de control emprendedor se utilizó la escala de Moriano et al. (2008). Esta escala contiene tres ítems puntuables en una escala tipo Likert con siete alternativas (1 a 7) en los que el participante ha de expresar el grado de interés que le suscitan conductas típicamente asociadas a un perfil cognitivo de persona emprendedora, caracterizada por un locus de control interno. El índice de consistencia interna observado en nuestro estudio es de .7 (ICsup 90% = .75, ICinf 90% = .64), pero en estudios previos esta escala mostró una consistencia interna de .77 (Moriano et al., 2008). Por su parte, para valorar el riesgo asociado a la creación de empresas los participantes fueron instados a responder tres ítems tipo Likert con cuatro alternativas de respuesta indicando el grado en que el contenido de cada ítem suscitaba riesgo percibido (desde nada o casi nada a mucho). Cada uno de los tres ítems estuvo referido al riesgo asociado a la actividad económica de la empresa, al riesgo económico personal y al riesgo para la carrera profesional personal. Los ítems no se agregaron en una única puntuación referida al riesgo global percibido, pero las respuestas que dieron los participantes a cada ítem se recodificaron en dos grupos: las dos opciones más altas de respuesta para indicar riesgo percibido y las opciones más bajas de respuesta para indicar ausencia de riesgo percibido. La mayor parte de las variables destinadas a medir componentes actitudinales fueron presentadas a los participantes de forma dicotómica. La intención emprendedora se evaluó con la pregunta: ¿Has pensado seriamente en crear tu propia empresa?, cuyas alternativas de respuesta fueron sí y no. La deseabilidad de crear una empresa también se valoró con una escala de respuesta dicotómica con las alternativas sí y no ante la pregunta: ¿Consideras deseable crear una empresa propia al finalizar tus estudios? Del mismo modo, se introdujeron dos preguntas para valorar la influencia de las normas sociales utilizando el mismo esquema de respuesta: una para considerar la influencia de la familia (¿Le gustaría a tu familia que creases tu propio negocio?) y otra para estudiar la influencia de los iguales (¿Le gustaría a tus amigos que creases tu propio negocio?). La actitud de los participantes hacia la creación de empresas se midió con la pregunta: ¿Crees que la creación de empresas es algo positivo o negativo?, donde las opciones de respuesta fueron algo positivo y algo negativo. La cantidad de obstáculos percibidos también fue valorado con una pregunta dicotómica (¿Qué cantidad de obstáculos crees que encontrarías si decidieses crear tu propio negocio?) en la que las alternativas de respuesta fueron muchos y pocos. Por último, la viabilidad de la creación de empresas se valoró a tres niveles: desde un punto de vista general, desde el punto de vista de la viabilidad de oportunidades y desde el punto de vista de la viabilidad entendida como recursos económicos (Cohen y Winn, 2007; McMullen y Shepherd, 2006; Shapero, 1981). La viabilidad (general) se midió utilizando un ítem tipo Likert con cuatro alternativas de respuesta que demandaba que el participante indicase el grado de viabilidad que percibía con relación a la creación de una empresa propia (desde muy poco viable a muy viable). Por su parte, la viabilidad de recursos (¿Qué importancia crees que tiene la disponibilidad de recursos (económicos, contactos, conocimientos, etc.) frente a la decisión de crear una empresa?) y la viabilidad de oportunidad (¿Qué importancia crees que tiene la detección de una oportunidad de negocio (una idea innovadora, detección de necesidades, nuevo método de producción, etc.) frente a la decisión de crear una empresa?) se evaluaron con tres alternativas de respuesta que hacían referencia a la importancia atribuida a cada aspecto de la viabilidad (ninguna, alguna y mucha). Procedimiento Para acceder a los participantes se diseñó un mensaje de correo electrónico de invitación que contenía una explicación de los objetivos de la investigación y el hiperenlace al formulario electrónico correspondiente. El mensaje de invitación se envió haciendo uso de la herramienta de correo electrónico integrada en la plataforma de enseñanza virtual que los alumnos utilizaban para seguir sus asignaturas de la universidad. Los participantes no recibieron Revista de Psicología 2014, 23(2), 71-87 75 López y Ruiz-Ruano ningún tipo de compensación económica ni crediticia por participar en la investigación y la única gratificación que recibieron por responder al cuestionario electrónico fue de carácter verbal. Análisis de datos La especificación de las estructuras gráficas fueron determinadas como pasos previos a la estimación paramétrica (Cowell et al., 1999). Se consideraron tres modelos gráficos: uno basado en la teoría existente en la literatura, otro basado en criterios metodológicos (el clasificador ingenuo, o simple, bayesiano) y, por último, uno basado en criterios estrictamente estadísticos (el TAN, Tree Augmented Naïve-Bayes o clasificador bayesiano optimizado). El modelo teórico testado en este estudio aparece representado en la figura 1, donde queda reflejado que la intención se relaciona con la viabilidad y la deseabilidad. Por su parte, la deseabilidad estaría asociada a tres tipos de riesgo (actividad de la empresa, económico personal y de carrera profesional), a la actitud hacia la creación de empresas y a las normas sociales (tanto desde el punto de vista familiar como desde la óptica de la interacción con los iguales). Por su parte, la viabilidad estaría asociada a dos componentes de la viabilidad (enfocados a los recursos económicos y a la oportunidad de negocio), a los obstáculos percibidos y al locus de control. El modelo TAN se estimó con el algoritmo implementado en la versión 5.12 de Netica (Norsys Software Corp., 2013), mientras que el clasificador simple bayesiano, dada su similitud con la regresión logística (Greiner et al., 2005; Greiner y Zhou, 2002; Shen et al., 2003), queda definido por la variable a predecir, antecedente o madre (en nuestro caso la intención emprendedora) y un conjunto de variables predictoras o hijas (el resto de variables implicadas en el estudio) y mostradas en la figura 1. La estimación paramétrica se llevó a cabo utilizando el procedimiento de máxima verosimilitud corregido con la ley de la sucesión de Laplace (Greiner et al., 2005). Para valorar la bondad de ajuste de los modelos se utilizaron parámetros de validación predictiva (López y García, 2011a), la pérdida logarítmica, la pérdida cuadrática y la compensación esférica (Pearl, 1978). Por último, para indagar en la influencia que tenía cada variable sobre el nodo predicho, se llevó a cabo un análisis de sensibilidad basado en el porcentaje de influencia sobre este nodo (Neapolitan, 1990). El resto de análisis de datos fue realizado utilizando la versión 3.1.2 del software de computación estadística R (R Core Team, 2014). Figura 1. Modelo de red bayesiana teórica. Riesgo AE: riesgo asociado a la actividad económica de la empresa; Riesgo EP: riesgo económico personal; Riesgo CP: riesgo en la carrera profesional. 76 Revista de Psicología 2014, 23(2), 71-87 Intención emprendedora y redes bayesianas Resultados La tabla 1 muestra que la deseabilidad, la viabilidad, la norma social vinculada a los iguales y la viabilidad de oportunidades, se asocian positivamente a la intención emprendedora. En lo que respecta al locus de control, los participantes de nuestro estudio que mostraron una mayor intención emprendedora se caracterizan por mayor locus de control interno (M = 13.97, SD = 3.99) si los comparamos con los que muestran menor intención emprendedora (M = 12.08, SD = 3.17), t(218.70) = 3.98, p < .001, d = 0.52, 1-β = .99, contraste unilateral. Como se puede apreciar en la figura 2, el algoritmo TAN genera un modelo de red bayesiana que contiene 11 enlaces adicionales al modelo basal definido por el clasificador simple baye- siano. Es destacable señalar que la deseabilidad global de la creación de una empresa aparece condicionada a la viabilidad percibida, mientras que esta segunda se muestra dependiente de las normas sociales derivadas del contexto familiar. La actitud hacia la creación de empresas también se muestra como dependiente de la viabilidad percibida y la influencia normativa del grupo de iguales aparece como dependiente del contexto social familiar. Es también destacable que el modelo identifica que la relación que se establece entre viabilidad y obstáculos percibidos estaría mediada por el locus de control. Según el modelo estimado por el TAN, la viabilidad entendida desde el punto de vista de la detección de oportunidades empresariales influiría sobre las normas sociales familiares y sobre la viabilidad de recursos. Y, por último, dos de estos enlaces Tabla 1 Frecuencias observadas (porcentajes entre paréntesis) para cada nivel de la variable y su relación con la intención emprendedora Alto/a Bajo/a χ2 b p Intención 116 (50.22) 115 (49.78) Riesgo AE 207 (87.71) 29 (12.29) 1.36 .24 Riesgo EP 197 (83.47) 39 (16.53) 1.05 .31 Riesgo CP 128 (54.24) 108 (45.76) 0.22 .64 Actitud 226 (97.84) 5 (2.16) 3.31 .07 NS (familia) 188 (81.39) 43 (18.61) 1.09 .30 NS (iguales) 206 (89.18) 25 (10.18) 14.71 <.01 Deseabilidad 126 (54.55) 105 (45.45) 48.04 <.01 Viabilidad 134 (58.01) 97 (41.99) 21.05 <.01 Viabilidad Ra 204 (88.31) 27 (11.69) 0.15 .67 Viabilidad Oa 192 (83.12) 39 (16.89) 8.06 <.01 Obstáculos 202 (87.45) 73 (28.97 - H) 29 (12.55) 179 (71.03 - M) 2.44 2.02 .12 .15 Sexo Nota: Riesgo AE: riesgo asociado a la actividad económica de la empresa; Riesgo EP: riesgo económico personal; Riesgo CP: riesgo en la carrera profesional; NS: norma social; Viabilidad R: viabilidad de recursos; Viabilidad O: viabilidad de oportunidades; H: hombre, M: mujer. a Estas variables se recodificaron en alta y baja dado que la alternativa de respuesta más baja fue elegida en una proporción muy pequeña de participantes. b Los grados de libertad para las χ2 es 1 en todos los casos. Revista de Psicología 2014, 23(2), 71-87 77 López y Ruiz-Ruano identificados por el algoritmo TAN generan un camino dirigido entre el riesgo asociado a la actividad económica y el riesgo asociado a la continuidad de la carrera profesional, pasando por el riesgo vinculado a la economía personal. La red que mejor predice la intención emprendedora es la generada automáticamente por el algoritmo TAN (tabla 2), que mejora en un 3.9% la clasificación correcta de casos que realiza el clasificador simple y en un 4.7% respecto de lo que lo hace la red teórica. La red TAN también es más sensible que los otros dos modelos al detectar la intención emprendedora, pero el clasificador simple tiene un mayor grado de especificidad. De manera general, el modelo generado automáticamente se comporta ligeramente mejor al tratar de predecir la intención emprendedora frente al modelo teórico y al modelo de clasificación simple. Sin embargo, no existen diferencias estadísticamente significativas entre la tasa de clasificaciones correctas realizadas por el modelo TAN cuando la comparamos con la de la red teórica, 2(1) = 2.745, p = .10, contraste bilateral o con el clasificador simple bayesiano, 2(1) = 1.795, p = .18, contraste bila- teral. Además, este incremento predictivo se produce a costa de una reducción en la parsimonia del modelo, entendida como el número de parámetros y de enlaces en el modelo. Así, mientras que tanto la red teórica como el clasificador simple bayesiano son capaces de realizar sus predicciones con 52 parámetros, el modelo TAN necesita más del doble para predecir la intención emprendedora. Del mismo modo, tanto el modelo teórico como el clasificador simple tienen 12 enlaces, mientras que la red TAN necesita casi el doble (24). Aunque podemos observar diferencias relativas en la influencia que ejerce cada variable sobre la intención emprendedora en función del modelo que estemos utilizando, todos los modelos coinciden al identificar que las dos variables que más influyen sobre la intención son la deseabilidad y la viabilidad (tabla 3). Adicionalmente, las otras dos variables que más variabilidad explican de la intención emprendedora son la influencia normativa de los iguales (amigos) y el locus de control. No obstante, el orden de importancia de estas variables depende de si utilizamos el modelo TAN, el teórico o el clasificador simple. Figura 2. Modelo de red bayesiana TAN estimado. Riesgo AE: riesgo asociado a la actividad económica de la empresa; Riesgo EP: riesgo económico personal; Riesgo CP: riesgo en la carrera profesional. 78 Revista de Psicología 2014, 23(2), 71-87 Intención emprendedora y redes bayesianas Tabla 2 Índices de bondad de ajuste de los modelos 24.5% (modelo TAN) y un 28% (clasificador simple). Si, adicionalmente, consideramos que la viabilidad percibida es alta, la probabilidad de que un estudiante universitario experimente una alta intención emprendedora aumenta entre un 28.1% (modelo TAN) y un 30.5% (modelo teórico). De manera antagónica, cuando la deseabilidad y la viabilidad son bajas, la probabilidad de que un estudiante manifieste intención de crear una empresa desciende entre un 29.5% (modelo TAN) y un 37.7% (clasificador simple). Red TAN Red teórica Clasificador simple Enlaces .23 .12 .12 Parámetros .106 .52 .52 Sensibilidad .819 .776 .681 Especificidad .739 .687 .800 PFP .261 .313 .200 Discusión y conclusiones PFN .181 .224 .319 VPP .760 .714 .775 VPN .802 .752 .713 TCC .779 .732 .740 Pérdida logarítmica .475 .563 .543 Pérdida cuadrática .313 .377 .362 Compensación esférica .825 .789 .797 En este trabajo hemos desarrollado, testado y utilizado tres modelos de red bayesiana para modelar la intención emprendedora en una muestra de estudiantes universitarios. Los tres modelos coinciden en indicar que la deseabilidad y la viabilidad son los predictores más importantes de la intención emprendedora. Por consiguiente, nuestros resultados apuntan en la misma línea trazada por los estudios previos que han sugerido, basándose en las ideas de Shapero y Shokol (1982), que la deseabilidad y la viabilidad son variables clave para entender el comportamiento de la intención emprendedora (Fitzsimmons y Douglas, 2011; Krueger y Brazeal, 1994; Krueger y Carsrud, 1993; Moriano et al., 2008). Además de la deseabilidad y la viabilidad, los tres modelos desarrollados y testados han mostrado que tanto la influencia de los iguales (amigos normalmente en este nivel educativo) como el locus de control son también variables relevantes para comprender la naturaleza de la intención emprendedora. Dado que las redes bayesianas son instrumentos estadísticos susceptibles de ser utilizados para orientar u optimizar la toma de decisiones (e.g., Edwards y Fasolo, 2001; López y García, 2011a), los modelos aquí desarrollados podrían ser de utilidad en el contexto aplicado para diseñar políticas o estrategias destinadas a la potenciación del emprendimiento entre los estudiantes universitarios. Así, como indican Smilor y Feeser (1991), herramientas como las que hemos desarrollado en este trabajo podrían ser de utilidad en la gestión del emprendimiento a nivel universitario donde, normalmente, se comienzan a desarrollar iniciativas de creación de empresas que, presumiblemente, tendrán efectos positivos en el tejido productivo. Nota: PFP: proporción de falsos positivos; PFN: proporción de falsos negativos; VPP: valor predictivo positivo; VPN: valor predictivo negativo; TCC: tasa de clasificaciones correctas. Las predicciones que hacen los tres modelos van en la misma dirección cuando consideramos la influencia secuencial de las cuatro variables que más impacto tienen sobre los mismos. En la tabla 4 aparecen representados, para cada uno de los modelos, los cambios que se producen en los niveles estimados de intención emprendedora cuando consideramos un escenario favorable (una respuesta positiva a cada una de las variables predictoras) y desfavorable (una respuesta negativa a cada una de las variables predictoras) a la intención emprendedora. Como se puede observar, la intención emprendedora aumenta entre un 17.8% (modelo TAN) y un 20.6% (clasificador simple) cuando se observa una alta deseabilidad en un estudiante universitario. Por el contrario, cuando la deseabilidad es baja, la intención emprendedora se reduce entre un Revista de Psicología 2014, 23(2), 71-87 79 López y Ruiz-Ruano Tabla 3 Porcentaje de influencia que ejerce cada variable sobre la intención emprendedora en cada modelo Nodos Red TAN Red Teórica Clasificador Simple Deseabilidad 15.60 (1) 15.60 (1) 15.00 (1) Viabilidad 6.68 (2) 6.84 (2) 4.88 (2) Norma social (iguales) 4.66 (3) 0.49 (4) 2.14 (4) Locus de control 3.61 (4) 0.61 (3) 3.14 (3) Viabilidad de oportunidades 2.66 (5) 0.16 (5) 1.71 (5) Actitud 0.94 (6) 0.02 (9) 0.08 (10) Obstáculos 0.84 (7) 0.11 (7) 0.06 (11) Riesgo AE 0.47 (8) 0.00 (12) 0.54 (7) Norma social (familia) 0.42 (9) 0.14 (6) 0.21 (9) Riesgo EP 0.36 (10) 0.05 (8) 1.38 (6) Riesgo CP 0.10 (11) 0.00 (11) 0.44 (8) Viabilidad de recursos 0.08 (12) 0.02 (10) 0.02 (12) Nota: Riesgo AE: riesgo asociado a la actividad económica de la empresa; Riesgo EP: riesgo económico personal; Riesgo CP: riesgo en la carrera profesional. Los números entre paréntesis indican la posición de influencia relativa que ocupa cada variable en un modelo concreto. Como se puede apreciar en la tabla 4, el cambio que experimenta la intención emprendedora cuando conocemos el estado de las cuatro variables más influyentes en cada uno de los modelos (deseabilidad, viabilidad, influencia social normativa de los iguales y locus de control) es sustancial. Para el modelo TAN y para el clasificador simple bayesiano, la probabilidad de que la intención emprendedora sea alta cuando se dan respuestas favorables a estas cuatro variables, aumenta entre un 32 y un 34 por ciento, respectivamente. A su vez, la reducción que se produce en la probabilidad de que la intención emprendedora sea alta cuando nos ponemos en el peor de los casos para las cuatro variables mencionadas, oscila entre un 45 y un 48 por ciento para los modelos TAN y de clasificación simple, respectivamente. Estos datos son más moderados si tenemos en cuenta el modelo teórico planteado inicialmente (en torno a un 30% de aumento cuando nos posicionamos en un escenario favorable y una reducción del 36% cuando suponemos el peor de los casos). 80 Revista de Psicología 2014, 23(2), 71-87 Estos resultados sugieren pautas estratégicas de acción que podrían orientarse a potenciar la intención emprendedora entre los estudiantes universitarios. Así, si se tratase de mostrar que la creación de una empresa propia es algo deseable para la sociedad y viable desde el punto de vista financiero, numerosos estudiantes universitarios podrían ver esta salida laboral como algo plausible al terminar sus estudios, del mismo modo a como lo ve la población general (Fitzsimmons y Douglas, 2011; Krueger y Brazeal, 1994). Así, de la misma manera a como han hecho notar Dreisler, Blenker y Nielsen (2003) para el caso danés, se podrían desarrollar programas de intervención orientados a estimular actitudes positivas hacia la creación de empresas en los estudiantes universitarios o, como señalan Liñan, Rodríguez-Cohard y RuedaCantuche (2011), comenzar a trabajar la orientación emprendedora a niveles preuniversitarios. Intención emprendedora y redes bayesianas Tabla 4 Cambio secuencial que experimenta la intención emprendedora en función de un escenario favorable (deseabilidad, viabilidad, influencia social y locus de control altos) o desfavorable (deseabilidad, viabilidad, influencia social y locus de control bajos) +28.1 Norma social (familia) +30.0 Locus de control +34.4 +19.9 +17.8 +30.5 +25.9 +27.4 +32.3 -24.5 -25.0 -28.0 -29.5 -36.4 -37.7 -45.0 -45.5 -47.4 -48.6 Escenario Modelo Deseabilidad Viabilidad Favorable Red TAN Red Teórica Clasificador Simple +20.6 Red TAN Red Teórica Clasificador Simple Desfavorable Nota: La probabilidad previa de que un estudiante muestre intención emprendedora es del 50.2%. Por otro lado, el modelo generado automáticamente (TAN) ha mostrado relaciones entre variables que deberían de ser estudiadas con más detenimiento en futuros estudios. Cabría destacar algunas relaciones interesantes que han sido puestas de manifiesto en la investigación sobre este fenómeno. Por un lado, se observa que tanto la actitud hacia la creación de empresas como la deseabilidad de este comportamiento dependerían de la viabilidad percibida, mientras que esta última dependería de la influencia que tienen las normas sociales asociadas al contexto familiar. Por consiguiente, el modelo generado muestra una compleja relación entre el emprendimiento y la influencia familiar en los emprendedores potenciales tal y como señalaron, por ejemplo, Stanworth et al. (1989) y Rogoff y Lee (1996). Así, en la medida en que la familia valore positivamente la creación de empresas propias, los hijos tenderán a decantarse por esta alternativa laboral y esto, según el modelo generado, podría explicar la viabilidad, la deseabilidad, la influencia social de los grupos de iguales y la actitud hacia la creación de empresas propias. Es particularmente interesante que el modelo TAN haya identificado un camino dirigido desde la influencia familiar hasta la actitud hacia la creación de empresas dado que, como señalaba Allport (1935) en el siglo pasado, uno de los cuatro pilares fundamentales de la creación de actitudes proviene de la influencia parental. El algoritmo de descubrimiento estructural automático también sugiere que el locus de control es una variable mediadora entre los obstáculos y la viabilidad percibida frente a la creación de una empresa. Esta relación tiene sentido desde un punto de vista teórico desde que se sabe que el locus de control interno es un rasgo que se asocia con las personas emprendedoras (e.g., Sánchez, 2003). Sin embargo, la relación que se establece entre influencia normativa social, locus de control e intención emprendedora parece ser más compleja de lo que se pensaba con anterioridad. Por ejemplo, la influencia de las normas sociales sobre la intención emprendedora, así como otras variables asociadas a la creación de empresas, parece ser más indirecta de lo que se creía anteriormente (e.g, Heuer y Liñán, 2013). En este sentido Siu y Lo (2013) han observado recientemente que la influencia de las normas sociales con relación a la intención emprendedora depende del grado en que una sociedad puede considerarse más colectivista o más individualista. Por su parte, los resultados de Zellweger, Sieger y Halter (2011) contrastan con la observación clásica que indica que el locus de control se asocia a la intención emprendedora. No obstante, tal y como indican en su trabajo, la relación entre locus de control e intención emprendedora podría estar mediada por la influencia familiar, de modo que los descenRevista de Psicología 2014, 23(2), 71-87 81 López y Ruiz-Ruano dientes de familias emprendedoras “se dan cuenta de que la capacidad de controlar su propio destino al optar por una carrera emprendedora puede ser mayormente una ilusión” (Zellweger et al., 2011, p. 531) si tenemos en cuenta el volumen de responsabilidades y presiones a la que están sometidos estos profesionales. Nuestros resultados arrojan luz sobre la compleja relación que podría existir entre estas variables; futuras investigaciones podrán poner a prueba hipótesis relacionadas con las mismas. Adicionalmente, el modelo TAN establece relaciones entre los diferentes componentes del riesgo y los diferentes formatos en los que se pueden entender la viabilidad de la creación de empresas que convendría estudiar en futuras investigaciones. Ello podría ser de utilidad, como hemos indicado más arriba, frente a la orientación de emprendedores potenciales con el ánimo de que evalúen sensatamente sus prospectivas de éxito en sus propuestas de negocio. De este modo, podrían reducir sensiblemente sus probabilidades de fracaso debidas a una mala gestión de sus creencias hacia los riesgos que tienen que asumir a lo largo del proceso (e.g., Cooper et al., 1988; Hmieleski y Baron, 2009; Lovallo y Kahneman, 2003). Por último, cabe mencionar el desfase que se observa en la capacidad predictiva del modelo TAN y su poder predictivo. Dado que el aumento que se produce en la bondad de ajuste de este modelo es marginal con relación a los otros dos y considerando que se reduce sensiblemente la parsimonia del modelo (en términos de número de parámetros y enlaces), futuros trabajos tendrían que profundizar en este aspecto para desarrollar modelos más aceptables desde el punto de vista metodológico. El uso y comparación de diferentes tipos de algoritmos de aprendizaje estructural podría ser una estrategia apropiada en este sentido. Para terminar, es conveniente destacar algunas limitaciones al trabajo realizado, así como futuras líneas de investigación que pudiesen arrojar 82 Revista de Psicología 2014, 23(2), 71-87 luz sobre el fenómeno del emprendimiento y que pudiesen servir de cara a la mejora del desarrollo social de nuestro entorno cercano. En primer lugar, sería deseable desarrollar estudios análogos a este en los que se utilizase una muestra de emprendedores reales, con el objetivo de comparar los resultados y las conclusiones que se han presentado en este trabajo. Esto podría servir para tratar de identificar diferencias y similitudes entre un perfil y otro con el ánimo de ahondar en la comprensión del fenómeno emprendedor. También sería deseable realizar más estudios destinados a clarificar las relaciones que el algoritmo TAN ha identificado entre las variables analizadas para tratar de ahondar en el entendimiento de la compleja relación que se establece entre las variables utilizadas. Como han intentado recientemente Ruiz-Ruano, López y Scutari (2014), utilizar otros algoritmos de búsqueda estructural podría ser de utilidad en este sentido. Desde el punto de vista metodológico podríamos criticar la generalización de nuestros resultados, dado el reducido tamaño muestral. Aunque, como hemos indicado anteriormente, el tamaño de muestra es un problema que las redes bayesianas parecen tolerar bien, un estudio con mayor tamaño muestral podría aportar evidencias sobre la validez cruzada del modelo estimado. A modo de conclusión, creemos que la potenciación de las actitudes emprendedoras tendría que enfocarse a la incipiente área del emprendimiento sostenible. 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Rigid limit for hypermultiplets and five-dimensional gauge theories
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To cite this version: Serguei Alexandrov, Sibasish Banerjee, Pietro Longhi. Rigid limit for hypermultiplets and five-dimensional gauge theories. Journal of High Energy Physics, 2018, 1801 (1), pp.156. ￿10.1007/JHEP01(2018)156￿. ￿hal-01627837￿ Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01627837 https://hal.science/hal-01627837v1 Submitted on 7 Jun 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Published for SISSA by Springer Received: December 16, 2017 Accepted: January 21, 2018 Published: January 31, 2018 Received: December 16, 2017 Accepted: January 21, 2018 Published: January 31, 2018 Open Access, c⃝The Authors. Article funded by SCOAP3. Rigid limit for hypermultiplets and five-dimensional gauge theories JHEP01(2018)156 JHEP01(2018)156 Sergei Alexandrov,a,b Sibasish Banerjeec,d and Pietro Longhie aLaboratoire Charles Coulomb (L2C), UMR 5221 CNRS-Universit´e de Montpellier, F-34095, Montpellier, France bTheoretical Physics Department, CERN, Geneva, Switzerland cIPhT, CEA, Saclay, Gif-sur-Yvette, F-91191, France dMax-Planck-Institut f¨ur Mathematik, Vivatsgasse 7, 53111 Bonn, Germany eDepartment of Physics and Astronomy, Uppsala University, Uppsala, Sweden E-mail: sergey.alexandrov@umontpellier.fr, sbanerje@mpim-bonn.mpg.de, pietro.longhi@physics.uu.se Sergei Alexandrov,a,b Sibasish Banerjeec,d and Pietro Longhie Sergei Alexandrov,a,b Sibasish Banerjeec,d and Pietro Longhie aLaboratoire Charles Coulomb (L2C), UMR 5221 CNRS-Universit´e de Montpellier, F-34095, Montpellier, France bTheoretical Physics Department, CERN, Geneva, Switzerland E-mail: sergey.alexandrov@umontpellier.fr, sbanerje@mpim-bonn.mpg.de, Abstract: We study the rigid limit of a class of hypermultiplet moduli spaces appearing in Calabi-Yau compactifications of type IIB string theory, which is induced by a local limit of the Calabi-Yau. We show that the resulting hyperk¨ahler manifold is obtained by performing a hyperk¨ahler quotient of the Swann bundle over the moduli space, along the isometries arising in the limit. Physically, this manifold appears as the target space of the non-linear sigma model obtained by compactification of a five-dimensional gauge theory on a torus. This allows to compute dyonic and stringy instantons of the gauge theory from the known results on D-instantons in string theory. Besides, we formulate a simple condition on the existence of a non-trivial local limit in terms of intersection numbers of the Calabi-Yau, and find an explicit form for the hypermultiplet metric including corrections from all mutually non-local D-instantons, which can be of independent interest. Keywords: D-branes, M-Theory, String Duality Keywords: D-branes, M-Theory, String Duality ArXiv ePrint: 1710.10665v2 ArXiv ePrint: 1710.10665v2 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP01(2018)156 Contents 1 Introduction 1 2 Rigid limit 5 2.1 Definition 6 2.2 Example: classical c-map 7 2.3 Rigid limit of the non-perturbative HM moduli space 11 2.3.1 Quantum corrections 11 2.3.2 D-instanton corrected HM metric 13 2.3.3 The limit 14 2.4 Geometric interpretation 15 2 Rigid limit 2.1 Definition 2.2 Example: classical c-map JHEP01(2018)156 2.4 Geometric interpretation 3 Physical interpretation: 5d gauge theory on a torus 3.1 String dualities and rigid limit 3.2 Low energy description of 5d gauge theories 3.3 Torus compactification 3.4 BPS spectrum and modular invariance 1 Introduction String theory plays a prominent role in extracting the non-perturbative dynamics of super- symmetric gauge theories. Indeed, due to the existence of various dualities, sometimes it is easier to solve a problem in string theory and then to take the so called rigid limit, in which gravity decouples and one recovers a gauge theory description [1, 2]. A particularly fruitful playground for this are theories with 8 supercharges corresponding to N = 2 supersymme- try in 4 dimensions. In such case, the dynamics of compactified string theory is captured at low energies by effective supergravity which comprises, besides the gravitational multiplet, also vector and hypermultiplets. The kinetic couplings of the former are encoded in the vector multiplet moduli space MV , which is a projective (also called local) special K¨ahler manifold. In the rigid limit it directly reduces to a simpler rigid special K¨ahler manifold, whose prepotential contains all information about the solution of the corresponding gauge theory. Due to this, previous works mostly concentrated on the vector multiplet sector of string compactifications [3–6], and one can say that the procedure of extracting the rigid limit there is understood fairly well (see [7] for a recent discussion). JHEP01(2018)156 Let us recall that MV is only one component of the moduli space of N = 2 supergravity. The second one is the hypermultiplet (HM) moduli space MH, and it is natural to ask what happens to this space after decoupling gravity. The local supersymmetry restricts MH to be quaternion-K¨ahler (QK) [8], i.e. a 4n real dimensional manifold with holonomy group SU(2) × Sp(n). For such manifolds the Riemann curvature tensor decomposes as Rµνρσ = κ2 ˆRµνρσ + Wµνρσ, (1.1) (1.1) where κ2 = 8πM−2 Pl is the gravitational coupling, ˆRµνρσ is the dimensionless SU(2) part of the curvature, and Wµνρσ is the Weyl tensor. Thus, one can expect that in the rigid limit only the second contribution survives and one ends up with a Ricci-flat manifold with holonomy group Sp(n), i.e. a hyperk¨ahler (HK) manifold. This is indeed a very natural expectation because such manifolds are known to play an important role in the low energy description of theories with global supersymmetry. For instance, they appear as Higgs branches of 4d N = 2 gauge theories. However, the metric on these Higgs branches is classically exact. 4 Examples 4 Examples 26 4.1 Elliptic fibrations and SU(2) gauge theory 26 4.1.1 Hirzebruch surfaces 27 4.1.2 Del Pezzo surfaces 29 4.2 Two large moduli 30 4.3 SU(3) gauge theory 32 4.4 No local limit 34 5 Conclusions 35 A Special geometry in the classical approximation 37 B Derivation of the D-instanton corrected HM metric 38 B.1 Twistorial description of QK manifolds 38 B.2 D-instantons in twistor space 40 B.3 Computation of the metric 40 B.4 The last step 44 C Metric on M′ H 47 C.1 Scaling behavior 47 C.2 Evaluation of the limit 48 C.3 HK structure 50 D Torus reduction of 5d gauge theory 53 E Toric data 54 4.2 Two large moduli 4.3 SU(3) gauge theory A Special geometry in the classical approximation D Torus reduction of 5d gauge theory – i – 1 Introduction For this reason, and since MH does receive quantum corrections, we do not expect them to be relevant in our context. A more interesting and, as we will see, relevant example is provided by target spaces of N = 4 non-linear sigma models in 3 dimensions [9], some of which can also be viewed as circle compactifications of 4d N = 2 gauge theories [10]. Unfortunately, it turns out that the naive decoupling leads to a flat hyperk¨ahler ge- ometry, and to get a non-trivial limit it is necessary to introduce an additional mass scale, which is kept finite as κ →0. As a result, no general treatment of the rigid limit for QK spaces exists in the literature, and a non-trivial limit was produced only in a number of particular cases [11–15]. At the same time, the rigid limit, used to extract information about gauge theories from the vector multiplet sector of string compactifications, usually has a geometric realization as a local limit on the compactification manifold Y where one zooms in on the region near some singularity in the moduli space [4, 5]. Since the met- – 1 – ric on both moduli spaces, MV and MH, is completely determined by the geometry of Y, it is natural to ask whether this zooming procedure is sufficient to induce the rigid limit of the HM moduli space. This is the question that we investigate in this paper for compactifications of type IIB string theory on a Calabi-Yau (CY) threefold Y. The advantage of considering this type of compactifications is that in recent years substantial progress has been made towards understanding the complete non-perturbative description of the corresponding HM moduli space (see [16, 17] for reviews). As a result, we now have access to the metric on MH which includes most of the non-perturbative correc- tions. In the type IIB formulation, the latter include Dp-brane instantons (with p = −1, 1, 3 and 5) and NS5-brane instantons. Only contributions of five-branes remain not well un- derstood (although some partial results can be found in [18–20]), whereas all D-instantons have been incorporated [21, 22] using a twistorial description of QK geometry [23, 24]. For- tunately, it turns out that in any local limit the unknown five-brane contributions always decouple and one remains with a metric which is completely under our control. JHEP01(2018)156 The last statement however needs a refinement. 1 Introduction Geometric construction of the rigid limit via the Swann bundle and hyperk¨ahler quotient. JHEP01(2018)156 gure 1. Geometric construction of the rigid limit via the Swann bundle and hyperk¨ahler quotient. that n′ coincides with the dimension of the intersection of the kernels for the matrices MA,ab = κabcvc A. Note that the possibility of having a non-empty common kernel is a very non-trivial condition on both the vectors ⃗vA and the triple intersection numbers, so that far from any CY allows for a non-trivial rigid limit even with n∞= 1. Furthermore, we prove that M′ H is an HK manifold and can be constructed from MH in a pure geometric way (see figure 1). To this end, one should first note that the local limit induces on MH a set of n −n′ commuting isometries where n = h1,1(Y) + 1 is the quaternionic dimension of MH. These isometries are present in the perturbative metric, but are broken in general by instanton corrections. However, the relevant corrections vanish exponentially fast in our limit and thus can be ignored. Next, one constructs a canonical C2/Z2 bundle SM, known as Swann bundle [26] or hyperk¨ahler cone in the physics litera- ture [27]. SM is an HK manifold, which immediately brings us in the realm of hyperk¨ahler geometry with all its available methods. Finally, M′ H is obtained by performing n −n′ + 1 hyperk¨ahler quotients along the set of commuting isometries, which include those men- tioned above plus one additional isometry corresponding to a U(1) symmetry on the fiber of the Swann bundle. Interestingly, at an intermediate step of this quotient construction, one finds the HK manifold Mcor H which is associated with MH by the so called QK/HK correspondence. This correspondence establishes a one-to-one map between, on one hand, QK spaces with a quaternionic isometry and, on the other hand, HK spaces of the same dimension with a rotational isometry, equipped with a hyperholomorphic line bundle [28–30]. Its physical interpretation is in fact very close to the subject of this paper: it translates into a formal correspondence between the D-instanton corrected HM moduli space MH and the moduli space of a 4d N = 2 gauge theory compactified on a circle, described by the same holomor- phic prepotential as the CY. In particular, the D-instantons are mapped into the gauge theory instantons produced by BPS particles wrapping the circle. 1 Introduction Although the twistorial description, used to obtain the cited results, is very powerful, it is also somewhat implicit because it encodes the QK metric into the contact structure on the twistor space ZM, a CP 1 bundle over the original manifold, and it is not so easy to extract it. Recently this problem was solved [25] only for mutually local D-instantons, i.e. a subset of all D-instantons whose charges γ have vanishing symplectic products ⟨γ, γ′⟩. In this paper we seize on the oppor- tunity to improve the situation and calculate the explicit HM metric, which includes all mutually non-local D-instanton corrections and is parametrized by topological data on the CY, such as its triple intersection numbers κabc, Euler characteristic χY and generalized Donaldson-Thomas (DT) invariants Ωγ. Having at hand the explicit metric, we can study its behavior in the local limit. To define it, we fix a set of n∞vectors ⃗vA belonging to the boundary of the K¨ahler cone of Y. They correspond to the directions in the moduli space along which some of the (dimensionless) K¨ahler moduli are sent to infinity, thereby introducing a new scale Λ. Geometrically, they fix a set of 2-cycles which shrink in the local limit and have vanishing intersection with the divisors defined by ⃗vA. Then, evaluating the HM metric in the so-defined limit, we show that, besides a non- trivial finite part, it also features a divergent part. This leads to the freezing of some moduli, including those which are sent to infinity. As a result, all moduli can be split into 3 groups: • moduli appearing only in the vanishing part of the metric and thus dropping out in the limit; • moduli appearing only in the vanishing part of the metric and thus dropping out in the limit; • frozen moduli; • moduli appearing in the finite, but not in the divergent parts of the metric and thus remaining dynamical. • moduli appearing in the finite, but not in the divergent parts of the metric and thus remaining dynamical. Only the latter moduli parametrize the limiting manifold M′ H, which therefore has al- ways a smaller dimension than the original MH. More precisely, the dimension of M′ H is given by 4n′ where n′ is the number of K¨ahler moduli remaining dynamical. We show – 2 – Figure 1. 1 Introduction In a sense, our rigid – 3 – Figure 2. Duality map and rigid limit of moduli spaces in string and gauge theories. JHEP01(2018)156 Figure 2. Duality map and rigid limit of moduli spaces in string and gauge theories. limit is a close analogue of this formal mathematical correspondence, with the additional property that both sides realize concrete physical systems. One should note that a similar geometric prescription for the rigid limit was already given in [31] for a particular compactification on an elliptically fibered CY. Here we extend it to the full non-perturbative level, prove it by carefully analyzing the metric, and generalize it to a generic CY. The work [31] also suggests a physical interpretation of the HK manifold M′ H: it is expected to describe the non-perturbative moduli space of a 5d N = 1 gauge theory compactified on a torus, where the complex structure of the torus is identified with the frozen axio-dilaton of compactified type IIB string theory. Indeed, the chain of dualities, shown on figure 2 and explained in detail in section 3.1, demonstrates that MH is the same moduli space which is obtained by first compactifying M-theory on the same CY Y and then compactifying its vector multiplet sector on a torus. Since the torus compactification is expected to commute with the rigid limit, the alternative way to get M′ H is to start from 5d supergravity obtained from M-theory on Y, take the rigid limit in its vector multiplet sector, and only then compactify on T 2. Then the above gauge theory interpretation immediately follows. This interpretation opens the possibility to derive non-perturbative effects in compact- ified 5d gauge theory, such as dyonic and stringy instantons, from the known results on – 4 – D-instantons in CY string theory compactifications. Although we leave the detailed study of this problem to a future research, here we discuss various implications of this possibility. The organization of the paper is as follows. In the next section we study the rigid limit of the HM moduli space MH. First, in section 2.1 we provide the definition of the limit. Then in section 2.2 we show how it works on the example of the classical moduli space where the derivation is particularly explicit, but contains all the features of the general construction. 1 Introduction In section 2.3 we present the rigid limit for the full non-perturbative moduli space and in section 2.4 provide its geometric interpretation. The physical interpretation is elaborated in section 3, which starts from a discussion of string dualities suggesting the interpretation in terms of 5d gauge theories (section 3.1), proceeds with a brief review of these theories (section 3.2), their compactification on a torus (section 3.3), and finishes with a discussion of implications for dyonic and stringy instantons (section 3.4). In section 4 we provide several examples of our construction and in section 5 discuss the results of the paper. A few appendices contain details on special geometry (section A), calculations of the D-instanton corrected HM metric (section B), of the rigid limit (section C) and of compactification on a torus (section D), and toric data for the examples presented in section 4 (section E). JHEP01(2018)156 2 Rigid limit In this section we study the rigid limit of the HM moduli space MH of type IIB string theory compactified on a CY threefold Y. We recall that the moduli space comprises • the axio-dilaton τ ≡τ1 + iτ2 = c0 + i/gs; • the axio-dilaton τ ≡τ1 + iτ2 = c0 + i/gs; • the K¨ahler moduli za = ba + ita (a = 1, . . . , h1,1(Y)) parametrizing the deformations of the complexified K¨ahler structure of Y; • the K¨ahler moduli za = ba + ita (a = 1, . . . , h1,1(Y)) parametrizing the deformations of the complexified K¨ahler structure of Y; • the RR-fields ca, ˜ca, ˜c0, corresponding to periods of the RR 2-form, 4-form and 6-form on a basis of Heven(Y, Z); • the RR-fields ca, ˜ca, ˜c0, corresponding to periods of the RR 2-form, 4-form and 6-form on a basis of Heven(Y, Z); • the RR-fields ca, ˜ca, ˜c0, corresponding to periods of the RR 2-form, 4-form and 6-form on a basis of Heven(Y, Z); • and the NS-axion ψ dual to the Kalb-Ramond two-form B in four dimensions. • and the NS-axion ψ dual to the Kalb-Ramond two-form B in four dimensions. We will use Ca and Da to denote a basis in the space of curves H2(Y, Z) and divisors H4(Y, Z), respectively, and ωa for the basis of harmonic 2-forms dual to Da so that the expansion of the K¨ahler form reads J = taωa. These objects satisfy Ca ∩Db = Z Ca ωb = δa b , Da ∩Db ∩Dc = Z Y ωa ∧ωb ∧ωc = κabc. (2.1) (2.1) Finally, note that in this paper we work in terms of dimensionless moduli. Therefore, the dimensionful volumes are obtained by dividing integrals of the K¨ahler form by a mass (squared) scale Λ. For instance, for 2-cycles one has Finally, note that in this paper we work in terms of dimensionless moduli. Therefore, the dimensionful volumes are obtained by dividing integrals of the K¨ahler form by a mass (squared) scale Λ. For instance, for 2-cycles one has Vol(Ca) = Λ−1 Z Ca J = Λ−1 ta. (2.2) (2.2) In the rigid limit, this scale is sent to infinity together with MPl so that the shrinking cycles correspond to the finite K¨ahler parameters, whereas the cycles of finite volume correspond to the moduli scaling as Λ. 2.1 Definition Our aim here is to provide a definition of a local limit of the CY manifold. Usually, this is done by specifying either a set of shrinking 4-cycles or 2-cycles. On the other hand, to apply it to the metric on the moduli space, we need a workable definition in terms of the K¨ahler moduli. Therefore, instead of shrinking cycles, let us start from a set of n∞linearly independent vectors ⃗vA belonging to the K¨ahler cone of Y. Given these vectors, we define a set of matrices (2.3) MA,ab = κabcvc A (2.3) which in turn allow to introduce another set of vectors ⃗vI — a basis for the common kernel of MA, i.e. linearly independent vectors satisfying which in turn allow to introduce another set of vectors ⃗vI — a basis for the common kernel of MA, i.e. linearly independent vectors satisfying JHEP01(2018)156 MA,ab vb I = 0. (2.4) (2.4) We denote their number (i.e. the number of values taken by index I) by n′. We assume that n′ > 0 and that the two sets, ⃗vA and ⃗vI, are linearly independent. Already at this point it becomes clear that ⃗vA must belong to the boundary of the K¨ahler cone because it is well known that for any vector inside the cone its contraction with the intersection numbers defines a non-degenerate matrix of signature (1, h1,1(Y) −1). Thus, to have n′ > 0, all vectors ⃗vA must belong to the boundary.1 Finally, we complete these sets to a basis in H2(Y, R), which can be done by providing an additional set of h1,1 −n∞−n′ ≡nfr vectors ⃗vX. This allows to expand the K¨ahler moduli in the new basis ta = va A ˆtA + va X ˆtX + va I ˆtI ≡va b ˆtb, (2.5) (2.5) where we combined three indices A, X and I into one index b. Then our local limit is defined by taking the moduli ˆtA to scale as Λ, whereas ˆtX and ˆtI to stay finite (see the comment below (2.2)). It is important that this definition of the limit does not depend on the choice of ⃗vX. Indeed, changing ⃗vX in (2.5) can at most shift ˆtA and ˆtI by a combination of ˆtX. But this does not affect which variables grow with Λ and which of them do not. 2 Rigid limit – 5 – – 5 – 2We consider a generic point in the moduli space so that no accidental cancellations are possible due to contraction with ˆtA. 1This has a simple physical explanation. In a local limit one usually zooms in around a point in the moduli space where CY becomes singular, and the vectors ⃗vA are supposed to point towards such singularity. But CY can develop a singularity only when its moduli approach the boundary of the K¨ahler cone, which implies the condition on ⃗vA. 2.1 Definition Let us show that the above definition is equivalent to the usual one in terms of shrinking cycles. First, we define a rotated basis of divisors ˆDa = vb aDb. It is easy to see that ˆDI are the divisors shrinking in the limit, whereas the divisors ˆD ˆ A, where we introduced a combined index ˆA = (A, X), remain with a finite volume. Indeed, Vol( ˆDI) = 1 2Λ2 Z ˆDI J ∧J = 1 2Λ2 va I κabctbtc ∼Λ−2, Vol( ˆD ˆ A) = 1 2Λ2 Z ˆD ˆ A J ∧J = 1 2Λ2 va ˆ A κabctbtc ≈ 1 2Λ2 va ˆ A MB,abvb CˆtBˆtC ∼1, (2.6) (2.6) where the first result follows from (2.4), whereas the second is due to that none of vectors ⃗v ˆ A belongs to the common kernel of MA.2 1This has a simple physical explanation. In a local limit one usually zooms in around a point in the moduli space where CY becomes singular, and the vectors ⃗vA are supposed to point towards such singularity. But CY can develop a singularity only when its moduli approach the boundary of the K¨ahler cone, which implies the condition on ⃗vA. 2We consider a generic point in the moduli space so that no accidental cancellations are possible due to contraction with ˆtA. – 6 – Second, we define a rotated basis of curves ˆCa = (v−1)a bCb. Their volumes are given by Λ−1ˆta and therefore ˆCA has a finite volume, whereas C ˆI, where we introduced another combined index ˆI = (I, X), are shrinking. It is important to note that all shrinking curves can be characterized by their orthogonality to the divisors ˆDA, ˆC ˆI ∩ˆDA = 0, (2.7) (2.7) due to (2.1) the l.h.s. is evaluated to (v−1)ˆI ava A = 0. since due to (2.1) the l.h.s. is evaluated to (v−1)ˆI ava A = 0. Thus, our definition of the local limit is equivalent to specifying either the set of shrink- ing divisors ˆDI or the set of shrinking curves ˆC ˆI. Both sets are in one-to-one correspondence with vectors ⃗vA, and both their definitions ˆDI = va I Da as well as the orthogonality rela- tion (2.7) do not depend on ⃗vX. Of course, to talk about a local limit, one must have at least one shrinking divisor, which gives the condition n′ > 0. 2.1 Definition Thus, the condition of having a non-trivial limit is that the common kernel of MA is non-empty. JHEP01(2018)156 Finally, we impose an additional condition on the vectors ⃗vA that κabcva Avb Bvc C is non- zero at least for some A, B, C. It ensures that the volume of the CY, V = 1 6 κabctatbtc, scales as Λ3 in the local limit. As we will show below, under these conditions the three sets of moduli appearing in (2.5) acquire in the limit a very different status: • ˆtA become frozen and do not enter the finite part of the metric; • ˆtX are also frozen, but appear in the finite part; • ˆtI remain dynamical. Correspondingly, their physical interpretation in the dual gauge theory will also be different: while ˆtI are associated with the Coulomb branch moduli, ˆtX provide its physical parameters such as masses and the gauge coupling. In the following, to simplify notations, we assume that the rotation of the basis (2.5) has already been done and drop hats on the moduli adapted to the limit, i.e. consider tA to be of order Λ, whereas tX and tI as finite variables. Then (2.4) implies that in this basis the intersection numbers possess the following property κaAI = 0, (2.8) (2.8) whereas the matrix M ˆ A ˆB = κ ˆ A ˆBCtC is non-degenerate. In section 4 we will return back to the original basis and discuss in more detail the conditions for the existence of a non-trivial limit. 2.2 Example: classical c-map Before attacking the problem of taking the rigid limit of the non-perturbative HM moduli space, let us consider how it works at the classical level where all quantum corrections in α′ and gs are ignored. In this approximation the metric on MH is given by the local c- map [32, 33] which is a QK manifold constructed in a canonical way from the holomorphic prepotential F(XΛ) (Λ = 0, . . . , h1,1(Y)) on the K¨ahler moduli space of Y. It has the simplest form in terms of the fields of type IIA string theory compactified on the mirror – 7 – CY ˆY, which comprise the four-dimensional dilaton r = eφ, the complex structure moduli za, the RR-scalars ζΛ, ˜ζΛ corresponding to periods of the RR 3-form on a basis of H3( ˆY, Z), and the NS-axion σ dual to the B-field. In these coordinates the metric reads CY ˆY, which comprise the four-dimensional dilaton r = eφ, the complex structure moduli za, the RR-scalars ζΛ, ˜ζΛ corresponding to periods of the RR 3-form on a basis of H3( ˆY, Z), and the NS-axion σ dual to the B-field. In these coordinates the metric reads ds2 Mcl H = dr2 r2 −1 2r Im N ΛΣ  d˜ζΛ −NΛΛ′dζΛ′  d˜ζΣ −¯ NΣΣ′dζΣ′ + 1 16r2  dσ + ˜ζΛdζΛ −ζΛd˜ζΛ 2 + 4Ka¯bdzad¯z ¯b, (2.9) (2.9) where K is the K¨ahler potential on the special K¨ahler space of complex structure deforma- tions of ˆY (we set zΛ = (1, za)) K = −log  i(¯zΛFΛ −zΛ ¯FΛ)  , (2.10) JHEP01(2018)156 (2.10) FΛ, Ka¯b, etc. denote derivatives of the corresponding quantities without indices, and NΛΣ is the matrix of the gauge couplings defined in (A.1). We refer to appendix A for the details on the special geometry encoded by the prepotential F. To return to the type IIB fields, which we used to define the rigid limit, one should apply the mirror map. In the classical approximation it was found in [34] and identifies the complex structure moduli za with the complexified K¨ahler moduli as well as r = τ 2 2 2 V, ζ0 = τ1, ζa = −ca + τ1ba, ˜ζa = ˜ca + 1 2 κabcbb(cc −τ1bc), ˜ζ0 = ˜c0 −1 6 κabcbabb(cc −τ1bc), σ = −2  ψ + 1 2 τ1˜c0  + ˜ca(ca −τ1ba) −1 6 κabcbacb(cc −τ1bc). 2.2 Example: classical c-map (2.11) (2.11) The classical prepotential to be used in (2.9) is completely determined by the triple inter- section numbers of Y b F cl(X) = −1 6 κabc XaXbXc X0 . (2.12) (2.12) Let us now plug in this prepotential and the change of variables (2.11) into the c-map metric. Then, using the expressions (A.7) for the gauge coupling matrix and its inverse, after straightforward, but a bit tedious manipulations the metric can be brought to the following form Let us now plug in this prepotential and the change of variables (2.11) into the c-map metric. Then, using the expressions (A.7) for the gauge coupling matrix and its inverse, after straightforward, but a bit tedious manipulations the metric can be brought to the following form ds2 Mcl H = (dr)2 r2 + dτ 2 1 τ 2 2 +4Ka¯b  dtadtb+ 1 τ 2 2  dca−τdba)(dcb−¯τdbb + Ka¯b 4τ 2 2 V 2  d˜ca+ 1 2 κacd  ccdbd−bcdcd d˜cb+ 1 2 κbfg  cfdbg−bfdcg + 1 τ 2 2 V 2  d˜c0+bad˜ca+ 1 6 κabcba(cbdbc−bbdcc) 2 + 1 τ 4 2 V 2  dψ+τ1d˜c0−(ca−τ1ba)  d˜ca−1 6κabc(bbdcc−cbdbc) 2 . (2.13) (2.13) Using (2.11) and (A.6), the first three terms can be rewritten as |dτ|2 τ 2 2 + 2  d log(V τ 3/2 2 ) 2 −κabctc τ2V d(√τ2ta)d(√τ2tb), (2.14) (2.14) – 8 – – 8 – whereas the last two terms can be reorganizied in the following way 1 τ 4 2 V 2 " |dψ+τd˜c0|2+ (ca−¯τba)  d˜ca−1 6κabc(bbdcc−cbdbc)  2# (2.15) − 1 τ 4 2 V 2  (dψ+τd˜c0)(ca−¯τba)+(dψ+¯τd˜c0)(ca−τba)  d˜ca−1 6κabc(bbdcc−cbdbc)  . (2.15) This rewriting makes it manifest that the whole metric is invariant under the SL(2, R) isometry group acting on the type IIB fields as τ 7→aτ + b cτ + d , ta 7→ta|cτ + d| , ˜ca 7→˜ca , ca ba ! 7→ a b c d ! ca ba ! , ˜c0 ψ ! 7→ d −c −b a ! ˜c0 ψ ! , (2.16) JHEP01(2018)156 (2.16) where a, b, c, d are the parameters of the transformation a b c d  ∈SL(2, R) with ad −bc = 1. This symmetry descends from the S-duality group of type IIB supergravity in 10 dimen- sions, but is broken to the discrete subgroup SL(2, Z) by quantum corrections [35]. 3The overall minus sign comes from that we work in the ‘most plus’ signature (−, +, +, +) 2.2 Example: classical c-map It is this symmetry that fixed the form of the mirror map (2.11) and it will play an important role in the physical interpretation of the rigid limit. To extract this limit from the metric (2.13), it is enough to understand the behavior of the special K¨ahler metric Ka¯b and its inverse. This can be done using the representa- tion (A.6) valid in the classical approximation. It involves the matrix κab = κabctc and its inverse, so first we establish the scaling for them. Using notations for indices from the pre- vious subsection, the restriction on intersection numbers (2.8), the matrix M ˆ A ˆB introduced below it, the matrix gIJ = −κIJ ˆ Kt ˆ K and their inverse M ˆ A ˆB and gIJ, one finds κab ≈ M ˆ A ˆB κ ˆ AJ ˆ Kt ˆ K κI ˆB ˆ Kt ˆ K −gIJ ! ∼ Λ 1 1 1 ! , (2.17) κab ≈ M ˆ A ˆB M ˆ AXκXK ˆLtˆLgKJ gIKκXK ˆLtˆLMX ˆB −gIJ ! ∼ Λ−1 Λ−1 Λ−1 1 ! . (2.18) (2.17) (2.18) Plugging these results into (A.6), one obtains Plugging these results into (A.6), one obtains 4V K ˆ A ¯ˆB ≈−M ˆ A ˆB+ 1 4V M ˆ AAtAM ˆBBtB ∼Λ, 1 4V K ˆ A ¯ˆB ≈−M ˆ A ˆB+δ ˆ A Aδ ˆB B tAtB 2V ∼Λ−1 4V KI ¯B ≈ 1 4V MBCtCκI ˆ K ˆLt ˆ KtˆL ∼Λ−1, 1 4V KI ¯ˆB ≈−gIJκJX ˆ Kt ˆ KMX ˆB ∼Λ−1, 4V KI ¯ˆJ ≈−κI ˆJ ˆ Kt ˆ K ∼1, 1 4V KI ¯J ≈gIJ ∼1. (2 19) (2.19) ( ) On the basis of these scaling results, the bosonic Lagrangian defined by the met- ric (2.13) can be split into three contributions3 ( ) On the basis of these scaling results, the bosonic Lagrangian defined by the met- ric (2.13) can be split into three contributions3 Lbos = − √−g 2κ2V τ 3/2 2 (L+ + L0 + L−) , (2.20) (2.20) 3The overall minus sign comes from that we work in the ‘most plus’ signature (−, +, +, +). 3The overall minus sign comes from that we work in the ‘most plus’ signature (−, +, +, +). 2.2 Example: classical c-map – 9 – where L+ = V 2τ 1/2 2  2∂µτ2+ τ2 2V κabctatb∂µtc2 +(∂µτ1)2  +2τ 3/2 2 V K ˆ A ˆB  ∂µt ˆ A∂µt ˆB+ 1 τ 2 2  ∂µc ˆ A−τ∂µb ˆ A ∂µc ˆB−¯τ∂µb ˆB , (2.21a) L0 = 4τ 3/2 2 V KI ˆB  ∂µtI∂µt ˆB+ 1 τ 2 2 ∂µcI −τ∂µbI ∂µc ˆB−¯τ∂µb ˆB +2τ 3/2 2 V KIJ  ∂µtI∂µtJ + 1 τ 2 2 ∂µcI −τ∂µbI)(∂µcJ −¯τ∂µbJ + KI ¯J 8τ 1/2 2 V yIµyJ µ, (2.21b) L−= 1 8τ 1/2 2 V ( K ˆ A ˆBy ˆ Aµy ˆB µ+2KI ˆByIµy ˆB µ+4  ∂µ˜c0+ba∂µ˜ca+ 1 6 κabcba(cb∂µbc−bb∂µcc) 2 + 4 τ 2 2  ∂µψ+τ1∂µ˜c0−(ca−τ1ba)  ∂µ˜ca−1 6κabc(bb∂µcc−cb∂µbc) 2) , (2.21c) (2.21a) JHEP01(2018)156 and we denoted and we denoted yaµ = ∂µ˜ca + 1 2 κabc  cb∂µbc −bb∂µcc . (2.22) (2.22) Let us take the gravitational coupling κ2 scaling as Λ−3 so that κ2V remains constant. Then, as the notations suggest, L+ corresponds in our limit to the divergent part of the Lagrangian, L0 stays finite, and L−vanishes. As a result, the fields ψ, ˜c0 and ˜c ˆ A, appearing only in L−, simply drop out from the theory, whereas the divergent part imposes its equations of motion as strong constraints. These leads to the freezing of the moduli τ, t ˆ A, b ˆ A and c ˆ A, which means that their fluctuations vanish or at least scale as Λ−1, and thus these fields can be considered as constant. Taking this into account in L0, one obtains that its non-vanishing part is determined by the following metric ds2 M′cl H = 1 2 τ 3/2 2 gIJ  dtIdtJ + 1 τ 2 2 dcI −τdbI)(dcJ −¯τdbJ (2.23) + gIJ 2τ 1/2 2  d˜cI + 1 2 κIK ˆL  c ˆLdbK −b ˆLdcK d˜cJ + 1 2 κJM ˆ N  c ˆ NdbM −b ˆ NdcM . (2.23) Note that it is manifestly SL(2, R) invariant. It is to keep this invariance we included the factor τ 3/2 2 into the rescaling of the Lagrangian in (2.20). The metric (2.23) describes the rigid limit of the classical HM moduli space. 2.2 Example: classical c-map The space M′cl H where it leaves on is parametrized by 4n′ coordinates tI, bI, cI and ˜cI, whereas τ, tX, bX and cX also appearing in the metric play the role of fixed parameters. The geometric meaning of this metric can be elucidated by going back to the analogue of the type IIA variables. Using the inverse mirror map relations (2.11), the metric can be rewritten as ds2 M′cl H = 1 2√τ2 h τ 2 2 Imfcl IJdzId¯zJ +(Imfcl)IJ  d˜ζI −fcl JKdζK d˜ζJ −¯fcl JLdζLi , (2.24) (2.24) – 10 – where the new prepotential is fcl(zI) = −1 6 κˆI ˆJ ˆ Kz ˆIz ˆJz ˆ K. (2.25) (2.25) One recognizes in (2.24) the well known rigid c-map [32], which describes an HK space con- structed as a canonical bundle over the rigid special K¨ahler base with the holomorphic pre- potential fcl(zI). Typically, it arises as the classical target space of three-dimensional non- linear σ-models obtained by compactifications of gauge theories with eight supercharges. The parameter τ2 controls the radius of compactification, but can be absorbed by the redefinition uI = τ2 2 zI. One recognizes in (2.24) the well known rigid c-map [32], which describes an HK space con- structed as a canonical bundle over the rigid special K¨ahler base with the holomorphic pre- potential fcl(zI). Typically, it arises as the classical target space of three-dimensional non- linear σ-models obtained by compactifications of gauge theories with eight supercharges. The parameter τ2 controls the radius of compactification, but can be absorbed by the redefinition uI = τ2 2 zI. JHEP01(2018)156 Furthermore, it is easy to see that M′cl H can be obtained from a larger rigid c-map space, which we call Mcl,cor H and which is determined by the prepotential fcl(uΛ) = F cl(uΛ). The space Mcl,cor H has quaternionic dimension n = h1,1(Y)+1, and its metric is given by exactly the same metric (2.24) (after the rescaling mentioned above) where however the indices I, J, . . . should be replaced by Λ, Σ, . . . running over 0, . . . , h1,1(Y). As any rigid c-map, Mcl,cor H has a set of commuting isometries acting by shifts of ˜ζΛ, with the triplet of moment maps given in the chiral basis by (ρΛ +, ρΛ −, ρΛ 3 ) = (uΛ, ¯uΛ, ζΛ). 2.2 Example: classical c-map Then performing n −n′ hyperk¨ahler quotients along ˜ζ0 and ˜ζ ˆ A fixes the moment maps ⃗ρ 0 and ⃗ρ ˆ A and gives us back the manifold M′cl H . The decoupling of the variables fixed by ⃗ρ A is ensured by the condition (2.8). In particular, the prepotential F cl(uΛ), up to an overall factor and an irrelevant constant contribution, reduces to (2.25) after identifying the moment maps of the first isometry as ( τ2 2 , τ2 2 , τ1).4 In turn, the rigid c-map Mcl,cor H is known to be related to the local c-map Mcl H by the QK/HK correspondence [29]. It proceeds via construction of the Swann bundle SM over the QK space with an isometry and subsequent hyperk¨ahler quotient along the isometry inherited on SM. In the case of the local c-map (2.9), the role of such isometry is played by shifts of the NS-axion σ. As a result, we arrive at the precise realization of the geometric scheme shown on figure 1. 4Mcl,cor H has an isometry which acts by multiplying all uΛ by a phase. It can be used to cancel the phase of the moment maps ρ0 ±, this is why it is always possible to choose them to be real. 2.3.1 Quantum corrections To extract the rigid limit of the full non-perturbative moduli space MH, let us first recall what kinds of quantum corrections affect the classical c-map metric considered in the previous subsection. There are two classes of such corrections: one comes from quantum effects on the string worldsheet and is weighted by α′, and the other comes from physics in the target space and is weighted by gs. All α′-corrections are captured as corrections to the holomorphic prepotential, and therefore the α′-corrected HM metric still falls into the class of metrics given by the local c-map. However, the prepotential is now a deformation – 11 – of the simple classical function (2.12), which is known to have the following form [36, 37]5 l ζ(3)(X0)2 (X0)2 X (0)  2 ik Xa/X0 of the simple classical function (2.12), which is known to have the following form [36, 37]5 F(X) = F cl(X) −χY ζ(3)(X0)2 2(2πi)3 −(X0)2 (2πi)3 X kaCa∈H+ 2 (Y) n(0) ka Li3  e2πikaXa/X0 , (2.26) the simple classical function (2.12), which is known to have the following form [36, 37]5 F(X) = F cl(X) −χY ζ(3)(X0)2 2(2πi)3 −(X0)2 (2πi)3 X kaCa∈H+ 2 (Y) n(0) ka Li3  e2πikaXa/X0 , (2.26) (2.26) where the second term describes a perturbative α′-correction, whereas the third term, parametrized by genus zero Gopakumar-Vafa invariants n(0) ka , corresponds to the contribu- tion of worldsheet instantons wrapping effective curves kaCa. The situation with gs-corrections is more complicated. At the perturbative level, the corrections appear only at one-loop [38, 39] and the corresponding metric, which is already not in the c-map class, is explicitly known [40]. At the non-perturbative level, there are two sources of gs-corrections: D-branes wrapping non-trivial cycles on the CY and NS5-branes wrapping the whole CY. How to include the contributions of the former, to all orders in the instanton expansion, has been understood (in the type IIA formulation) in [21, 22], but only partial results are accessible for the latter [18–20]. JHEP01(2018)156 Given such incomplete understanding of the HM moduli space, it is natural to ask whether it is possible to find the exact rigid limit of MH or only its approximation? It turns out that the lack of knowledge of the exact description of NS5-brane instantons does not pose a problem for evaluating the rigid limit because these instantons necessarily decouple. 6In fact, the charges are not integer due to two reasons. First, they have rational shifts because of the symplectic rotation mentioned in footnote 5. And second, our charge lattice is already a result of rotation (2.5) to the basis adapted for the rigid limit in which, in particular, the intersection numbers satisfy the condition (2.8). 5In fact, the prepotential also has a quadratic contribution 1 2 AΛΣXΛXΣ where AΛΣ is real so that this term does not affect the K¨ahler potential K and is often omitted. However, it becomes important when one extends mirror symmetry to the non-perturbative level [18]. Nevertheless, it is still possible to remove this term by a symplectic transformation. One should just take into account that this transformation affects the integrality of D-brane charges which become rational. This is the symplectic frame that is accepted in this work. 5In fact, the prepotential also has a quadratic contribution 1 2 AΛΣXΛXΣ where AΛΣ is real so that this term does not affect the K¨ahler potential K and is often omitted. However, it becomes important when one extends mirror symmetry to the non-perturbative level [18]. Nevertheless, it is still possible to remove this term by a symplectic transformation. One should just take into account that this transformation affects the integrality of D-brane charges which become rational. This is the symplectic frame that is accepted in this work. 6In fact, the charges are not integer due to two reasons. First, they have rational shifts because of the symplectic rotation mentioned in footnote 5. And second, our charge lattice is already a result of rotation (2.5) to the basis adapted for the rigid limit in which, in particular, the intersection numbers satisfy the condition (2.8). 2.3.1 Quantum corrections • D5-instantons (p0 ̸= 0): • D5 instantons (p ̸= 0): ∼|p | V ; • D3-instantons (p0 = 0, pa ̸= 0): ∼|paκabCtbtC| = |Mabpatb|; • D1-instantons (p0 = pa = 0, qa ̸= 0): ∼|qAtA|. • D1-instantons (p0 = pa = 0, qa ̸= 0): ∼|qAtA|. Thus, D5-instantons are always exponentially suppressed, and the same is true for D3- instantons with charges having at least one non-vanishing component p ˆ A and D1-instantons with charges having at least one non-vanishing component qA. On the other hand, it is easy to check that the D-instantons with charges γ = (0, pI, qˆI, q0) have a finite instanton action and do not decouple. We denote the lattice of the remaining charges by Γrig. Note that these results are in perfect agreement with the discussion in section 2.1 because Γrig precisely corresponds to the set of shrinking cycles, whereas for large K¨ahler moduli the instanton action coincides with the volume of the cycle wrapped by the brane. JHEP01(2018)156 Finally, it is clear that the worldsheet instantons wrapping curves CA also decouple since their instanton action is proportional to |kAtA|. As a result, to extract the rigid limit, it is enough to consider the HM metric corrected by worldsheet instantons with charges kˆI and D-instantons with charges γ ∈Γrig. 2.3.1 Quantum corrections Indeed, they are known to have the following leading contribution [41] ∼e−2π|k|V/g2 s−iπkσ. (2.27) (2.27) At the same time, in a any local limit the (dimensionless) volume of the CY V diverges and thus the NS5-instantons are exponentially suppressed and can be ignored. At the same time, in a any local limit the (dimensionless) volume of the CY V diverges and thus the NS5-instantons are exponentially suppressed and can be ignored. Furthermore, some of D-instantons decouple too. Let us look as above at their leading contribution, which in the type IIA variables has the following form [41] ∼e−2π|Zγ|/gs−2πi(qΛζΛ−pΛ ˜ζΛ), (2.28) ∼e−2π|Zγ|/gs−2πi(qΛζΛ−pΛ ˜ζΛ), (2.28) where Zγ(z) = qΛzΛ −pΛFΛ(z) (2.29) (2.29) is the central charge function determined by the prepotential and the charge vector γ = (pΛ, qΛ). In the type IIA formulation, γ picks out an element of H3(Y, Z) wrapped by a D2-brane, whereas in type IIB it decomposes as γ = (p0, pa, qa, q0) and defines an element6 of Heven(Y, Z) corresponding to a D5-D3-D1-D(-1) bound state. Substituting the 5In fact, the prepotential also has a quadratic contribution 1 2 AΛΣXΛXΣ where AΛΣ is real so that this term does not affect the K¨ahler potential K and is often omitted. However, it becomes important when one extends mirror symmetry to the non-perturbative level [18]. Nevertheless, it is still possible to remove this term by a symplectic transformation. One should just take into account that this transformation affects the integrality of D-brane charges which become rational. This is the symplectic frame that is accepted in this work. 6In fact, the charges are not integer due to two reasons. First, they have rational shifts because of the symplectic rotation mentioned in footnote 5. And second, our charge lattice is already a result of rotation (2.5) to the basis adapted for the rigid limit in which, in particular, the intersection numbers satisfy the condition (2.8). – 12 – prepotential (2.26) into the central charge, one finds that in the local limit the leading part of the D-instanton action behaves as • D5-instantons (p0 ̸= 0): ∼|p0| V ; • D3-instantons (p0 = 0, pa ̸= 0): ∼|paκabCtbtC| = |Mabpatb|; • D1-instantons (p0 = pa = 0, qa ̸= 0): ∼|qAtA|. 2.3.2 D-instanton corrected HM metric As explained above, all of the instantons needed for the rigid limit are in principle known. But do we know them in practice? In fact, in the case of D-instantons we do not. In [21, 22] these instanton effects have been implemented at the level of the twistor space ZM, a canon- ical CP 1 bundle over MH, as deformations of its contact structure. More precisely, this contact structure can be encoded in a set of holomorphic Darboux coordinates (ξΛ, ˜ξΛ, α) on ZM expressed as functions of coordinates on MH and a holomorphic coordinate on the CP 1 fiber (see appendix B for details). The instantons modify these functions and, as a re- sult, the Darboux coordinates become determined by a system of integral equations which has the form of thermodynamic Bethe ansatz. Not only these equations cannot be solved in full generality, but also the procedure to get the metric out of the Darboux coordinates is quite complicated and involves several non-trivial steps. Recently, the problem of deriving the explicit metric corrected by D-instantons has been solved for a subset of them [25], which can be characterized as instantons with charges all having vanishing symplectic products ⟨γ, γ′⟩= qΛp′Λ −q′ ΛpΛ (2.30) (2.30) and called usually mutually local. A crucial simplification arising in this case is that the above mentioned integral equations become solvable. However, this result is not sufficient for our purposes because the effective charge lattice Γrig does contain mutually non-local charges. These are, for instance, D3-instantons with charges pI and D1-instantons with charges qI. Thus, we need a generalization of the result presented in [25]. – 13 – In appendix B, we solve this problem and derive the HM metric including all D- instanton corrections. 2.3.2 D-instanton corrected HM metric The result is given by ds2 MH = 2 r2  1 −8r τ 2 2 U  (dr)2 −1 r  N ΛΣ −τ 2 2 8r zΛ¯zΣ  YΛ ¯YΣ −2 r X γ,γ′ (vM−1)γγ′Yγ ¯Yγ′ + 1 rU X γ  (zM−1)γYγ + τ2 4π WγdZγ  2 + τ2 r X γ,γ′,γ′′ M−1 γγ′ h v(+,1) γγ′′ dZγ′′ −U−1Zγ′′∂e−K ¯Yγ′ + Yγ′v(−,1) γγ′′ d ¯Zγ′′ −U−1 ¯Zγ′′ ¯∂e−Ki + τ 2 2 4r " U−1|∂e−K|2 −NΛΣdzΛd¯zΣ − 1 2πU X γ  WγdZγ ¯∂e−K + ∂e−K ¯ Wγd ¯Zγ # (2.31) + τ 2 2 2r X γ,γ′ v(+) γγ′ dZγ′d ¯Zγ −τ 2 2 r X γ,γ′ (M−1Q)γγ′ X ˜γ v(+,1) γ˜γ dZ˜γ X ˜γ′ v(−,1) γ′˜γ′ d ¯Z˜γ′ + 1 32r2  1 − 8r τ 2 2 U   dσ + ˜ζΛdζΛ −ζΛd˜ζΛ + 1 64π4 X γ,γ′ ΩγΩγ′⟨γ, γ′⟩J (1) γ dJ (1) γ′ + V   2 . JHEP01(2018)156 (2.31) We refer to the appendix for the explanation of all the notations appearing in (2.31). Here we just note that this result is only semi-explicit because all the functions appearing in the metric are defined by a solution of the integral equations which is supposed to be found as a perturbative series in the number of instantons. Besides, the result involves two other expansions. One is used to define the matrices (B.30) entering the definition of other quantities such as vγγ′ and v(±) γγ′ . The other is due to the inverse of matrix Mγγ′ which also can be found only as a perturbative series. However, to every given order, both series can be easily evaluated and the metric follows by a direct substitution. More importantly, this does not represent any obstacle for finding the rigid limit. 2.3.3 The limit The first step to be done for taking the rigid limit of the metric (2.31) is to pass to the IIB fields. However, at the non-perturbative level this becomes problematic because the mirror map itself gets quantum corrections. Fortunately, as we argue now, this step is not really necessary and all calculations can be done in the type IIA variables. Indeed, the limit is defined as tA →∞keeping all other type IIB fields finite. In the classical mirror map (2.11) tA appear only in the imaginary part of zA (and the four- dimensional dilaton r which we assume to be always expressed through τ2 as in (2.11) or (B.21)). Thus, in the classical approximation the limit can equally be defined as Im zA →∞keeping all other type IIA fields finite. At quantum level, the mirror map relations acquire additional terms which make all type IIA fields tA-dependent. Neverthe- less, we can still define the limit in terms of these fields if all such tA-dependent terms are exponentially suppressed as tA →∞. In other words, it is possible if the tA-dependence of the mirror map drops out when one restricts to worldsheet instantons with charges kˆI and D-instantons with charges from Γrig. In fact, the quantum corrected mirror map is known only in the presence of worldsheet and D1-instantons [42, 43] and D3-instantons in the – 14 – large volume limit [44, 45] (i.e. when all K¨ahler moduli are taken to be large). Although these cases do not cover all what we need (because of the large volume approximation used for D3-instantons), the inspection shows that all known corrections to the mirror map respect the above property. We assume that it continues to hold beyond the large volume approximation for D3-instantons as well, and thus the rigid limit can be evaluated using the type IIA variables. We do this evaluation in appendix C. It is very similar to the one presented in section 2.2 for the classical c-map because the leading behavior of the most important quantities, such as the K¨ahler potential and the gauge coupling matrix, is correctly captured by the classical contributions. As a result, we find that: JHEP01(2018)156 • The divergent part of the metric leads to the freezing of τ, z ˆ A and ζ ˆ A. 2.3.3 The limit • The fields σ, ˜ζ0 and ˜ζ ˆ A appear only in the vanishing part of the metric and drop out after taking the limit. This becomes possible because the dependence of quantum corrections on σ, ˜ζΛ and ζΛ arises only through the axionic couplings in the instanton contributions (2.27) and (2.28), but due to the decoupling of NS5-instantons and the restriction to Γrig the dependence on σ, ˜ζ0 and ˜ζ ˆ A disappears. • The finite part of the metric describes a space M′ H parametrized by zI, ζI and ˜ζI and depends on τ, zX and ζX as fixed parameters. • The finite part of the metric describes a space M′ H parametrized by zI, ζI and ˜ζI and depends on τ, zX and ζX as fixed parameters. Explicitly, the limiting metric is given by Explicitly, the limiting metric is given by Explicitly, the limiting metric is given by ds2 M′ H = 1 2√τ2  τ 2 2 gIJdzId¯zJ +gIJY′ I ¯Y′ J −4 X γ,γ′∈Γrig (vM−1)γγ′Y′ γ ¯Y′ γ′   +√τ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ d′Zγ′′ ¯Y′ γ′ +v(−,1) γγ′′ d′ ¯Zγ′′Y′ γ′ i (2.32) + τ 3/2 2 2 X γ,γ′∈Γrig v(+) γγ′ d′Zγ′d′ ¯Zγ−τ 3/2 2 X γ,γ′∈Γrig (M−1Q)γγ′ X ˜γ∈Γrig v(+,1) γ˜γ d′Z˜γ X ˜γ′∈Γrig v(−,1) γ′˜γ′ d′ ¯Z˜γ′. = 1 2√τ2  τ 2 2 gIJdzId¯zJ +gIJY′ I ¯Y′ J −4 X γ,γ′∈Γrig (vM−1)γγ′Y′ γ ¯Y′ γ′   +√τ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ d′Zγ′′ ¯Y′ γ′ +v(−,1) γγ′′ d′ ¯Zγ′′Y′ γ′ i (2.32) 2√τ2  X γ,γ′∈Γrig γ γ  +√τ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ d′Zγ′′ ¯Y′ γ′ +v(−,1) γγ′′ d′ ¯Zγ′′Y′ γ′ i (2. (2.32) + τ 3/2 2 2 X γ,γ′∈Γrig v(+) γγ′ d′Zγ′d′ ¯Zγ−τ 3/2 2 X γ,γ′∈Γrig (M−1Q)γγ′ X ˜γ∈Γrig v(+,1) γ˜γ d′Z˜γ X ˜γ′∈Γrig v(−,1) γ′˜γ′ d′ ¯Z˜γ′. Here gIJ = Im FIJ, d′ denotes the differential on M′ H, i.e. acting only on the dynamical fields, and we refer to the appendix for all other notations. Here gIJ = Im FIJ, d′ denotes the differential on M′ H, i.e. acting only on the dynamic fields, and we refer to the appendix for all other notations. Here gIJ = Im FIJ, d′ denotes the differential on M′ H, i.e. acting only on the dynamical fields, and we refer to the appendix for all other notations. 2.4 Geometric interpretation One can check that these two steps lead directly to the twistorial construction of an HK space whose metric coincides with the rigid limit (2.32). Essentially, this is the way which we use to prove that M′ H carries the HK structure. One can check that these two steps lead directly to the twistorial construction of an HK space whose metric coincides with the rigid limit (2.32). Essentially, this is the way which we use to prove that M′ H carries the HK structure. Given the twistorial description of M′ H, it is easy to see that, as it was in the case of the classical c-map, it can be obtained by a series of hyperk¨ahler quotients from a larger HK space Mcor H which is also of the type described by [46]. This larger space has quaternionic dimension n and is defined by the original prepotential F. Although the space is larger, the BPS states are restricted to belong to the same charge lattice Γrig as before. As a result, the metric on Mcor H has the same form as in (2.32) (after the rescaling of zI by τ2 2 to absorb this factor except the overall τ −1/2 2 ) where indices I, J, . . . taking n′ values are replaced by Λ, Σ, . . . running over n values, but the charges run over the same lattice Γrig. Due to the restriction of charges to Γrig, the Darboux coordinates η0, η ˆ A and µA do not receive instanton corrections and are given by quadratic polynomials in the coordinate t parametrizing the CP 1 fiber of the twistor space, e.g. η ˆ A = u ˆ At−1 + ζ ˆ A −¯u ˆ At. (2.34) (2.34) Besides, it leads to the existence of n −n′ commuting isometries acting by shifts of ˜ζ0 and ˜ζ ˆ A for which the Darboux coordinates η0 and η ˆ A play the role of moment maps. Whereas on the twistor space they are the usual moment maps with respect to the holomorphic symplectic structure, on Mcor H they encode the whole triplet of moment maps: their 3 coefficients in the t-expansion (2.34) provide the moment maps with respect to the triplet of K¨ahler forms on Mcor H . Performing the hyperk¨ahler quotients along these isometries, one freezes their moment maps and gets back M′ H. 2.4 Geometric interpretation It is important to understand what kind of manifold is described by the metric (2.32). In appendix C.3 we prove that M′ H is an HK manifold. This is done by showing that the metric (2.32) comes from a holomorphic symplectic structure on the trivial CP 1 bundle over M′ H, which thus gets interpretation of the associated twistor space. This symplectic structure encodes the triplet of K¨ahler structures on M′ H and, similarly to the contact structure on ZM, can itself be encoded in a set of holomorphic Darboux coordinates (ηI, µI) satisfying certain integral equations. The equations which we find (see (C.27)) turn out to be identical to the ones describing the non-perturbative moduli space of 4d N = 2 gauge theories compactified on a circle [46], for the specific choice of the charge lattice – 15 – Γrig labeling 4d BPS states, with q0 and qX playing the role of flavor charges, and the holomorphic prepotential given by Γrig labeling 4d BPS states, with q0 and qX playing the role of flavor charges, and the holomorphic prepotential given by f(zI) = −1 6 κˆI ˆJ ˆ Kz ˆIz ˆJz ˆ K − 1 (2πi)3 X kˆIC ˆI∈H+ 2 (Y) n(0) kˆI Li3  e2πikˆIz ˆI . (2.33) (2.33) This already establishes a connection to gauge theories with eight supercharges. A more precise relation will be discussed in the next section. Note that the twistor formalism provides us with an extremely simple way of taking the rigid limit. As explained above, the QK geometry of MH is encoded in the Darboux coordinates ξΛ, ˜ξΛ, α. Due to the decoupling of some of the instantons, the non-trivial integral equations determining these coordinates involve only ξI and ˜ξI, whereas other Darboux coordinates either have a simple classical form (as e.g. (C.26)) or can be obtained from the solution for this pair. Then to obtain M′ H, it is enough JHEP01(2018)156 1. to declare that the Darboux coordinates on its twistor space, ηI and µI, satisfy the same equations as ξI and ˜ξI; 1. to declare that the Darboux coordinates on its twistor space, ηI and µI, satisfy the same equations as ξI and ˜ξI; 2. to replace the prepotential entering the classical parts of Darboux coordinates by (2.33). 2.4 Geometric interpretation Besides, it leads to the existence of n −n′ commuting isometries acting by shifts of ˜ζ0 and ˜ζ ˆ A for which the Darboux coordinates η0 and η ˆ A play the role of moment maps. Whereas on the twistor space they are the usual moment maps with respect to the holomorphic symplectic structure, on Mcor H they encode the whole triplet of moment maps: their 3 coefficients in the t-expansion (2.34) provide the moment maps with respect to the triplet of K¨ahler forms on Mcor H . Performing the hyperk¨ahler quotients along these isometries, one freezes their moment maps and gets back M′ H. – 16 – On the other hand, Mcor H is the HK manifold related to the non-perturbative HM moduli space MH (where NS5-instantons have been dropped and the charges of worldsheet and D-instantons are restricted as above) by the QK/HK correspondence [29]. The easiest way to see this is to compare the two sets of Darboux coordinates, (ξΛ, ˜ξΛ) and (ηΛ, µΛ), and to note that they are related as (cf. step 1 above or (C.24)) ηΛ(t) = ξΛ(t e−iθ′), µΛ(t) = ˜ξΛ(t e−iθ′), (2.35) (2.35) provided uΛ = τ2 2 eiθ′zΛ, i.e. θ′ is the phase7 of the complex coordinate u0. The isometry needed for the correspondence is ensured by the absence of NS5-instantons and is again realized by shifts of the NS-axion σ in (2.31). This proves the geometric scheme presented on figure 1 and, in particular, allows to obtain the rigid limit of MH as n−n′+1 hyperk¨ahler quotients of its Swann bundle. JHEP01(2018)156 7It parametrizes the isometry direction mentioned in footnote 4. After the hyperk¨ahler quotient along ˜ζ0, it can be set to zero. This is why it does not appear in (C.24) and in the relation between uI and zI on M′ H. 3.1 String dualities and rigid limit In the previous section, taking the rigid limit of the HM moduli space appearing in CY compactifications of type IIB string theory, we arrived at an HK manifold M′ H. The HK structure is an indication that this manifold should play a role in a physical theory with rigid supersymmetry. Indeed, quantum corrected HK manifolds typically arise as moduli spaces, or more precisely target spaces of 3d N = 4 non-linear σ models. But what class of σ-models are we describing? We already saw that the twistorial description of M′ H makes it clear that it fits into the mathematical framework of [46] developed for describing the class of σ-models arising as circle compactifications of 4d N = 2 gauge theories. However, we can still ask how to characterize the subclass corresponding to M′ H. In this section we propose an answer to this question. Our reasoning mainly follows the reverse of the one presented in [31] and is based on a chain of string dualities, which allow to establish a connection between M′ H and 5d N = 1 gauge theories compactified on a torus. The appearance of the torus compactification should not come as a surprise because M′ H is expected to carry an isometric action of the torus modular group SL(2, Z). We saw this explicitly in the classical approximation in section 2.2, where the symmetry group was enhanced to SL(2, R), but this should remain true even in the presence of quantum corrections. The reason for this expectation is that, on one hand, the initial HM moduli space MH does carry such an isometry and, on the other hand, its action on the K¨ahler parameters used to define the limit is a simple rescaling (see (2.16)), which implies that the rigid limit should commute with the SL(2, Z) action. To begin with, let us note that under compactification on a circle the HM sector does not change and the corresponding moduli space carries the same metric in both dimensions. In contrast, each four-dimensional N = 2 vector multiplet gives rise to a hypermultiplet in – 17 – three dimensions. Indeed, each vector gives rise to two scalars: one is the vector component along the circle and the second appears after dualization of the three-dimensional vector field. 3.1 String dualities and rigid limit Combining them with the complex scalar from the 4d multiplet, one finds four real scalars representing the bosonic content of a hypermultiplet. As a result, if we consider type IIB string theory compactified down to three dimensions on Y×S1, its moduli space is a direct product of two QK manifolds MB H × ˜ MB H: one is identical to the HM moduli space in 4d and the second comes from the vector multiplet sector of the intermediate 4d theory. Now let us perform T-duality along S1. Then type IIB string theory on Y × S1 R is mapped to type IIA string theory on Y×S1 1/R. Hence the moduli spaces of the two theories should also be identical. Since MB H and ˜ MB H involve K¨ahler and complex structure moduli of Y, respectively, whereas MA H and ˜ MA H involve them in the opposite way, T-duality exchanges the two factors and we have JHEP01(2018)156 MB H = ˜ MA H, ˜ MB H = MA H. (3.1) (3.1) Note that this fact is heavily used in the physical derivation of the c-map metric [32, 33] and is responsible for the identification of the instanton degeneracies Ωγ with degeneracies of BPS black holes [21]. Next, one realizes that since type IIA string theory can be viewed as compactification of M-theory on a circle, the same moduli spaces arise by considering M-theory on Y×T 2. But let us stop in five dimensions after compactification on the CY. The corresponding 5d N = 1 supergravity contains the HM sector with the moduli space MA H and the vector multiplet sector. Taking the rigid limit of the latter, one arrives at a 5d N = 1 gauge theory. Finally, assuming that the rigid limit commutes with compactification on a torus, one concludes that the rigid limit of MB H = ˜ MA H should be the same as the torus compactification of this five-dimensional gauge theory. All these dualities and limits are shown in detail in figure 2 in the introduction. Below we review some basic aspects of 5d N = 1 gauge theories, their torus compact- ifications and discuss some implications of their relation with the non-perturbative HM moduli space of string theory. 3.2 Low energy description of 5d gauge theories A 5d supersymmetric gauge theory with the gauge group G is specified by a coupling of the vector multiplet with a number of hypermultiplets representing the matter fields. The on-shell vector multiplet includes a vector field Aˆµ, a real scalar ϕ and a Dirac spinor ψ, all taking values in the Lie algebra of G, where ˆµ = 0, . . . , 4 will denote 5-dimensional spacetime indices. On the Coulomb branch of the moduli space the real scalar field ϕ takes non-vanishing vacuum expectation values in the Cartan subalgebra, and at a generic point of this branch the gauge group G is broken to its maximal torus U(1)r where r = rank(G). Thus, the fields from the Cartan subalgebra, ϕI and AI with I = 1, . . . , r, remain massless, whereas the fields associated with other generators of the Lie algebra form massive vector multiplets with masses determined by the expectation values of ϕI. – 18 – In the low energy limit the effective Lagrangian for the massless fields takes the fol- lowing general form which includes, in particular, the Chern-Simons (CS) coupling L5d bos = −FIJ(ϕ) 2π 1 4 F I ˆµˆνF J ˆµˆν + 1 2 ∂ˆµϕI∂ˆµϕJ  −FIJK 48π ǫˆµˆνˆλˆρˆσAI ˆµF J ˆνˆλF K ˆρˆσ (3.2) (3.2) and is completely determined by the prepotential F(ϕ), a real function on the Coulomb branch. The prepotential gets one-loop contributions from all dynamical fields, but is at most cubic in ϕI [47, 48] and is completely determined by the prepotential F(ϕ), a real function on the Coulomb branch. The prepotential gets one-loop contributions from all dynamical fields, but is at most cubic in ϕI [47, 48] F(ϕ) = π g2 0 hIJϕIϕJ + ccl 12π dIJKϕIϕJϕK + 1 24π  X r |r·ϕ|3− Nf X i=1 X wi |wi·ϕ+mi|3  + cIϕI 2π , (3 3) JHEP01(2018)156 (3.3) where g0 is the bare gauge coupling, r are the roots of G, wi are the weights of G in the representation Ri, hIJ = trF (TITJ), dIJK = 1 2 trF TI(TJTK + TKTJ), (3.4) (3.4) and trF denotes the trace in the fundamental representation.8 Note that dIJK are non-zero only for SU(N) theories with N > 2. In such case, ccl is the CS level in the ultraviolet Lagrangian. We also allow for a non-vanishing linear term specified by coefficients cI. 8Comparing to [48], we accept the same normalization for the generators trF T 2 I = 2 and take m0 = 4π2g−2 0 . Besides, we divide the whole prepotential by 2π so that our normalizations are consistent with the quantization of the Chern-Simons coupling in (3.2). 3.2 Low energy description of 5d gauge theories Such term is not seen in the Lagrangian (3.2), but contributes to the tension of magnetic strings discussed below. The important feature of the quantum corrected prepotential (3.3) is that it is not smooth at loci where wi ·ϕ+mi = 0, which physically correspond to some charged matter fields becoming massless. As a result, the Coulomb branch is divided into several chambers where the prepotential takes different forms. For future reference, let us specialize (3.3) for the SU(2) gauge theory with Nf hyper- multiplets in the fundamental representation, in which case one has 2πFSU(2) = 4π2 g2 0 ϕ2 + 4 3 ϕ3 −1 12 Nf X i=1 |ϕ −mi|3 −1 12 Nf X i=1 |ϕ + mi|3 + cϕ, (3.5) (3.5) and for the pure SU(3) theory, which gives 2πFSU(3) = 4π2 g2 0 ϕ2 1−ϕ1ϕ2+ϕ2 2  + ccl 2 ϕ2 1ϕ2−ϕ1ϕ2 2  + 1 6 8ϕ3 1−3ϕ2 1ϕ2−3ϕ1ϕ2 2+8ϕ3 2  +cIϕI. (3 6) ) = 4π2 g2 0 ϕ2 1−ϕ1ϕ2+ϕ2 2  + ccl 2 ϕ2 1ϕ2−ϕ1ϕ2 2  + 1 6 8ϕ3 1−3ϕ2 1ϕ2−3ϕ1ϕ2 2+8ϕ3 2  +cIϕI. (3.6) (3.6) ( ) Although five-dimensional gauge theories are non-renormalizable, they can have non- trivial fixed points at strong coupling and thus be ultraviolet complete [47]. Conditions on the matter content which ensure the existence of such a fixed point were studied in detail in [48] where they have been derived by requiring that the second derivatives of – 19 – the prepotential form a positive definite matrix in all chambers of the Coulomb branch. Recently, it has been noticed that this excludes some of the gauge theories, including in particular quiver gauge theories, which can be obtained from string or brane constructions and therefore have to be ultraviolet complete [5, 49–51]. This led to a proposal to relax the criterion of [48] and to require only that FIJ is positive definite in the regions of the Coulomb branch where all non-perturbative degrees of freedom remain massive [52]. These non-perturbative degrees of freedom are given by BPS states which, besides the usual electrically charged particles with masses determined by the central charge Z⃗e = eIϕI + ei fmi (3.7) (3.7) JHEP01(2018)156 where eI, ei f are gauge and flavor charges, respectively, include dyonic instantons [53] (see also [54, 55]) and magnetic monopole strings [56]. 3.2 Low energy description of 5d gauge theories The former are four-dimensional instantons lifted to solitons in 4 + 1 dimensions. They are charged under both local gauge symmetry and an additional global U(1)I symmetry. This symmetry has the current j = ⋆tr(F ∧F) (3.8) (3.8) which is always conserved in five dimensions and the corresponding charge is equal to the instanton winding number k [47]. The central charge of dyonic instantons is given by Zk,⃗e = k 8π2 g2 0 + βIϕI  + Z⃗e , (3.9) (3.9) where the additional term βIφI arises at quantum level due to a mixing between the gauge symmetries and the global U(1)I symmetry which can be traced back to the presence of the CS coupling in the bosonic Lagrangian (3.2). The monopole strings are magnetic dual to the electric particles and have tensions determined by derivatives of the prepotential where the additional term βIφI arises at quantum level due to a mixing between the gauge symmetries and the global U(1)I symmetry which can be traced back to the presence of the CS coupling in the bosonic Lagrangian (3.2). The monopole strings are magnetic dual to the electric particles and have tensions determined by derivatives of the prepotential (3.10) Z⃗p = pIFI(ϕ). (3.10) All these central charges are real functions which must be positive in the physical region of the Coulomb branch. All these central charges are real functions which must be positive in the physical region of the Coulomb branch. 3.3 Torus compactification Let us now compactify the 5d gauge theory considered above on a torus. To this end, we choose spacetime to have topology R3 × T 2 and to carry the metric Let us now compactify the 5d gauge theory considered above on a torus. To this end, we choose spacetime to have topology R3 × T 2 and to carry the metric gˆµˆν = ηµν 0 0 ̺mn ! , ̺mn = V τ2 |τ|2 τ1 τ1 1 ! , (3.11) (3.11) where Greek indices µ, ν label coordinates on the flat three-dimensional Minkowski space- time, Latin indices m, n correspond to directions along the torus, V is its volume and τ is its complex structure. where Greek indices µ, ν label coordinates on the flat three-dimensional Minkowski space- time, Latin indices m, n correspond to directions along the torus, V is its volume and τ is its complex structure. At classical level the compactified theory is given by the Kaluza-Klein reduction of the Lagrangian (3.2). This reduction is straightforward and we perform it in appendix D gener- alizing (and correcting a few sign errors) the procedure presented in [31]. The result (D.7) – 20 – represents a 3d non-linear sigma model with the target space parametrized by the 5d real scalars ϕI, the components of the 5d vector fields along the torus ϑI 1 and ϑI 2 (D.1), which can be combined in complex fields ϑI τ (D.2), and scalars λI dual to the 3d vector fields. The metric on this target space obtained by the Kaluza-Klein reduction has the following form ds2 3d = FIJ  π τ2 dϑI τd¯ϑJ τ + V 4π dϕI dϕJ  (3.12) + 4π3 V FIJ  dλI + 1 2 FIKL ϑK 2 dϑL 1 −ϑK 1 dϑL 2  dλJ + 1 2 FJMN ϑM 2 dϑN 1 −ϑM 1 dϑN 2  . (3.12) It is immediate to see that the metric is invariant under the action of SL(2, Z) group which simultaneously transforms the torus modular parameter τ by the usual fractional transformation and the three-dimensional fields as JHEP01(2018)156 ϕI 7→ϕI, λI 7→λI, ϑI 1 ϑI 2 ! 7→ a b c d ! ϑI 1 ϑI 2 ! . (3.13) (3.13) Since any theory on a torus must possess such invariance, it can be seen as a consistency check of the derived metric. 3.3 Torus compactification Let us now show that this is indeed the case. First, we claim that the contributions from perturbative α′ and gs-corrections as well as from D(-1)-instantons, which are known to correct the metric on MH, do not appear on M′ H. The easiest way to see this is to look at the twistorial formulation of the rigid limit. On MH these corrections are encoded by the second term in the prepotential (2.26), the logarithmic term parametrized by coefficient c in the Darboux coordinate α (B.13), and D-instantons with charges γ = (q0, 0, 0, 0), respectively. In particular, the latter affect only the Darboux coordinates ˜ξ0 and α, as can be seen from the integral equations (B.10). But going to M′ H, these Darboux coordinates drop out from the twistorial formulation and the prepotential (2.33) does not contain the perturbative correction term anymore. Thus, the twistorial formulation of M′ H does not contain all these contributions.9 JHEP01(2018)156 Next, let us consider the contributions of worldsheet and D1-instantons. Combining them together, one can perform a resummation which turns them into (p, q)-instantons with the instanton action of the following form [35, 42] S⃗q, m,n = 2π|mτ + n| |qˆIt ˆI| −2πiqˆI(mc ˆI + nb ˆI), (3.16) (3.16) where we took into account that due to the restriction to Γrig the only non-vanishing com- ponents, which D1-instanton charge can have, are qˆI. We would like to identify these (p, q)- instantons with dyonic instantons wrapping one-dimensional cycles of the torus. Expressing the real part of the instanton action in terms of the gauge theory variables, one finds Re S⃗q, m,n = r V τ2 |mτ + n| |Z⃗q|, Z⃗q = qI ϕI + bI XmX + qX bX I ϕI + bX Y mY  , (3.17) (3.17) where we denoted mX = (8π2g−2 0 , mi) and encoded the identification between the K¨ahler moduli tˆI and the gauge theory variables ϕI and mX in a matrix bˆI ˆJ with bI J = δJ I . The factor in front of Z⃗q has a clear interpretation: this is the volume of the one-dimensional closed cycle on the torus, labeled by two integers (m, n), which is wrapped by the instan- ton. Then the second factor should be identical to the dyonic central charge (3.9). 3.3 Torus compactification Furthermore, comparing this metric with the rigid c-map (2.23), which we obtained as the rigid limit of the classical HM moduli space, one finds that the two metrics coincide up to the multiplicative factor 2πV−1/2 provided FIJ = rτ2 V gIJ (3.14) (3.14) and the two sets of coordinates are identified as follows and the two sets of coordinates are identified as follows and the two sets of coordinates are identified as follows ϕI = 2π rτ2 V tI + βI XtX , ϑI 1 = cI, λI = ˜cI + 1 2 κIJX bJcX −cJbX , ϑI 2 = bI, (3.15) (3.15) where βI X are some constant coefficients. Note that these identifications are perfectly con- sistent with the SL(2, Z) transformations (2.16) and (3.13). They imply FIJK = −1 2π κIJK and that the gauge theory parameters 1/g2 0 and mi are given by linear combinations of the frozen K¨ahler parameters tX. The concrete form of these relations depends, on one hand, on the intersection numbers of the CY and, on the other hand, on the gauge group and matter content of 5d theory. Matching these data allows to determine which particular 5d theory is captured by the rigid limit of a given Calabi-Yau manifold. We consider several examples of this in section 4. However, the metric (3.12) is only the classical approximation to an exact result which includes contributions from instantons originating from BPS states wrapping the torus. The simplest type of BPS states are electrically charged particles. In particular, in the case of pure SU(2) theory, the contribution from the W-bosons to the quantum corrected metric of the 3d σ-model was computed in [31] by integrating out the tower of massive Kaluza- Klein states in the one-loop approximation. But as we saw in the previous subsection, – 21 – there are two other types of BPS states which can generate instantons: dyonic instantons and magnetic strings. Their contributions are much more difficult to calculate, and only a few partial results on stringy instantons are available at the moment [31, 57]. On the other hand, the argument presented in section 3.1 implies that the full non- perturbative metric including contributions from all instantons should coincide with the metric (2.32) describing the rigid limit of the non-perturbative HM moduli space. In particular, the instantons on the string theory side should match those on the gauge theory side. 3.3 Torus compactification Setting eˆI = b ˆJ ˆI q ˆJ, one obtains that Z⃗q = eIϕI + eXmX = 8π2e0 g2 0 + Z⃗e. (3.18) (3.18) 9Heuristically, this can be understood as follows. In the type IIB formulation, these quantum corrections can be resumed into modular functions represented typically by τ-dependent non-holomorphic Eisenstein series [35]. Since in our case τ is a fixed parameter, all such contributions are constant and can be absorbed into a redefinition of variables. A similar phenomenon happens when one applies the QK/HK correspon- dence to the one-loop corrected local c-map: the resulting HK space coincides with the standard rigid c-map and is independent of the parameter controlling the one-loop correction [29]. – 22 – This coincides with (3.9) upon identifying e0 with the instanton charge k, up to the shift of the bare gauge coupling g−2 0 . Of course, for vanishing e0 one reproduces the central charge of the usual electrically charged BPS particles. To reproduce the shift of the gauge coupling in (3.9), one should note two facts. First, only the rational part of the coefficients βI is unambiguously defined since their integer part can be absorbed into a redefinition of the charge lattice which can be done, for instance, by eI 7→eI −k[βI]. Second, the rotation of the charge lattice induced by b ˆJ ˆI generically does not preserve its integrality. Furthermore, the lattice of charges qˆI was already a result of the rotation to the basis adapted for taking the rigid limit (see section 2.1), which also can spoil the integrality. Taking this into account, the naive identification of eˆI with the set of electric, flavor and instanton charges of gauge theory suggested by (3.18) may not be correct, and a more careful analysis is required. We will see in section 4 on a concrete example how such analysis allows to get a non-trivial shift of the gauge coupling in the dyonic central charge. JHEP01(2018)156 It is worth also to note that the identification of (p, q) and dyonic instantons implies that the definition of a 5d gauge theory at the non-perturbative level involves new param- eters in addition to masses and the gauge coupling. These are cX and bX appearing as θ-angle terms in (3.16). We obtained them as frozen periods of the RR 2-form and the B-field along curves CX on the CY. What is their origin in gauge theory? 3.4 BPS spectrum and modular invariance In the previous subsections we argued that the non-perturbative moduli space of a 5d gauge theory compactified on a torus is captured by the metric on M′ H, the rigid limit of the HM moduli space of type IIB string theory on the appropriate CY threefold. Expanding this metric around the classical rigid c-map allows to read offvarious instanton corrections which, as we saw, can all be identified either with instantons from electrically charged BPS particles, or dyonic, or stringy instantons. Thus, string theory provides us with concrete predictions for the instanton contributions in compactified 5d gauge theory. JHEP01(2018)156 In practice, all that we need in order to get these contributions is to know the BPS spectrum and a relation between the frozen moduli and the gauge theory parameters. The latter can be found by matching the classical prepotentials. The BPS spectrum, however, represents a much more serious problem. On the gauge theory side, only some partial results about the spectrum of dyonic instantons are available [59, 60], which have been obtained using the technique of string web diagrams [61], and even less is known about the spectrum of magnetic strings. On the string theory side, the former spectrum is captured by Gopakumar-Vafa invariants of the CY, whereas the latter spectrum is encoded in more complicated generalized DT invariants. Here we would like to bring attention to unexpected constraints on the spectrum of bound states of magnetic strings and dyonic instantons arising as a consequence of the SL(2, Z) modular invariance. On the gauge theory side, this symmetry appears to be just an artefact of compactification on a torus, and it is not clear how it can constrain the spectrum in five-dimensions. But in string theory, it is a duality playing a fundamental role. In particular, imposing it as an isometry of the HM moduli space, one arrives at the condition that the D3-D1-D(-1) bound states form an SL(2, Z) invariant subsector. To put this condition in a clear mathematical form, let us consider the four-dimensional dilaton r = eφ which is known to transform under SL(2, Z) as r 7→ r |cτ + d|. (3.22) (3.22) Classically, r has a simple expression thorough the volume of the CY given in (2.11), but at quantum level it gets various corrections and can be expressed as in (B.21). 3.3 Torus compactification To answer this question, let us recall that the gauge theory parameters can be thought as background gauge superfields related to gauging global symmetries associated with these parameters [58]. In particular, the flavor masses can be identified with the scalar components of the vector superfields gauging the flavor symmetry, whereas the gauge coupling appears as the scalar component of the superfield for the U(1)I symmetry discussed around (3.8). Once the theory is put on a torus, each background vector field gives rise to two new parameters given by holonomies around the basis of one-dimensional cycles on the torus, which are precisely cX and bX.10 The last type of the instanton effects contributing to the metric on M′ H comes from D3-branes wrapping divisors DI. Their instanton action is given by S⃗p = 2πτ2|pIfcl I | −2πipI  ˜cI + 1 2 κI ˆJ ˆ Kb ˆJ(c ˆ K −τ1b ˆ K)  . (3.19) (3.19) Let us set for simplicity bˆI = 0. Then if the relation (3.14) can be integrated to fcl I |bˆI=0 = −V 2πτ2 FI, (3.20) (3.20) which can always be achieved by tuning the coefficients cI of the linear term in (3.3), then the instanton action takes the simple form which can always be achieved by tuning the coefficients cI of the linear term in (3.3), then the instanton action takes the simple form S⃗p|bˆI=0 = V Z⃗p −2πipIλI. (3.21) (3.21) 10It is amusing to note that background fields can be thought of as dynamical fields whose kinetic terms have infinite coefficients [58]. This remark closes the circle of ideas since it returns us back to the origin of the additional parameters in the rigid limit. – 23 – – 23 – Its real part coincides with the instanton action of a stringy instanton given by the volume of the torus wrapped by a magnetic string multiplied by its tension. The imaginary part is also natural since λI are the scalars dual to the vector fields of the gauge theory and therefore should be sourced by magnetic objects. Thus, all non-perturbative effects surviving in the rigid limit find their interpretation in the supersymmetric gauge theory compactified on a torus. 3.4 BPS spectrum and modular invariance Then the consistency with (3.22) requires that the D3-instanton contribution to r transforms as a non-holomorphic modular form of weight (−1 2, −1 2). This turns out be a non-trivial requirement which leads to certain constraints on the spectrum of these instantons, i.e. DT invariants Ωγ with charges γ = (0, pa, qa, q0), some of which descend in the rigid limit to the BPS degeneracies of bound states of magnetic strings and dyonic instantons. – 24 – Such constraints are typically formulated in terms of modular properties of a gener- ating function of these invariants. More precisely, let us introduce the so called MSW invariants [62], ¯ΩMSW γ = ¯Ωγ(za ∞(γ)), given by the rational DT invariants ¯Ωγ = X d|γ 1 d2 Ωγ/d (3.23) (3.23) evaluated at the ‘large volume attractor point’, za ∞(γ) = lim λ→+∞(−qa + iλpa). We recall that the specification of the complex moduli za is important because the DT invariants are only piecewise constant on the moduli space due to the wall-crossing phenomenon [63]. An important property of the MSW invariants is that they do not change under the spectral flow symmetry acting on charges as [64, 65] JHEP01(2018)156 qa 7→qa −κabcpbǫc, q0 7→q0 −ǫaqa + 1 2 κabcpaǫbǫc, ǫa ∈Z. (3.24) (3.24) As a result, they only depend on pa, the charge ˆq0 = q0 −1 2 κabqaqb invariant under the spectral flow, and a residue class µa which takes into account that not all integer charges qa can be obtained by varying ǫa in (3.24). This allows to write ¯ΩMSW γ = ¯Ω⃗p,⃗µ(ˆq0). Fur- thermore, since the invariant charge ˆq0 is bounded from above by ˆqmax 0 = 1 24(p3 + c2,apa), it is possible to define the generating function of the MSW invariants h⃗p,⃗µ(τ) = X ˆq0≤ˆqmax 0 ¯Ω⃗p,⃗µ(ˆq0) e−2πiˆq0τ (3.25) (3.25) with fixed magnetic charge and residue class. It is this function that must have an appro- priate modular behavior. In particular, in the one-instanton approximation, i.e. when a D3-brane wraps an irreducible divisor of Y, it must be a vector-valued holomorphic mod- ular form of negative weight − 1 2 h1,1(Y) + 1  [66–68]. Even more interesting behavior appears if one goes beyond the one-instanton approximation, i.e. considers branes on re- ducible divisors, in which case h⃗p,⃗µ was shown to be a vector-valued holomorphic mock modular form [69]. 3.4 BPS spectrum and modular invariance Very similar constraints should arise in the gauge theory setup. These constraints can be derived either by taking the rigid limit of the above construction or by studying the constraints of modular invariance directly for the metric (2.32). In either case, one expects to find non-trivial restrictions on the modular behavior of a generating function of BPS degeneracies of magnetic strings so that their spectrum will be severely constrained. Note that the mock modularity of the generating function (3.25) evaluated for reducible divisors takes its roots in the wall crossing of the DT invariants. This raises the question about the wall crossing in 5d gauge theories. The reality of the moduli and the central charges, (3.9) and (3.10), represents an essential difference from the more familiar four- dimensional case. Nevertheless, the relation to string theory indicates that the central charge of a bound state is complex and given by Z⃗p,k,⃗e = Z⃗p + iZk,⃗e. (3.26) Z⃗p,k,⃗e = Z⃗p + iZk,⃗e. (3.26) – 25 – Thus, one might have a non-trivial wall crossing even if the moduli space is real. Here we restrict ourselves just to these comments and postpone the study of five-dimensional wall crossing and modularity constraints to future research. Thus, one might have a non-trivial wall crossing even if the moduli space is real. Here we restrict ourselves just to these comments and postpone the study of five-dimensional wall crossing and modularity constraints to future research. 4 Examples In this section we present several examples illustrating our rigid limit for different types of Calabi-Yau manifolds and its relation to five-dimensional N = 1 gauge theories. 4.1 Elliptic fibrations and SU(2) gauge theory JHEP01(2018)156 We start with the most studied example of a family of elliptically fibered CYs which are well known to be related to SU(2) gauge theories with Nf < 8 flavors [70]. The elliptic fibrations π : Y →B, where B is a complex two-dimensional base, can locally be described by a Weierstrass form (4.1) y2 = 4x3 −g2(u1, u2)xw4 −g3(u1, u2)w6, (4.1) where u1, u2 are coordinates on the base. We assume that the fibration is smooth with a single section σ represented by the base B. This implies that singularities on the fiber can only be of Kodaira type I1, which means that the discriminant ∆= g3 2 −27g2 3 of (4.1) has only simple zeros on B. This restricts the possible choice of B to smooth almost Fano twofolds which include the Hirzebruch surfaces Fm, m = 0, 1, 2, del Pezzo surfaces dPm, m = 0, . . . , 8, and the toric surfaces described by the 16 reflexive two-dimensional polytopes. Here we consider only the first two possibilities, Fm and dPm. Their geometric description can be found, for instance, in [57, 71, 72]. For all smooth elliptic fibrations a basis of H1,1(Y) generating the K¨ahler cone is given by {ωe, π⋆ωα}, α = 1, . . . , h1,1(B), where ωe = σ + π⋆c1(B) (4.2) (4.2) and ωα are the generators of the K¨ahler cone on the base. We denote the corresponding basis of dual divisors by {De, Dα}. The divisor De is dual to the elliptic fiber curve E in the sense that it does not intersect any curve in B and obeys De ∩E = 1. Let us expand the first Chern class of the base in the basis of ωα c1(B) = cα 1 ωα, (4.3) (4.3) and denote by Cαβ the intersection matrix on B and denote by Cαβ the intersection matrix on B Z B ωα ∧ωβ = Cαβ, (4.4) (4.4) which has signature (1, h1,1(B) −1). Then, using the adjunction formula which leads to the relation σ2 = −c1σ, the triple intersection numbers of Y can be shown to be καβγ = 0, κe αβ = Cαβ, κee α = Cαβcβ 1, κeee = Cαβcα 1 cβ 1. (4.5) (4.5) – 26 – – 26 – Thus, all intersection numbers are determined by the intersection numbers of the base and its first Chern class. 4.1 Elliptic fibrations and SU(2) gauge theory A crucial property of the intersection numbers (4.5) is that the matrix Mab = κeab is degenerate, i.e. det M = 0. This suggests that the vector va 1 = δa e, playing the role of ⃗vA of section 2.1, defines a non-trivial local limit. Indeed, it belongs to the K¨ahler cone, the kernel of the matrix (2.3), coinciding with Mab defined above, is non-empty, and its self-intersection number κeee given in (4.5) is non-vanishing for all the bases under consideration. The kernel of Mab is one-dimensional and described by the vector11 ⃗vn−1 = (−1, cα 1 ), (4.6) (4.6) JHEP01(2018)156 playing the role of ⃗vI of section 2.1. Remarkably, the corresponding shrinking divisor ˆDn−1 = ⃗va n−1Da can be expressed using (4.2) as playing the role of ⃗vI of section 2.1. Remarkably, the corresponding shrinking divisor ˆDn−1 = ⃗va n−1Da can be expressed using (4.2) as ˆDn−1 = −De + ca 1Da = −B (4.7) (4.7) and thus it coincides with the base of the elliptic fibration. nd thus it coincides with the base of the elliptic fibration. and thus it coincides with the base of the elliptic fibration. Finally, one can complete the two vectors ⃗v1 and ⃗vn−1 to a basis in H2(Y, R) by choosing ⃗vX with X = 2, . . . , n −2. This structure indicates that in the local limit one K¨ahler modulus grows, one remains dynamical and nfr = n −3 = h1,1(B) −1 moduli become frozen. This is consistent with the expectation that such limit produces an SU(2) 5d gauge theory with nfr −1 flavors since one of the frozen moduli should play the role of the gauge coupling, whereas others can be associated with flavor masses. To verify this claim and establish a precise relation between the moduli and the gauge theory variables, we need to specify the choice of the base B and to analyze its homology lattice. 4.1.1 Hirzebruch surfaces First, we choose B = Fm. The Hirzebruch surface Fm is a P1 bundle over P1 of the form P(O ⊕O(m)) for m ≥0. The Mori cone, dual to the K¨ahler cone, is generated by two effective curves, the isolated section S of the bundle and the fiber F. These curves have the following intersections S ∩S = −m, S ∩F = 1, F ∩F = 0. (4.8) (4.8) The dual generators of the K¨ahler cone, Dα, are given by D1 = F, D2 = S + mF (4.9) D1 = F, D2 = S + mF (4.9) and have the following intersection matrix Cαβ = 0 1 1 m ! . (4.10) (4.10) 11In this section we accept the convention that the indices A corresponding to the large moduli run over 1, . . . , n∞, the indices I corresponding to the moduli remaining dynamical run over n −n′, . . . , n −1, and the indices X labeling the frozen moduli run in-between. We recall that n = h1,1(Y) + 1. – 27 – Finally, the first Chern class is known to be Finally, the first Chern class is known to be Finally, the first Chern class is known to be (4.11) c1(Fm) = 2S + (2 + m)F = (2 −m)D1 + 2D2, (4.11) where we used Poincar´e duality to write it in terms of divisors. Computing the intersection numbers of a smooth elliptic fibration over Fm using (4.5), one obtains that the classical cubic prepotential (2.12) is given by Computing the intersection numbers of a smooth elliptic fibration over Fm using (4.5), one obtains that the classical cubic prepotential (2.12) is given by F cl = − 4 3 (ze)3 + (ze)2z1 +  1 + m 2  (ze)2z2 + zez1z2 + m 2 ze(z2)2  . (4.12) (4.12) Next, we perform the rotation (2.5) to the basis adapted for taking the local limit. As explained above, the rotation is generated by the vectors ⃗vA,⃗vX,⃗vI which in this case are taken as JHEP01(2018)156 ⃗v1 = (1, 0, 0), ⃗v2 = (0, 1, 0), ⃗v3 = (−1, 2 −m, 2). (4.13) (4.13) ⃗v3 = (−1, 2 −m, 2). It brings the prepotential to the form F cl = −4 3 (ˆz1)3 −(ˆz1)2ˆz2 + ˆz2(ˆz3)2 + 4 3 (ˆz3)3. 4.1.2 Del Pezzo surfaces JHEP01(2018)156 Our second choice of the base is B = dPm. The del Pezzo surface dPm is a blowup of P2 at m points. It can be viewed as a fibration over P1 where the generic fiber is also P1, but degenerates over m−1 points into two P1’s intersecting at a point. The number of blow-up points can vary from 0 to 9, but we restrict to 1 ≤m ≤8 to present a uniform description.12 A standard choice of basis for H2(dPm, Z) is given by the hyperplane class H of P2 and by the exceptional divisors Ei, i = 1, . . . , m, of the blow-ups. Their intersections are H ∩H = 1, Ei ∩Ej = −δi,j, H ∩Ei = 0. (4.19) (4.19) However, this basis is not a basis of the K¨ahler cone. The latter can be obtained by choosing (4.20) Di = H −Ei, Dm+1 = H. (4.20) In fact, for m > 2 this choice is not unique because the K¨ahler cone is non-simplicial and the number of its generators exceeds the dimension of H2(dPm, Z). All K¨ahler generators can be found in [72] and different choices of the basis correspond to different sub-cones. In the basis (4.20), the first Chern class is given by In fact, for m > 2 this choice is not unique because the K¨ahler cone is non-simplicial and the number of its generators exceeds the dimension of H2(dPm, Z). All K¨ahler generators can be found in [72] and different choices of the basis correspond to different sub-cones. In the basis (4.20), the first Chern class is given by c1(dPm) = 3H − m X i=1 Ei = m X i=1 Di + (3 −m)Dm+1. (4.21) (4.21) Substituting these data into equations (4.5) for the intersection numbers of the CY constructed over dPm, one obtains the following prepotential Substituting these data into equations (4.5) for the intersection numbers of the CY constructed over dPm, one obtains the following prepotential F cl = −  9−m 6 (ze)3+(ze)2 m X i=1 zi+ 3 2(ze)2zm+1+ze m+1 X i,j=1 i<j zizj+ 1 2 ze(zm+1)2  . 4.1.1 Hirzebruch surfaces (4.14) (4.14) Note that the prepotential in the new basis does not depend on m and that the moduli ˆz1 and ˆz3 are decoupled, which makes possible to define the local limit as ˆt1 →∞. Then from our general discussion it follows that the limit is described by the prepotential Note that the prepotential in the new basis does not depend on m and that the moduli ˆz1 and ˆz3 are decoupled, which makes possible to define the local limit as ˆt1 →∞. Then from our general discussion it follows that the limit is described by the prepotential fcl = ˆz2(ˆz3)2 + 4 3 (ˆz3)3, (4.15) (4.15) where ˆz3 is dynamical, whereas ˆz2 is fixed to be constant. Since we expect that the local limit of the elliptic fibration over Fm corresponds to the pure SU(2) gauge theory, the prepotential (4.15) is to be compared to (3.5) with Nf = 0, i.e. Since we expect that the local limit of the elliptic fibration over Fm corresponds to the pure SU(2) gauge theory, the prepotential (4.15) is to be compared to (3.5) with Nf = 0, i.e. 2πFNf=0 SU(2) = 4π2 g2 0 ϕ2 + 4 3 ϕ3 + cϕ. (4.16) (4.16) It is immediate to see that this implies c = 0 and leads to the following identification of the moduli and the gauge theory variables ˆt2 = 4π2a g2 0 , ˆt3 = aϕ, (4.17) (4.17) where a is a proportionality coefficient which, according to (3.15), is given by a = 1 2π q V τ2 . q Furthermore, computing the central charge introduced in (3.17), which is supposed to encode the mass of dyonic instantons, in terms of the charges defined with respect to the original basis (4.9), one finds Z⃗q = qe(a−1ˆt1 −ϕ) + q1 4π2 g2 0 + (2 −m)ϕ  + 2q2ϕ. (4.18) (4.18) – 28 – – 28 – Here for completeness we included also the charge qe associated with the elliptic fiber, which is set to zero in the rigid limit. Comparing with the dyonic central charge (3.9), we see that the instanton winding number k can be identified with charge q1, whereas the function multiplying it receives a ϕ-dependent contribution, which can be traced back to the rotation of the basis induced by (4.13). In particular, we deduce that β = 2(2 −m). 4.1.1 Hirzebruch surfaces However, this coefficient is not uniquely defined as it can be changed by shifting the elec- tric charge e by a multiple of k, which shows that the identification of e with q2 suggested by (4.18) is also ambiguous. 12For instance, dP9 is qualitatively different since it is a rational elliptic surface with infinite dimensional Mori and K¨ahler cones. 4.1.2 Del Pezzo surfaces (4.22) (4.22) The vectors performing the rotation (2.5) to the basis adapted to the local limit can be chosen as The vectors performing the rotation (2.5) to the basis adapted to the local limit can be chosen as ⃗v1 = (1, 0, · · · , 0), ⃗vi = (0, 0, · · · , 0 | {z } i−1 , −1, 0, · · · , 0 | {z } m−i , 1), i = 2, . . . , m, ⃗vm+1 = (0, −1, 0, · · · , 0, 2), ⃗vm+2 = (−1, 1, · · · , 1, 3 −m). (4.23) (4.23) 12For instance, dP9 is qualitatively different since it is a rational elliptic surface with infinite dimensional Mori and K¨ahler cones. 12For instance, dP9 is qualitatively different since it is a rational elliptic surface with infinite dimensional Mori and K¨ahler cones. – 29 – After the rotation, the prepotential becomes After the rotation, the prepotential becomes After the rotation, the prepotential becomes F cl = 9 −m 6 (ˆzm+2)3 −(ˆz1)3 + 1 2 (ˆzm+2)2 −(ˆz1)2 m X i=2 ˆzi + 4ˆzm+1 ! −1 2 (ˆzm+2 −ˆz1) m X i=2 (ˆzi)2. (4.24) (4.24) Again the moduli ˆz1 and ˆzm+2 are decoupled and in the limit ˆt1 →∞the relevant part of the prepotential is given by Again the moduli ˆz1 and ˆzm+2 are decoupled and in the limit ˆt1 →∞the relevant part of the prepotential is given by JHEP01(2018)156 fcl = 9 −m 6 (ˆzm+2)3 + 1 2(ˆzm+2)2 m X i=2 ˆzi + 4ˆzm+1 ! −1 2 ˆzm+2 m X i=2 (ˆzi)2, (4.25) (4.25) where ˆzm+2 is dynamical and all other moduli are frozen. where ˆzm+2 is dynamical and all other moduli are frozen. We compare the prepotential (4.25) with the one of the SU(2) gauge theory with Nf = m−1 flavors (3.5) where we consider the chamber of the moduli space with ϕ±mi > 0. In this chamber the gauge theory prepotential takes the form 2πFNf=m−1 SU(2) = 9 −m 6 ϕ3 + 4π2 g2 0 ϕ2 + " c −1 2 m−1 X i=1 m2 i !# ϕ. (4.26) (4.26) Then again we should set c = 0, whereas the other variables are identified as follows ˆti = ami−1, ˆtm+1 = a 2π2 g2 0 −1 4 m−1 X i=1 mi ! , ˆtm+2 = aϕ. (4.27) (4.27) 4.2 Two large moduli In the elliptic fibrations considered so far, the local limit was obtained by sending only one K¨ahler modulus to infinity. However, one may expect that this is a very restricted set of examples because shrinking some of the divisors is a local procedure, which should not affect the cycles which are “far away” from them. Thus, in general, in the local limit several 2-cycles can stay finite and therefore several moduli are taken to infinity. In the elliptic fibrations considered so far, the local limit was obtained by sending only one K¨ahler modulus to infinity. However, one may expect that this is a very restricted set of examples because shrinking some of the divisors is a local procedure, which should not affect the cycles which are “far away” from them. Thus, in general, in the local limit several 2-cycles can stay finite and therefore several moduli are taken to infinity. In our language, this will happen whenever there exist two or more linearly independent vectors ⃗vA such that the intersection of the respective kernels (2.4) is non-empty. To give a concrete example of such situation, we consider one of the toric hypersurfaces constructed from the Kreuzer-Skarke list of reflexive polytopes [73, 74]. A useful database containing information about these CY varieties can be found in [75], and we will make extensive use of the data analyzed by these authors. We provide some details about these data and toric geometry in appendix E, whereas some relevant background can also be found in [75, 76]. Let us consider CY which corresponds to geometry 1 of polytope 337 in the database [75]. It is defined by the data given in (E.1) and (E.2) and has h1,1 = 4. We choose the following four generators as a basis of H4(Y, Z) D1 = D5, D2 = D6, D3 = D7, D4 = D8, (4.28) (4.28) – 30 – where we expressed them in terms of divisors Di of the ambient toric space. Extracting the triple intersection numbers in the basis from the database, the classical cubic prepotential can be written as where we expressed them in terms of divisors Di of the ambient toric space. 4.2 Two large moduli Extracting the triple intersection numbers in the basis from the database, the classical cubic prepotential can be written as F cl = −1 6 h −(z1)3 + 3(z1)2z2 + 3(z1)2z4 −3z1(z2)2 + 12z1z2z4 −6z1(z3)2 + 18z1z3z4 −3z1(z4)2 −5(z2)3 −18(z2)2z3 + 12(z2)2z4 −18z2(z3)2 + 36z2z3z4 −6z2(z4)2 −13(z3)3 + 27(z3)2z4 −9z3(z4)2 + (z4)3i . (4.29) (4.29) The K¨ahler moduli here are constrained by the requirement that the volumes of all Mori generators Ci must be positive The K¨ahler moduli here are constrained by the requirement that the volumes of all Mori generators Ci must be positive JHEP01(2018)156 JHEP01(2018)156 Z Ci J = taDa ∩Ci ≥0 . (4.30) (4.30) The matrix of intersections of Mori generators with the basis divisors is as follows The matrix of intersections of Mori generators with the basis divisors is as follows Ci ∩Da =          0 −1 −1 1 0 0 1 0 0 1 0 0 1 2 3 −1 1 0 0 0 −1 1 0 1          (4.31) (4.31) and leads to the following inequalities −t2 −t3 + t4 ≥0 , t3 ≥0 , t2 ≥0 , t1 + 2t2 + 3t3 −t4 ≥0 , t1 ≥0 , −t1 + t2 + t4 ≥0. (4.32) (4.32) The intersection numbers encoded by the prepotential (4.29) and the K¨ahler cone conditions provide the starting point for defining the local limit. Let us choose the following two vectors ⃗v1 = (1, 0, 0, 1), ⃗v2 = (0, 1, 0, 2). (4.33) (4.33) It is easy to check that their components satisfy the inequalities (4.32), saturating some of them, so that both vectors belong to the boundary of the K¨ahler cone. They give rise to the following intersection matrices (2.3) It is easy to check that their components satisfy the inequalities (4.32), saturating some of them, so that both vectors belong to the boundary of the K¨ahler cone. 4.2 Two large moduli Comparing with the prepotential (4.16) of the pure SU(2) gauge theory, one finds the following identifications ˆt3 = 2π2a g2 0 , ˆt4 = 2aϕ, c = 4π4a2 g4 0 . (4.41) (4.41) Note that in this model we obtain a non-vanishing coefficient of the linear term which contributes to the tension of magnetic strings. This contribution however still vanishes at the SCFT point where the gauge coupling is sent to infinity. Note that in this model we obtain a non-vanishing coefficient of the linear term which contributes to the tension of magnetic strings. This contribution however still vanishes at the SCFT point where the gauge coupling is sent to infinity. 4.2 Two large moduli They give rise to the following intersection matrices (2.3) M1,ab =      0 3 3 0 3 3 6 0 3 6 7 0 0 0 0 0     , M2,ab =      3 3 6 0 3 3 6 0 6 6 12 0 0 0 0 0     , (4.34) (4.34) whose kernels have dimensions 1 and 2, respectively. The two kernels overlap along the real line generated by ⃗v4 = (0, 0, 0, −1), (4.35) (4.35) – 31 – – 31 – which is linearly independent from the vectors (4.33). We complete all three vectors to a basis by taking which is linearly independent from the vectors (4.33). We complete all three vectors to a basis by taking ⃗v3 = (0, 0, 1, 2). (4.36) (4.36) Changing the basis as in (2.5), one arrives at the new form of K¨ahler cone conditions Changing the basis as in (2.5), one arrives at the new form of K¨ahler cone conditions ˆt1 + ˆt2 + ˆt3 −ˆt4 ≥0, ˆt3 ≥0, ˆt2 ≥0, ˆt3 + ˆt4 ≥0, ˆt1 ≥0, 3ˆt2 + 2ˆt3 −ˆt4 ≥0 (4.37) (4.37) and the classical prepotential and the classical prepotential JHEP01(2018)156 F cl = −1 6 h 9(ˆz1)2ˆz2 + 9ˆz1(ˆz2)2 + 3(ˆz2)3 + 9(ˆz1)2ˆz3 + 36ˆz1ˆz2ˆz3 + 18(ˆz2)2ˆz3 + 21ˆz1(ˆz3)2 + 36ˆz2(ˆz3)2 + 13(ˆz3)3 −3(ˆz3)2ˆz4 −3ˆz3(ˆz4)2 −(ˆz4)3i . (4.38) (4.38) As it should be, ˆz4 is decoupled from ˆz1 and ˆz2. Thus, in the local limit ˆt1 and ˆt2 are sent to infinity, ˆt3 becomes frozen, and ˆt4 remains dynamical. As a result, the prepotential reduces to 13 1 1 1 As it should be, ˆz4 is decoupled from ˆz1 and ˆz2. Thus, in the local limit ˆt1 and ˆt2 are sent to infinity, ˆt3 becomes frozen, and ˆt4 remains dynamical. As a result, the prepotential reduces to fcl = −13 6 (ˆz3)3 + 1 2 (ˆz3)2ˆz4 + 1 2 ˆz3(ˆz4)2 + 1 6 (ˆz4)3. (4.39) (4.39) Note that the K¨ahler cone conditions (4.37) ensure that the effective gauge coupling Note that the K¨ahler cone conditions (4.37) ensure that the effective gauge coupling Im ∂2fcl (∂ˆz4)2 = ˆt3 + ˆt4 ≥0 (4.40) (4.40) is positive definite. Comparing with the prepotential (4.16) of the pure SU(2) gauge theory, one finds the following identifications is positive definite. 4.3 SU(3) gauge theory Another variation on the models considered in section 4.1 are geometries whose local limits give rise to higher-rank gauge theories. To illustrate this possibility, in this subsection we explore a Calabi-Yau admitting a local limit which leads to the pure 5d N = 1 SU(3) gauge theory. Let us consider a toric hypersurface described by geometry 2 of polytope 1439 in [75]. Its defining data can be found in (E.3) and (E.4), and it has h1,1 = 4. In terms of toric divisors Di, the basis of H4(Y, Z) is chosen as D1 = D4, D2 = D5, D3 = D6, D4 = D7. (4.42) (4.42) – 32 – In this basis, the classical prepotential encoding the triple intersection numbers is given by the polynomial In this basis, the classical prepotential encoding the triple intersection numbers is given by the polynomial F cl = −1 6 h 3(z1)2z2 + 6(z1)2z3 + 3(z1)2z4 −9z1(z2)2 + 18z1(z3)2 −9z1(z4)2 + 8(z2)3 + 16(z3)3 + 9(z4)3i . (4.43) (4.43) The intersections of Mori generators with the basis divisors are given by the matrix The intersections of Mori generators with the basis divisors are given by the matrix Ci ∩Da =      1 0 0 −3 0 −1 0 0 0 1 1 0 0 0 0 1     , (4.44) (4.44) JHEP01(2018)156 JHEP01(2018)156 so that the K¨ahler moduli are subject to the following constraints t1 −3t4 ≥0, t2 ≤0, t2 + t3 ≥0, t4 ≥0. (4.45) (4.45) To take the local limit, we choose To take the local limit, we choose ⃗v1 = (3, 0, 0, 1), (4.46) ⃗v1 = (3, 0, 0, 1), ⃗v1 = (3, 0, 0, 1), (4.46) (4.46) which trivially satisfies the K¨ahler cone conditions (4.45). Its intersection matrix (2.3) which trivially satisfies the K¨ahler cone conditions (4.45). Its intersection matrix (2.3) Mab =      1 3 6 0 3 −9 0 0 6 0 18 0 0 0 0 0      (4.47) (4.47) has a two-dimensional kernel spanned by the following basis vectors has a two-dimensional kernel spanned by the following basis vectors ⃗v3 = (−6, −2, 2, −2), ⃗v4 = (3, 1, −1, 0). 13In fact, the vectors ⃗va, a = 2, 3, 4, may be chosen in a simpler form. Our choice instead allows to have simpler relations to the gauge theory variables, which are found below in (4.55). 4.3 SU(3) gauge theory (4.48) (4.48) The three vectors are linearly independent and can be completed to a basis by13 The three vectors are linearly independent and can be completed to a basis by13 ⃗v2 = (1, 1, 0, 0). (4.49) (4.49) The rotation of the basis (2.5) modifies the K¨ahler cone conditions to ˆt2 + 3ˆt4 ≥0, 2ˆt3 −ˆt2 −ˆt4 ≥0, ˆt2 ≥0, ˆt1 −2ˆt3 ≥0, (4.50) (4.50) and brings the prepotential to the following form and brings the prepotential to the following form F cl = −3 2 (ˆz1)3 −6(ˆz1)2ˆz2 + ˆz1(ˆz2)2 −1 3 (ˆz2)3 −(ˆz2)2ˆz3 + (ˆz2)2ˆz4 + 2ˆz2(ˆz3)2 −2ˆz2ˆz3ˆz4 + 2ˆz2(ˆz4)2 + 4 3 (ˆz3)3 −2(ˆz3)2ˆz4 + ˆz3(ˆz4)2 + 4 3 (ˆz4)3. (4.51) (4.51) 13In fact, the vectors ⃗va, a = 2, 3, 4, may be chosen in a simpler form. Our choice instead allows to have simpler relations to the gauge theory variables, which are found below in (4.55). – 33 – As is expected, ˆz3 and ˆz4 are decoupled from ˆz1. Thus, in the local limit where ˆt1 is large, we find two dynamical moduli, ˆt3 and ˆt4, whereas ˆt2 is frozen. The effective prepotential is fcl = −1 3 (ˆz2)3 −(ˆz2)2ˆz3 + (ˆz2)2ˆz4 + 2ˆz2(ˆz3)2 −2ˆz2ˆz3ˆz4 + 2ˆz2(ˆz4)2 + 4 3 (ˆz3)3 −2(ˆz3)2ˆz4 + ˆz3(ˆz4)2 + 4 3 (ˆz4)3 (4.52) (4.52) and gives rise to the following matrix of the effective gauge couplings gIJ = Im ∂2fcl ∂ˆzI ∂ˆzJ = 2 2(ˆt2 + 2ˆt3 −ˆt4) −ˆt2 −2ˆt3 + ˆt4 −ˆt2 −2ˆt3 + ˆt4 2ˆt2 + ˆt3 + 4ˆt4 ! . (4.53) (4.53) JHEP01(2018)156 The trace and the determinant of this matrix are given by Tr g = 2 4ˆt2 + 5ˆt3 + 2ˆt4 = 5(2ˆt3 −ˆt2 −ˆt4) + 3(ˆt2 + 3ˆt4) + 10ˆt2, det g = 12 ˆt2 + 2ˆt3 −ˆt4 ˆt2 + 3ˆt4 . (4.54) (4.54) It follows immediately from the K¨ahler cone conditions (4.50) that both of them are pos- itive, which ensures the positive definiteness of (4.53). Comparing (4.52) with the prepo- tential (3.6) of the pure SU(3) gauge theory, one obtains the following dictionary ˆt2 = 2π2a g2 0 , ˆt3 = aϕ1, ˆt4 = aϕ2, ccl = −3, c1 = −4π4a2 g4 0 , c2 = 4π4a2 g4 0 . (4.55) (4.55) 4.4 No local limit The previous examples could make an impression that most of Calabi-Yau manifolds allow a non-trivial local limit in the sense of section 2.1. However, this is not so. It is easy to find examples which do not allow any such limit. For instance, let us consider a complete intersection Calabi-Yau (CICY) manifold defined by the following configuration matrix   2 2 1 0 2 1 0 2 2 0 2 1   −48 . (4.56)   2 2 1 0 2 1 0 2 2 0 2 1   −48 . (4.56) (4.56) It appears first in the list of CICYs studied in [77], which simultaneously has h1,1 = 3 and the property of being K¨ahler favourable (model 5299 in this database). The latter property means that its K¨ahler cone descends from the one of the ambient projective space. Thus, choosing a basis of divisors given by a subset of the divisors of the ambient space, the K¨ahler cone admits a particularly simple description as the positive orthant ta ≥0, a = 1, 2, 3. The intersection numbers can be computed from the configuration matrix (4.56) using the standard technique (see e.g. [78]) and give rise to the following cubic prepotential It appears first in the list of CICYs studied in [77], which simultaneously has h1,1 = 3 and the property of being K¨ahler favourable (model 5299 in this database). The latter property means that its K¨ahler cone descends from the one of the ambient projective space. Thus, choosing a basis of divisors given by a subset of the divisors of the ambient space, the K¨ahler cone admits a particularly simple description as the positive orthant ta ≥0, a = 1, 2, 3. The intersection numbers can be computed from the configuration matrix (4.56) using the standard technique (see e.g. [78]) and give rise to the following cubic prepotential F cl = − h (z1)2z2+2(z1)2z3+2z1(z2)2+9z1z2z3+z1(z3)2+(z2)2z3+2z2(z3)2i . (4.57) (4.57) – 34 – To have a non-trivial local limit, we have to find at least one vector ⃗v, belonging to the K¨ahler cone, such that the matrix Mab = κabcvc is degenerate. In particular, this implies that it must have vanishing determinant. 4.4 No local limit Calculating the determinant for a generic vector, one finds det M = 18 h 4 (v1)3 + (v2)3 + (v3)3 + 9 (v1)2v2 + v1(v3)2 + (v2)2v3 + 18 v1(v2)2 + (v1)2v3 + v2(v3)2 + 69v1v2v3i . (4.58) (4.58) Note that all coefficients are positive. Thus, the determinant can vanish only if some components va have opposite signs. An example of such vector is provided by JHEP01(2018)156 ⃗v = (1, −4, 0). (4.59) (4.59) But any such vector does not belong to the K¨ahler cone which requires the positivity of all coefficients. Therefore, we conclude that this Calabi-Yau does not admit a non-trivial local limit. 5 Conclusions In this paper we analyzed the rigid limit of the HM moduli space MH of type IIB string theory compactified on a CY threefold Y. Whereas for generic QK manifolds the rigid limit is not well defined, for the HM moduli space we suggested to induce it by a local limit of the CY. When such local limit exists, we showed that the original manifold reduces to a manifold M′ H of real dimension 4n′, where n′ is the number of shrinking divisors on Y, and computed the exact non-perturbative metric on it. To accomplish this, we significantly improved the understanding of the D-instanton corrected metric on MH by computing explicitly its exact expression for all mutually non-local charges. Furthermore, we proved that M′ H is an HK manifold and can be obtained by a series of HK quotients of the Swann bundle over MH. An intermediate step of this quotient proce- dure coincides with the HK manifold Mcor H related to MH by the QK/HK correspondence. All these relations become particularly simple in the twistor formalism where the metric on a quaternionic manifold is encoded in a set of Darboux coordinates on its twistor space. Then it turns out that the rigid limit simply reduces one system of Darboux coordinates to another by restricting to the charge lattice of shrinking cycles, whereas the HK quotient along an isometry just removes a symplectic pair of Darboux coordinates, one of which plays the role of the moment map. We would like to point out that our rigid limit is essentially different from the one considered, for instance, in [13–15]. In these papers the limiting HK manifold has the same dimension as the original QK manifold and the procedure heavily relies on the existence of continuous isometries. In our case, the dimension is always reduced because of the decoupling of the “universal hypermultiplet” containing the dilaton. Such decoupling is very natural since this multiplet has a gravitational origin and should not contribute to the gauge theory physics recovered in the limit. Besides, although some isometries do – 35 – appear at intermediate steps of our procedure, the original manifold is taken to be fully non-perturbative where all classical isometries are broken by instanton corrections. Our limit is also different from the rigid limit suggested in [7] which relies on a sim- ple rescaling. 5 Conclusions Although this allows to decouple some multiplets, including the universal hypermultiplet, and thus to reduce the effective dimension, the decoupled fields do not disappear, but just support the flat metric. In contrast, in our limit some fields do drop out and others become frozen. Besides, the procedure of [7] was performed only for the classical metric described by the c-map, and an inspection shows that its direct generaliza- tion to the instanton corrected metric does not appear to produce sensible results. At the same time, the instanton corrections to the metric on M′ H all turn out to have a physical interpretation. JHEP01(2018)156 This interpretation comes from a general relation of M′ H to the physics of five- dimensional N = 1 gauge theories. Following [31], we argued that this manifold coin- cides with the non-perturbative target space of the σ-model obtained by compactifying a 5d gauge theory on a torus. Which 5d gauge theory is recovered in the limit can be established by matching the classical prepotentials. We demonstrated this matching pro- cedure on several examples, including a family of elliptically fibered CYs and a few toric hypersurfaces. Note that in the usual notion of local limit, one zooms in around a point in the moduli space where the CY develops a singularity. The study of the relation between five-dimensional gauge theories and singularities in CY threefolds has a long history (see, for instance, [48, 70, 79] and [80] for a recent work). We hope that this paper can make at least two contributions to this subject. First, we suggest a very simple condition in terms of intersection numbers of Y for the existence of a local limit. It simply requires that there exists a set of vectors belonging to the boundary of the K¨ahler cone such that the intersection of kernels of certain matrices constructed from them and the triple intersections is non-empty. It would be interesting to understand the precise relation of this criterion to the mathematical conditions for the existence of CY singularities [81]. Second, our work extends the discussion to the setting of torus compactifications where BPS states of 5d gauge theory generate non-perturbative effects. Our results provide precise predictions from string theory for their contributions to the metric on the moduli space. 5 Conclusions In particular, some of (p, q)-instantons are identified with the dyonic instantons of gauge theory and D3-instantons correspond to the instantons generated by magnetic strings. None of them has been computed exactly, and this work fills in this essential gap. The compactification on a torus gives rise to the modular invariance of the effective three-dimensional theory, which can be identified with the SL(2, Z) symmetry of type IIB string theory surviving compactification on CY and the rigid limit. This symmetry severely restricts both the form of the metric and the BPS spectrum, which remains in our results as a necessary input data. We suggested how such constraints on the spectrum can be derived along the lines of [44, 69] which should result in specific modular properties of a generating function of BPS degeneracies of magnetic strings bound to dyonic instantons. This function is also expected to have a relation to the modular partition function studied in [57] in the same context. – 36 – Another interesting and related problem is to understand the wall crossing in 5d gauge theories. Although there are some important differences with wall crossing in four dimen- sions, it is natural to expect some relation between the two, as the theories can be related by compactification on a circle. Moreover, while 5d N = 1 gauge theories on a torus are richer than 4d N = 2 gauge theories on a circle considered in [46], their effective low energy descriptions are captured by the same mathematical framework, so that both of them appear to be just particular cases of a general structure which is built on the wall crossing formula discovered by Kontsevich and Soibelman [82]. It would be interesting to understand the role of this structure directly in five dimensions. JHEP01(2018)156 Acknowledgments We would like to thank Benjamin Assel, Seung-Joo Lee, Joe Minahan, Boris Pioline, Stefan Vandoren and Timo Weigand for correspondence and valuable discussions. The work of P.L. is supported by the grants “Geometry and Physics” and “Exact Results in Gauge and String Theories” from the Knut and Alice Wallenberg Foundation. A Special geometry in the classical approximation The local special geometry is determined by a prepotential F(X), a holomorphic function homogeneous of degree 2 in coordinates XΛ. It defines the two main quantities of interest: the K¨ahler potential K (2.10) and the matrix of the gauge couplings NΛΣ = ¯FΛΣ −i(Nz)Λ(Nz)Σ (zNz) , (A.1) (A.1) where NΛΣ = −2 Im FΛΣ. The imaginary part of NΛΣ plays a particularly important role. It is a negative definite matrix and for its inverse one can establish the following general result where NΛΣ = −2 Im FΛΣ. The imaginary part of NΛΣ plays a particularly important role. It is a negative definite matrix and for its inverse one can establish the following general result Im N ΛΣ = 2NΛΣ −2 eK zΛ¯zΣ + ¯zΛzΣ , (A.2) (A.2) where NΛΣ is the inverse of NΛΣ. where NΛΣ is the inverse of NΛΣ. In the particular case of the classical prepotential (2.12), it is possible to find more concrete representations for the above objects. First, it is straightforward to compute In the particular case of the classical prepotential (2.12), it is possible to find more concrete representations for the above objects. First, it is straightforward to compute NΛΣ = 2 3 κabc 3babbtc −tatbtc −2κbcdbctd −2κacdbctd 2κabctc ! . (A.3) (A.3) This implies (zNz) = (¯zN ¯z) = −8 3 κabctatbtc = −16V, e−K = 4 3 κabctatbtc = 8V. (A.4) (A.4) The inverse matrix NΛΣ can be found in terms of κab, the inverse of κab = κabctc. The result reads ! The inverse matrix NΛΣ can be found in terms of κab, the inverse of κab = κabctc. The result reads NΛΣ = −1 4V 1 bb ba −2V κab + babb ! . (A.5) (A.5) – 37 – Using these results and notation (vu)a = κabcvbuc, one then finds the K¨ahler metric and its inverse Ka¯b = −1 4V  κab −1 4V (tt)a(tt)b  , Ka¯b = −4V κab + 2 tatb, (A.6) (A.6) nd the real and imaginary parts of the gauge coupling matrix and the real and imaginary parts of the gauge coupling matrix and the real and imaginary parts of the gauge coupling matrix Re NΛΣ = −1 3(bbb) 1 2(bb)b 1 2(bb)a −κabcbc ! , Im NΛΣ = −V 1 + 4Ka¯bbabb −4Ka¯bbb −4Ka¯bba 4Ka¯b ! , (A.7) Im N ΛΣ = −V −1 1 bb ba babb + 1 4 Ka¯b ! . A Special geometry in the classical approximation JHEP01(2018)156 Im NΛΣ = −V 1 + 4Ka¯bbabb −4Ka¯bbb −4Ka¯bba 4Ka¯b ! , (A.7) Im N ΛΣ = −V −1 1 bb ba babb + 1 4 Ka¯b ! . (A.7) B.1 Twistorial description of QK manifolds QK manifolds represent a very complicated type of geometry. Although they carry a quaternionic structure given by the triplet of almost complex structures ⃗J, all these almost complex structures are non-integrable so that QK manifolds are not even complex [83]. A very efficient way to deal with such manifolds is to work with their twistor spaces ZM whose CP 1 fiber describes normalized linear combinations of Ji, i = 1, 2, 3. In contrast to the original manifold M, its twistor space is a K¨ahler manifold and, most importantly, it carries a holomorphic contact structure [23] defined as the kernel of the canonical (1,0)-form on ZM Dt = dt + p+ −ip3t + p−t2, (B.1) (B.1) where t is the standard stereographic coordinate parametrizing CP 1, ⃗p is the SU(2) part of the Levi-Civita connection on M, and we used the chiral components defined as p± = −1 2 (p1 ∓ip2). Rescaling Dt, one can make from it a holomorphic one-form14 where t is the standard stereographic coordinate parametrizing CP 1, ⃗p is the SU( part of the Levi-Civita connection on M, and we used the chiral components defin as p± = −1 2 (p1 ∓ip2). Rescaling Dt, one can make from it a holomorphic one-form14 1 ∓ip2). Rescaling Dt, one can make from it a holomorphic one-form14 X = 4 it eφ Dt (B.2) (B.2) such that X ∧(dX)n is the non-vanishing holomorphic top form. The rescaling function φ is called the contact potential. The properties of X imply that locally, by a proper choice of coordinates, it can always be trivialized as such that X ∧(dX)n is the non-vanishing holomorphic top form. The rescaling function φ is called the contact potential. The properties of X imply that locally, by a proper choice of coordinates, it can always be trivialized as X = dα[i] + ξΛ [i]d˜ξ[i] Λ , (B.3) (B.3) 14In general, the rescaling factor may depend holomorphically on the fiber coordinate t and is different in different patches of an open covering of the twistor space, which implies that the contact one-form is not globally defined and has different local realizations X [i]. However, we will not need such generic construction which becomes relevant only after inclusion of NS5-brane instantons. B.1 Twistorial description of QK manifolds – 38 – – 38 – where the index [i] labels open patches of an atlas, Z = ∪Ui, and (ξΛ [i], ˜ξ[i] Λ , α[i]) is the set of Darboux coordinates in Ui. These coordinates is the central element of this construction because knowing them as functions on the base M and of the fiber coordinate t is, in principle, equivalent to knowing the metric on M. Let us spell out the steps necessary to compute it [24]: 1. First, one finds the Laurent expansion of the Darboux coordinates near t = 0. Denot- ing by [+] the patch surrounding the north pole of CP 1, we assume that the expansion has the following general form 1. First, one finds the Laurent expansion of the Darboux coordinates near t = 0. Denot- ing by [+] the patch surrounding the north pole of CP 1, we assume that the expansion has the following general form ξΛ [+] = ξΛ,−1 [+] t−1 + ξΛ,0 [+] + O(t), ˜ξ[+] Λ = ˜ξ[+] Λ,0 + O(t), α[+] = 4ic log t + α[+] 0 + O(t), (B.4) JHEP01(2018)156 (B.4) which is consistent with the form of Darboux coordinates in the case of the D-instanton corrected HM moduli space (see the next subsection). which is consistent with the form of Darboux coordinates in the case of the D-instanton corrected HM moduli space (see the next subsection). which is consistent with the form of Darboux coordinates in the case of the D-instanton corrected HM moduli space (see the next subsection). 2. One specifies the almost complex structure J3 by providing a basis of (1,0) forms on M. Such a basis was found in [24] and, after some simplifications, it takes the following form 2. One specifies the almost complex structure J3 by providing a basis of (1,0) forms on M. Such a basis was found in [24] and, after some simplifications, it takes the following form πa = d  ξa,−1 [+] /ξ0,−1 [+]  , ˜πΛ = d˜ξ[+] Λ,0, ˜πα = 1 2i dα[+] 0 + 2c d log ξ0,−1 [+] . (B.5) πa = d  ξa,−1 [+] /ξ0,−1 [+]  , ˜πΛ = d˜ξ[+] Λ,0, ˜πα = 1 2i dα[+] 0 + 2c d log ξ0,−1 [+] . (B.5) (B.5) 3. B.1 Twistorial description of QK manifolds Substituting the expansions (B.4) into the contact one-form X (B.3) and comparing it with the canonical form Dt (B.1) using (B.2), one finds the contact potential φ and the components of the SU(2) connection 3. Substituting the expansions (B.4) into the contact one-form X (B.3) and comparing it with the canonical form Dt (B.1) using (B.2), one finds the contact potential φ and the components of the SU(2) connection 3. Substituting the expansions (B.4) into the contact one-form X (B.3) and comparing it with the canonical form Dt (B.1) using (B.2), one finds the contact potential φ and the components of the SU(2) connection p+ = i 4 e−φ ξΛ,−1 [+] d˜ξ[+] Λ,0, p3 = −1 4 e−φ  dα[+] 0 + ξΛ,0 [+] d˜ξ[+] Λ,0 + ξΛ,−1 [+] d˜ξ[+] Λ,1  . (B.6) (B.6) 4. The SU(2) connection ⃗p can then be used to compute the triplet of quaternionic two- forms ⃗ω which are defined by the metric and the triplet of almost complex structures as ⃗ω(X, Y ) = g( ⃗JX, Y ), but are known to be proportional to the curvature of the SU(2) connection [83]. In particular, for ω3 the formula reads 4. The SU(2) connection ⃗p can then be used to compute the triplet of quaternionic two- forms ⃗ω which are defined by the metric and the triplet of almost complex structures as ⃗ω(X, Y ) = g( ⃗JX, Y ), but are known to be proportional to the curvature of the SU(2) connection [83]. In particular, for ω3 the formula reads 4. The SU(2) connection ⃗p can then be used to compute the triplet of quaternionic two- forms ⃗ω which are defined by the metric and the triplet of almost complex structures as ⃗ω(X, Y ) = g( ⃗JX, Y ), but are known to be proportional to the curvature of the SU(2) connection [83]. In particular, for ω3 the formula reads ω3 = −2dp3 + 4ip+ ∧p−. (B.7) (B.7) 5. Finally, the metric is recovered as g(X, Y ) = ω3(X, J3Y ). To do this in practice, one should rewrite ω3, computed by (B.7) in terms of differentials of (generically real) coordinates on M, in the form which makes explicit that it is of (1,1) Dolbeault type. Using for this purpose the basis πX = (πa, ˜πΛ, ˜πα) given in (B.5), the final result should look like 5. B.1 Twistorial description of QK manifolds Finally, the metric is recovered as g(X, Y ) = ω3(X, J3Y ). To do this in practice, one should rewrite ω3, computed by (B.7) in terms of differentials of (generically real) coordinates on M, in the form which makes explicit that it is of (1,1) Dolbeault type. Using for this purpose the basis πX = (πa, ˜πΛ, ˜πα) given in (B.5), the final result should look like 5. Finally, the metric is recovered as g(X, Y ) = ω3(X, J3Y ). To do this in practice, one should rewrite ω3, computed by (B.7) in terms of differentials of (generically real) coordinates on M, in the form which makes explicit that it is of (1,1) Dolbeault type. Using for this purpose the basis πX = (πa, ˜πΛ, ˜πα) given in (B.5), the final result should look like ω3 = igX ¯Y πX ∧¯πY , (B.8) (B.8) from which the metric readily follows as ds2 = 2gX ¯Y πX ⊗¯πY . Technically, this is the most non-trivial step, which we realize for the D-instanton corrected HM moduli space in section B.4. – 39 – B.2 D-instantons in twistor space As we saw above, a QK manifold can be specified by a system of Darboux coordinates on its twistor space. For the D-instanton corrected HM moduli space this was done in [21, 22] where it was shown that Darboux coordinates ξΛ and ˜ξΛ are determined by a system of integral equations. To write it explicitly, it is convenient to introduce the exponentiated version of the Darboux coordinates labeled by charge Xγ = σγ e−2πi(qΛξΛ−pΛ ˜ξΛ), (B.9) (B.9) where σγ is a sign function, known as quadratic refinement, which satisfies σγσγ′ = (−1)⟨γ,γ′⟩σγ+γ′ and can be chosen as σγ = (−1)qΛpΛ. Then the equations read where σγ is a sign function, known as quadratic refinement, which satisfies σγσγ′ = (−1)⟨γ,γ′⟩σγ+γ′ and can be chosen as σγ = (−1)qΛpΛ. Then the equations read JHEP01(2018)156 Xγ(t) = X sf γ (t) exp  1 4πi X γ′ Ωγ′ ⟨γ, γ′⟩ Z ℓγ′ dt′ t′ t + t′ t −t′ log 1 −Xγ′(t′)   , (B.10) where where X sf γ (t) = exp h −2πi  Θγ + τ2 2 Zγ t−1 −¯Zγ t i , (B.11) (B.11) Θγ = qΛζΛ −pΛ˜ζΛ is a combination of RR-fields, Zγ is the central charge (2.29), Ωγ′ is the generalized DT invariant, ⟨γ, γ′⟩is the skew-symmetric product (2.30), and ℓγ is the so-called BPS ray on CP 1 joining t = 0 and t = ∞along the direction determined by the phase of the central charge ℓγ = {t : Zγ(z)/t ∈iR−}. (B.12) (B.12) In the perturbative approximation where the D-instantons are ignored, the Darboux coor- dinates are given by X sf γ , whereas the D-instantons are incorporated by the integral contri- bution in (B.10) weighted by DT invariants. Given a solution of these integral equations, the remaining Darboux coordinate α can be found by simple integration α(t) = 4iclogt−1 2 σ−τ2 4 t−1W−t ¯ W  − i 16π3 X γ Ωγ Z ℓγ dt′ t′ t+t′ t−t′ Lγ(t′)−1 2 ξΛ ˜ξΛ , (B.13) where the parameter c = χY 192π encodes the one-loop gs-correction, Lγ(t) = Li2 (Xγ) + 1 2 log σ−1 γ Xγ  log (1 −Xγ) (B.14) (B.14) is a variant of the Rogers dilogarithm and W = FΛζΛ −zΛ˜ζΛ + 1 8π2 X γ Ωγ Zγ Z ℓγ dt t log (1 −Xγ) . (B.15) (B.15) B.3 Computation of the metric Now we will follow the procedure outlined in section B.1 towards evaluation of the metric corresponding to the twistorial construction of the previous subsection. All equations given below are a direct generalization of the ones which can be found in [25] where the additional – 40 – restriction ⟨γ, γ′⟩= 0 has been imposed. Under this restriction the integral equations (B.10) are trivially solved and the one-instanton approximation to the Darboux coordinates be- comes exact, which simplifies the derivation of the metric. However, as we will show below, this derivation can be done even avoiding the assumption of mutual locality. Before we start, let us introduce a few useful notations: two measures D(1) γ [t] = dt t log (1 −Xγ(t)) , D(2) γ [t] = dt t Xγ(t) 1 −Xγ(t), (B.16) (B.16) JHEP01(2018)156 JHEP01(2018)156 and integrals J (1) γ = Z ℓγ D(1) γ [t], J (2) γ = Z ℓγ D(2) γ [t], J (1,±) γ = ± Z ℓγ t∓1D(1) γ [t], J (2,±) γ = ± Z ℓγ t∓1D(2) γ [t], (B.17) (B.17) which appear in the expansion around t = 0 of the t-dependent integrals in (B.10) and similar equations. Note that they satisfy the reality properties J (n) γ = J (n) −γ , J (n,+) γ = J (n,−) −γ . (B.18) (B.18) Besides, by partial integration one can find the following identity ZγJ (n,+) γ −¯ZγJ (n,−) γ + 1 8π2 X γ′ Ωγ′⟨γ, γ′⟩ Z ℓγ D(n) γ [t] Z ℓγ′ D(1) γ′ [t′] tt′ (t −t′)2 = 0, (B.19) which takes a very simple form for n = 1 and after summing over charges X γ Ωγ  ZγJ (1,+) γ −¯ZγJ (1,−) γ  = 0. (B.20) (B.20) These notations become already useful when one writes the result for the contact potential, the rescaling factor appearing in (B.2) These notations become already useful when one writes the result for the contact potential, the rescaling factor appearing in (B.2) eφ = τ 2 2 16 e−K −c −iτ2 64π2 X γ Ωγ  ZγJ (1,+) γ + ¯ZγJ (1,−) γ  . (B.21) (B.21) Comparing the first term with the first relation in (2.11), one observes that the contact potential provides a generalization of the four-dimensional dilaton to quantum level [24]. This partially explains the important role played by this function both in physics and mathematics [84]. B.3 Computation of the metric As for the dilaton, we will also use for it the notation r = eφ. Comparing the first term with the first relation in (2.11), one observes that the contact potential provides a generalization of the four-dimensional dilaton to quantum level [24]. This partially explains the important role played by this function both in physics and mathematics [84]. As for the dilaton, we will also use for it the notation r = eφ. 1. The first step is to find the expansion of the Darboux coordinates around t = 0. How- ever, the Darboux coordinates defined by (B.10) and (B.13) live in a patch of CP 1 which does not include its north and south poles. This is seen from the presence of ad- ditional poles compared to (B.4) in the perturbative part and an essential singularity – 41 – in the instanton part at t = 0. The additional singularities can be removed by per- forming a holomorphic contact transformation, i.e. a change of Darboux coordinates preserving the contact one-form (B.3). Such contact transformation is given by in the instanton part at t = 0. The additional singularities can be removed by per- forming a holomorphic contact transformation, i.e. a change of Darboux coordinates preserving the contact one-form (B.3). Such contact transformation is given by ξΛ [+] = ξΛ + ∂˜ξΛH[+], ˜ξ[+] Λ = ˜ξΛ −∂ξΛH[+], α[+] = α −H[+] + ξΛ [+]∂ξΛH[+], (B.22) (B.22) where the holomorphic function H[+] was found in [22] to have the following form JHEP01(2018)156 H[+] = F(ξ[+]) + G(ξ[+], ˜ξ). (B.23) (B.23) Here the second term is a complicated, but irrelevant function for us because, as was shown in [22], it affects only O(t2) terms in the Laurent expansion of the Darboux coordinates. Thus, we can safely ignore it for our purposes, and this allows to replace ξΛ [+] on the r.h.s. of (B.22) by ξΛ. B.3 Computation of the metric Furthermore, it turns out to be convenient to add to ˜πα the term −i 2 ξΛ,0 [+] ˜πΛ. As a result, one arrives at the following basis πa = dza in other basis elements. Furthermore, it turns out to be convenient to add to ˜πα the term −i 2 ξΛ,0 [+] ˜πΛ. As a result, one arrives at the following basis dza, YΛ = d˜ζΛ −FΛΣdζΣ − 1 8π2 X γ Ωγ qΛ −pΣFΛΣ  dJ (1) γ , (B.26) Σ = dr + 2c d log τ2 2 + i 4  dσ + ˜ζΛdζΛ −ζΛd˜ζΛ  + i 32π2 X γ Ωγ  J (1,−) γ d τ2 ¯Zγ  −τ2ZγdJ (1,+) γ + 1 8π2 X γ′ Ωγ′⟨γ, γ′⟩J (1) γ dJ (1) γ′  . (B.26) JHEP01(2018)156 3. Substituting the Laurent coefficients (B.24) into (B.6), one obtains the explicit expression for the components of the SU(2) connection: 3. Substituting the Laurent coefficients (B.24) into (B.6), one obtains the explicit expression for the components of the SU(2) connection: p+ = iτ2 8r zΛYΛ = iτ2 8r " zΛ  d˜ζΛ−FΛΣdζΣ −1 8π2 X γ ΩγZγdJ (1) γ # , p3 = 1 8r  dσ+˜ζΛdζΛ−ζΛd˜ζΛ+ iτ 2 2 2 e−KAK −τ2 8π2 X γ Ωγ  J (1,+) γ dZγ−J (1,−) γ d ¯Zγ  + 1 64π4 X γ,γ′ ΩγΩγ′⟨γ,γ′⟩J (1) γ dJ (1) γ′  , (B.27) + 1 64π4 X γ,γ′ ΩγΩγ′⟨γ,γ′⟩J (1) γ dJ (1) γ′  , (B.27) (B.27) where we introduced the K¨ahler connection on the complex structure moduli space AK = i 2 (Kadza −K¯ad¯za) = i 2 eKNΛΣ zΛd¯zΣ −¯zΣdzΛ . (B.28) (B.28) 4. The SU(2) connection allows to find the quaternionic 2-form (B.7): 4. The SU(2) connection allows to find the quaternionic 2-form (B.7): ω3 = 1 4r2 dr∧  dσ+˜ζΛdζΛ−ζΛd˜ζΛ−τ2 8π2 X γ Ωγ  J (1,+) γ dZγ−J (1,−) γ d ¯Zγ  + 1 64π4 X γ,γ′ ΩγΩγ′⟨γ,γ′⟩J (1) γ dJ (1) γ′  + τ 2 2 8r e−Kdlog r τ 2 2 ∧AK + 1 2r  dζΛ∧d˜ζΛ−iτ 2 2 4 NΛΣdzΛ∧d¯zΣ+ iτ 2 2 8r zΛ¯zΣYΛ∧¯YΣ  + 1 32π2r X γ Ωγ  dJ (1,+) γ ∧d(τ2Zγ)−dJ (1,−) γ ∧d(τ2 ¯Zγ)  − 1 256π4r X γ,γ′ ΩγΩγ′⟨γ,γ′⟩dJ (1) γ ∧dJ (1) γ′ . B.3 Computation of the metric Then one finds the following coefficients of the Laurent expansion of the Darboux coordinates: ξΛ,−1 [+] = τ2 2 zΛ, ξΛ,0 [+] = ζΛ − 1 8π2 X γ ΩγpΛJ (1) γ , ˜ξ[+] Λ,0 = ˜ζΛ −FΛΣζΣ − 1 8π2 X γ ΩγVγΛJ (1) γ , ˜ξ[+] Λ,1 = −iτ2 2 ¯zΣNΛΣ −1 τ2 FΛΣΘζΣζΘ − 1 4π2 X γ Ωγ " VγΛJ (1,+) γ −1 τ2 FΛΣΘpΣζΘJ (1) γ + 1 16π2τ2 FΛΣΘpΣJ (1) γ X γ′ Ωγ′p′ΘJ (1) γ′ # , (B.24) α[+] 0 = −1 2  σ + ζΛ˜ζΛ −FΛΣζΛζΣ + 2i (r + c) − 1 8π2 X γ Ωγ " 1 2πi Z ℓγ dt t Li2 (Xγ) −VγΛζΛJ (1) γ −τ2 2 ZγJ (1,+) γ + 1 16π2 X γ′ Ωγ′ pΛJ (1) γ Vγ′ΛJ (1) γ′ −⟨γ, γ′⟩ Z ℓγ D(1) γ [t] Z ℓγ′ D(1) γ′ [t′] t + t′ t −t′ ! , ξΛ,−1 [+] = τ2 2 zΛ, ξΛ,0 [+] = ζΛ − 1 8π2 X γ ΩγpΛJ (1) γ , ˜ξ[+] Λ,0 = ˜ζΛ −FΛΣζΣ − 1 8π2 X γ ΩγVγΛJ (1) γ , [ ] iτ2 1 1 " ˜ξ[+] Λ,1 = −iτ2 2 ¯zΣNΛΣ −1 τ2 FΛΣΘζΣζΘ − 1 4π2 X γ Ωγ " VγΛJ (1,+) γ −1 τ2 FΛΣΘpΣζΘJ (1) γ + 1 16π2τ2 FΛΣΘpΣJ (1) γ X γ′ Ωγ′p′ΘJ (1) γ′ # , (B.24) α0 = −2  σ + ζ ζΛ −FΛΣζ ζ  + 2i (r + c) − 1 8π2 X γ Ωγ " 1 2πi Z ℓγ dt t Li2 (Xγ) −VγΛζΛJ (1) γ −τ2 2 ZγJ (1,+) γ + 1 16 2 X Ωγ′ pΛJ (1) γ Vγ′ΛJ (1) γ′ −⟨γ, γ′⟩ Z D(1) γ [t] Z D(1) γ′ [t′] where we introduced a useful shorthand notation where we introduced a useful shorthand notation VγΛ = qΛ −FΛΣpΣ. (B.25) (B.25) 2. Using these coefficients, it is straightforward to compute the basis of (1,0) forms (B.5). However, it can be further simplified since one can drop all terms proportional to 2. Using these coefficients, it is straightforward to compute the basis of (1,0) forms (B.5). However, it can be further simplified since one can drop all terms proportional to – 42 – – 42 – πa = dza in other basis elements. B.3 Computation of the metric (B.29) (B.29) The last step, which is supposed to bring ω3 into the form (B.8), is technically very com- plicated. Therefore, we relegate it into the next separate subsection. The last step, which is supposed to bring ω3 into the form (B.8), is technically very com- plicated. Therefore, we relegate it into the next separate subsection. – 43 – B.4 The last step The main complication arising due to mutual non-locality is that it is impossible to get Xγ in a closed form. However, what is crucial for the derivation of the metric is not Xγ itself, but its differential. From (B.10) one can derive an integral equation which it satisfies. This equation is simpler than the original equation on Xγ because it is linear, and its solution can be given in terms certain t-dependent matrices on the charge lattice, i.e. matrices acting on the (infinite-dimensional) space of vectors whose components are enumerated by charges. More precisely, we define them as the following infinite series of nested integrals I(0) γγ′(t0) = δγγ′+ ∞ X n=1  i 4π n X γ1,...,γn−1 γ0=γ, γn=γ′ n Y k=1 " Ωγk⟨γk−1,γk⟩ Z ℓγk D(2) γk [tk] tk−1+tk tk−1−tk # , (B.30) I(±) γγ′ (t0) = δγγ′+ ∞ X n=1  i 4π n X γ1,...,γn−1 γ0=γ, γn=γ′ n Y k=1 " Ωγk⟨γk−1,γk⟩ Z ℓγk D(2) γk [tk] tk−1+tk tk−1−tk # t0 tn ±1 . JHEP01(2018)156 They can be checked to satisfy the simple conjugation properties I(0) γγ′(1/¯t) = I(0) −γ,−γ′(t), I(+) γγ′ (1/¯t) = I(−) −γ,−γ′(t). (B.31) (B.31) Applying the differential operator to the integral equation (B.10) and performing iterations, one finds that these matrices encode the differential of the Darboux coordinates: Applying the differential operator to the integral equation (B.10) and performing iterations, one finds that these matrices encode the differential of the Darboux coordinates: −1 2πi d log Xγ(t) = X γ′  I(0) γγ′(t)dΘγ′ + τ2 2  t−1I(+) γγ′ (t)dZγ′ −tI(−) γγ′ (t)d ¯Zγ′  + 1 2  t−1I(+) γγ′ (t)Zγ′ −tI(−) γγ′ (t) ¯Zγ′  dτ2  . (B.32) (B.32) Note that all the matrices (B.30) collapse to δγγ′ under the condition of mutual locality. This implies that most of our general results can be obtained from the equations in [25] by insertion of these matrices in proper places. Due to this reason we will not repeat the cal- culation which is similar to the one done in [25, appenidix B.3] and showing how to arrive at the representation (B.8) for ω3. Instead, we just give the final result which, upon substi- tution of all definitions, can be checked to reproduce the initial expression (B.29). But first we need to introduce several notations which allow to write the result in a readable form. B.4 The last step • First, we define a simple matrix constructed from the vector (B.25) Qγγ′ = VγΛNΛΣ ¯Vγ′Σ + i 2 ⟨γ, γ′⟩ = NΛΣ Re VγΛ Re Vγ′Σ + 1 4 NΛΣpΛp′Σ. (B.33) (B.33) • Next, we introduce an integrated version of the matrices (B.30) vγγ′ = Ωγ 4π Z ℓγ D(2) γ [t] I(0) γγ′(t), v(±n) γγ′ = (±1)n Ωγ 4π Z ℓγ D(2) γ [t] t∓nI(0) γγ′(t), v(±) γγ′ = Ωγ 4π Z ℓγ D(2) γ [t] I(±) γγ′ (t), v(±,n) γγ′ = (±1)n Ωγ 4π Z ℓγ D(2) γ [t] t∓nI(±) γγ′ (t). (B.34) (B.34) – 44 – – 44 – Not all of them are actually independent since it is easy to check that they satisfy the following properties: Not all of them are actually independent since it is easy to check that they satisfy the following properties: – the matrices vγγ′, v(+,2) γγ′ and v(−,2) γγ′ are symmetric; – the matrices vγγ′, v(+,2) γγ′ and v(−,2) γγ′ are symmetric; – the matrices vγγ′, v(+,2) γγ′ and v(−,2) γγ′ are symmetric; – the matrices vγγ′, v(+,2) γγ′ and v(−,2) γγ′ are symmetric; – identities under transposition – identities under transposition v(+) γγ′ = v(−) γ′γ , v(+,1) γγ′ = v(1) γ′γ, v(−,1) γγ′ = v(−1) γ′γ ; (B.35) (B.35) – identity involving the central charge – identity involving the central charge – identity involving the central charge X γ′  v(+,1) γγ′ Zγ′ −v(−,1) γγ′ ¯Zγ′  = X γ′  Zγ′v(1) γ′γ −¯Zγ′v(−1) γ′γ  = 0. (B.36) (B.36) JHEP01(2018)156 • Then we combine the matrices defined above into a new object • Then we combine the matrices defined above into a new object • Then we combine the matrices defined above into a new object Mγγ′ = δγγ′ −2 X γ′′ Qγγ′′vγ′′γ′. (B.37) (B.37) We are really interested in the inverse of this matrix which can always be found by an expansion treating the second term as a perturbation. Note that although Mγγ′ is not symmetric, the matrix product (vM)γγ′ is symmetric. In fact, the product (vM−1)γγ′ is also symmetric which can be shown by expanding M−1 so that We are really interested in the inverse of this matrix which can always be found by an expansion treating the second term as a perturbation. Note that although Mγγ′ is not symmetric, the matrix product (vM)γγ′ is symmetric. B.4 The last step In fact, the product (vM−1)γγ′ is also symmetric which can be shown by expanding M−1 so that (vM−1)γγ′ = vγγ′ + 2(vQv)γγ′ + 4(vQvQv)γγ′ + · · · . (B.38) (B.38) Analogously, (M−1Q)γγ′ is symmetric as well. Using this property, one can also find the following useful identity 2vM−1Q = 2(vM−1)T Q = 2M−T vQ = M−T δ −MT  = M−T −δ. (B.39) (B.39) • It is convenient also to introduce two vectors • It is convenient also to introduce two vectors zγ = X γ′  Zγ′v(1) γ′γ + ¯Zγ′v(−1) γ′γ  , Wγ = 4π X γ′  ¯Zγ′v(+) γ′γ − X ˜γ,˜γ′ z˜γM−1 ˜γ˜γ′Q˜γ′γ′v(+,1) γ′γ   (B.40) (B.40) γ Wγ = 4π X γ′  ¯Zγ′v(+) γ′γ − X ˜γ,˜γ′ z˜γM−1 ˜γ˜γ′Q˜γ′γ′v(+,1) γ′γ   (B.40) and a potential U = e−K −2 X γ,γ′ v(+) γγ′ ¯ZγZγ′ + X γ,γ′ zγ(M−1Q)γγ′zγ′, (B.41) (B.41) which is a real function due to the property (B.35). which is a real function due to the property (B.35). • Besides, we define several 1-forms. The first one is a certain linear combination of the differentials of the RR-fields • Besides, we define several 1-forms. The first one is a certain linear combination of the differentials of the RR-fields • Besides, we define several 1-forms. The first one is a certain linear combination of the differentials of the RR-fields Cγ = NΛΣ qΛ −Re FΛΞpΞ  d˜ζΣ −Re FΣΘdζΘ + 1 4 NΛΣ pΛ dζΣ, (B.42) (B.42) – 45 – – 45 – which is built in the way analogous to Qγγ′ (B.33). The second, which we call V, appears explicitly in the HM metric (2.31) as the quantum part of the connection on the circle bundle parametrized by the NS-axion σ. In terms of Cγ and the other quantities introduced above, it reads V = τ 2 2 2 e−K  1 −16r τ 2 2 U  AK + 16r τ2U X γ,γ′ zγ′M−1 γ′γ  Cγ − i 16π2 X γ′′ Ωγ′′⟨γ, γ′′⟩dJ (1) γ′′   + 2r πiU X γ  Wγ + τ2U 16πir ΩγJ (1,+) γ  dZγ −  ¯ Wγ + τ2U 16πir ΩγJ (1,−) γ  d ¯Zγ  . B.4 The last step (B.43) JHEP01(2018)156 (B.43) Finally, we introduce Finally, we introduce Finally, we introduce Yγ = iNΛΣ ¯VγΛ YΣ = iCγ−1 2 dΘγ−i 8π2 X γ′ Ωγ′  Qγγ′+ i 2 ⟨γ,γ′⟩  dJ (1) γ′ , (B.44) ˆΣ = 2  1−8r τ 2 2 U  dr+ i 4  dσ+˜ζΛdζΛ−ζΛd˜ζΛ+ 1 64π4 X γ,γ′ ΩγΩγ′⟨γ,γ′⟩J (1) γ dJ (1) γ′ +V  . Yγ = iNΛΣ ¯VγΛ YΣ = iCγ−1 2 dΘγ−i 8π2 X γ′ Ωγ′  Qγγ′+ i 2 ⟨γ,γ′⟩  dJ (1) γ′ , (B.44) (B.44) ˆΣ = 2  1−8r τ 2 2 U  dr+ i 4  dσ+˜ζΛdζΛ−ζΛd˜ζΛ+ 1 64π4 X γ,γ′ ΩγΩγ′⟨γ,γ′⟩J (1) γ dJ (1) γ′ +V  . Both these 1-forms are of (1,0) Dolbeault type. Whereas this is evident for Yγ, for ˆΣ this follows from the following representation ˆΣ = Σ + fΛdzΛ + gΛYΛ (B.45) (B.45) with fΛ = τ 2 2 8 −2r U  NΛΣ¯zΣ + 1 π X γ  τ2 16πi ΩγJ (1,+) γ + r U Wγ  VγΛ, gΛ = 4ir τ2U NΛΣ X γ,γ′ zγM−1 γγ′ ¯Vγ′Σ. (B.46) fΛ = τ 2 2 8 −2r U  NΛΣ¯zΣ + 1 π X γ  τ2 16πi ΩγJ (1,+) γ + r U Wγ  VγΛ, 4ir f  8 U  π X γ  16πi γ γ U γ  γ , gΛ = 4ir τ2U NΛΣ X γ,γ′ zγM−1 γγ′ ¯Vγ′Σ. (B.46) (B.46) In terms of all these notations, one can show that the quaternionic 2-form (B.29) can be rewritten as In terms of all these notations, one can show that the quaternionic 2-form (B.29) can be rewritten as ω3 = i ˆΣ∧¯ˆΣ 4r2  1−8r τ 2 2 U  −i 2r  N ΛΣ−τ 2 2 8r zΛ¯zΣ  YΛ∧¯YΣ−i r X γ,γ′ (vM−1)γγ′Yγ∧¯Yγ′ + i 2rU X γ  (zM−1)γYγ+ τ2 4π WγdZγ  ∧ X γ′  (zM−1)γ′ ¯Yγ′ + τ2 4π ¯ Wγ′d ¯Zγ′  + iτ2 2r X γ,γ′,γ′′ M−1 γγ′ h v(+,1) γγ′′ dZγ′′ −U−1Zγ′′∂e−K ∧¯Yγ′ +Yγ′ ∧v(−,1) γγ′′ d ¯Zγ′′ −U−1 ¯Zγ′′ ¯∂e−Ki + iτ 2 2 8r " U−1∂e−K∧¯∂e−K−NΛΣdzΛ∧d¯zΣ− 1 2πU X γ  WγdZγ∧¯∂e−K+∂e−K∧¯ Wγd ¯Zγ # + iτ 2 2 4r X γ,γ′ v(+) γγ′ dZγ′ ∧d ¯Zγ−iτ 2 2 2r X γ,γ′ (M−1Q)γγ′ X ˜γ v(+,1) γ˜γ dZ˜γ∧ X γ′′ v(−,1) γ′γ′′ d ¯Zγ′′. B.4 The last step (B.47) – 46 – All terms appearing in this representation are explicitly of (1,1) Dolbeault type. Therefore, one can apply the rule (B.8) which immediately produces the metric (2.31) given in the main text. All terms appearing in this representation are explicitly of (1,1) Dolbeault type. Therefore, one can apply the rule (B.8) which immediately produces the metric (2.31) given in the main text. C Metric on M′ H In this appendix we derive the rigid limit of the D-instanton corrected HM metric (2.31) and uncover its geometric structure. As discussed in section 2.3.1, in this limit some of worldsheet and D-instantons actually decouple and therefore we should restrict our attention only to the charges γ belonging to the lattice Γrig = {γ = (0, pI, qˆI, q0)}. This means that in all sums over charges appearing in (2.31) the condition γ ∈Γrig should be inserted. JHEP01(2018)156 C.1 Scaling behavior Let us first find the scaling behavior of various quantities entering the metric. Noticing that the quantum part of the prepotential (2.26) remains finite, for the real and imaginary parts of its second derivative one obtains Re FΛΣ ∼    Λ2 Λ2 1 Λ2 1 1 1 1 1   , NΛΣ ∼    Λ3 Λ 1 Λ Λ 1 1 1 1   , (C.1) (C.1) where the rows and columns correspond to the splitting of the index Λ = (0, ˆA, I). To get the scaling of the inverse matrix NΛΣ, one can split NΛΣ into its classical part Ncl given in (A.3) and the part Nq encoding the quantum corrections. After that the expansion N−1 = (Ncl)−1 −(Ncl)−1Nq(Ncl)−1 + · · · (C.2) (C.2) together with the explicit expression for the (Ncl)−1 (A.5) and the scaling (2.18), result in NΛΣ ∼    Λ−3 Λ−3 Λ−3 Λ−3 Λ−1 Λ−1 Λ−3 Λ−1 1   . (C.3) (C.3) Similarly, for the gauge coupling matrix one obtains Re NΛΣ ∼1, Im NΛΣ ∼    Λ3 Λ 1 Λ Λ 1 1 1 1   , Im N ΛΣ ∼    Λ−3 Λ−3 Λ−3 Λ−3 Λ−1 Λ−1 Λ−3 Λ−1 1   . (C.4) (C.4) In particular, the scaling of the inverse matrices implies that NIJ = −1 2 gIJ + O(Λ−1), IIJ = −gIJ + O(Λ−1), (C.5) (C.5) where gIJ is the inverse of gIJ = −1 2 NIJ. where gIJ is the inverse of gIJ = −1 2 NIJ. where gIJ is the inverse of gIJ = −1 2 NIJ. – 47 – – 47 – Combining the condition γ ∈Γrig with these results, it is easy to see that the central charge Zγ remains finite and unaffected by the limit so as the vectors VγΛ, zγ and the matrices vγγ′, v(±) γγ′ , v(±,n) γγ′ , Mγγ′. The matrix Qγγ′ also remains finite, but simplifies because some components of the matrix NΛΣ vanish. Qγγ′ = 1 4 NIJpIp′J + NIJ(qI −Re FIKpL)(q′ J −Re FJLp′L) + O(Λ−1). (C.6) (C.6) The four-dimensional dilaton (coinciding with the contact potential) r and the potential U (B.41) have the leading contributions scaling as Λ3. The four-dimensional dilaton (coinciding with the contact potential) r and the potential U (B.41) have the leading contributions scaling as Λ3. C.1 Scaling behavior An important role is played by the one-form YΛ (B.26). However, its components have too different scaling and, instead of working with them, it turns out to be more convenient to introduce two real one-forms, yΛ and wΛ, defined by JHEP01(2018)156 YΛ = yΛ −FΛΣwΣ. (C.7) (C.7) Their explicit expressions are Their explicit expressions are yΛ = d˜ζΛ − i 4π2 X γ∈Γrig Ωγ 1 4 NΛΣpΣ −Re FΛΛ′NΛ′Σ Re VγΣ  dJ (1) γ , wΛ = dζΛ + i 4π2 X γ∈Γrig ΩγNΛΣ Re VγΣdJ (1) γ , (C.8) (C.8) and it is easy to check that in the limit they both remain finite except one contribution in y0 which scales as Λ. It turns out that the one-forms Cγ (B.42) and Yγ (B.44) also have a divergent piece which is determined by the same quantity. Thus, the three one-forms can be written as and it is easy to check that in the limit they both remain finite except one contribution in y0 which scales as Λ. It turns out that the one-forms Cγ (B.42) and Yγ (B.44) also have a divergent piece which is determined by the same quantity. Thus, the three one-forms can be written as y0 = −i 4π2 X γ∈Γrig ΩγCγdJ (1) γ +y(0) 0 , Cγ = Cγdζ0 +C(0) γ , Yγ = iCγw0 +Y(0) γ , (C.9) where Cγ = −Re F0 ˆ AN ˆ AΛ qΛ −Re FΛΣpΣ (C.10) (C.10) diverges,15 whereas y(0) 0 , C(0) γ and Y(0) γ are all finite. diverges,15 whereas y(0) 0 , C(0) γ and Y(0) γ are all finite. diverges,15 whereas y(0) 0 , C(0) γ and Y(0) γ are all finite. 16Again the factor √τ2 is included to preserve the modular symmetry. C.2 Evaluation of the limit Before we apply the scaling results from the previous subsection to the metric (2.31), it is convenient to rewrite the second term on the first line using (C.7). Then it becomes −1 r  NΛΣ−τ 2 2 8r zΛ¯zΣ  YΛ ¯YΣ = −1 2r ImN ΛΣ  yΛ−ReNΛΛ′wΛ′ yΛ−ReNΛΛ′wΛ′ −1 2r ImNΛΣwΛwΣ+ τ 2 2 8r2  1−16r τ 2 2 eK  zΛYΛ 2 . (C.11) 15Note that not all terms in Cγ scale as Λ. Some of them stay finite or even decay, but we find convenient to combine all of them into one expression. What is important is the behavior of the leading contribution. 15Note that not all terms in Cγ scale as Λ. Some of them stay finite or even decay, but we find convenient to combine all of them into one expression. What is important is the behavior of the leading contribution. – 48 – Note that the coefficient of the last term is given by 1 −16r τ 2 2 eK = eK τ 2 2  χY 12π + iτ2 4π2 X γ∈Γrig Ωγ  ZγJ (1,+) γ + ¯ZγJ (1,−) γ    (C.12) (C.12) and scales as Λ−3. The Lagrangian based on the metric (2.31) can be represented as in (2.20)16 The Lagrangian based on the metric (2.31) can be represented as in (2.20) Lbos = − √−g√τ2 4κ2r (L+ + L0 + L−) , (C.13) (C.13) JHEP01(2018)156 where the three terms in the brackets correspond to divergent, finite and vanishing contri- butions, respectively. Taking the gravitation coupling κ2 to scale as Λ−3, one ensures that the prefactor is constant. Then the contribution L−drops out and we will not specify its form since it is completely irrelevant. On the other hand, the divergent part L+ imposes strongly classical equations of motion which lead to the freezing of some fields. To compute its effect, let us first denote A = −I00 −4 X γ,γ′∈Γrig (vM−1)γγ′CγCγ′, (C.14) B = I0 ˆ Aw ˆ A + iτ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ dZγ′′ −v(−,1) γγ′′ d ¯Zγ′′ i Cγ′ +4 X γ,γ′∈Γrig (vM−1)γγ′Cγ Im Y(0) γ′ (C.14) and redefine w0 as and redefine w0 as ˆw0 = w0 −A−1B. C.2 Evaluation of the limit (C.18) √τ2ds2 M′ H = τ 2 2 2 gIJdzId¯zJ + 1 2 gIJw′Iw′J + 1 2 gIJ y′ I −ReFIKw′Ky′ J −ReFJLw′L +τ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ d′Zγ′′Y′ γ′ +v(−,1) γγ′′ d′ ¯Zγ′′Y′ γ′ i −2 X γ,γ′∈Γrig (vM −1)γγ′Y′ γ ¯Y′ γ′ τ 2 2 X (+) ′ ′ ¯ 2 X (M 1Q) X (+ 1) ′ X (−1) ′ ¯ + τ 2 2 2 X γ,γ′∈Γrig v(+) γγ′ d′Zγ′d′ ¯Zγ−τ 2 2 X γ,γ′∈Γrig (M−1Q)γγ′ X ˜γ∈Γrig v(+,1) γ˜γ d′Z˜γ X ˜γ′∈Γrig v(−,1) γ′˜γ′ d′ ¯Z˜γ′. (C 18) JHEP01(2018)156 Here the differential d′ acts only on the fields zI, ζI and ˜ζI, and we defined y′ I = d˜ζI + i 8π2 X γ∈Γrig Ωγ gIJpJ −Re FIJ gJK qK −Re FKLpL d′J (1) γ , γ∈Γrig w′I = dζI − i 8π2 X γ∈Γrig ΩγgIJ qJ −Re FJKpK d′J (1) γ , Y′ γ = −i 2 gIJ(qI −FIKpK)Y′ J , (C.19) w′I = dζI − i 8π2 X γ∈Γrig ΩγgIJ qJ −Re FJKpK d′J (1) γ , (C.19) (C.19) Y′ γ = −i 2 gIJ(qI −FIKpK)Y′ J , Y′ γ = −i 2 gIJ(qI −FIKpK)Y′ J , Y′ γ = −i 2 gIJ(qI −FIKpK)Y′ J , where, similarly to (C.7), we have Y′ I = y′ I −FIJw′J = d˜ζI −FIJdζJ − 1 8π2 X γ∈Γrig Ωγ(qI −FIJpJ)d′J (1) γ . (C.20) (C.20) Then, using the identity gIJw′Iw′J + gIJ(y′ I −Re FIKw′K)(y′ J −Re FJLw′L) = gIJY′ I ¯Y′ J , (C.21) gIJw′Iw′J + gIJ(y′ I −Re FIKw′K)(y′ J −Re FJLw′L) = gIJY′ I ¯Y′ J , (C.21) (C.21) which coincides with the finite part of (C.11), one can rewrite the metric (C.18) precisely as in (2.32). C.2 Evaluation of the limit (C.15) (C.15) One can show that in terms of these notations, L+ can be represented as √τ2L+ = 2r  1−8r τ 2 2 U  (∂µ logr)2+ A 2 ( ˆw0 µ)2−1 2 I ˆ A ˆBw ˆ A µ w ˆBµ−τ 2 2 4 N ˆ A ˆB∂µz ˆ A∂µ¯z ˆB (C.16) and leads to very simple equations of motion and leads to very simple equations of motion ∂µ log r = 2∂µ log τ2 + O(Λ−1) = O(Λ−3), ˆw0 µ = ∂µζ0 + O(Λ−2) = O(Λ−3), w ˆ A µ = ∂µζ ˆ A + O(Λ−1) = O(Λ−1). ∂µz ˆ A = O(Λ−1). (C.17) (C.17) The non-vanishing r.h.s. of these equations correspond to the omitted contributions coming from L0 + L−, and the power of Λ is determined by the growth rate of the coefficients in (C.16), which follows from the results of the previous subsection and that A ∼Λ3, B ∼Λ. Thus, in the leading approximation the fields τ2, ζ0 = τ1, ζ ˆ A and z ˆ A become frozen and have vanishing variations. Furthermore, substituting (C.17) back into L+ (C.16), one finds that it behaves as O(Λ−1) and thus does not contribute to L0. – 49 – Finally, let us turn to the finite part of the Lagrangian. Imposing the equations (C.17) and taking into account that Re NIJ = Re FIJ + O(Λ−1), the metric corresponding to L0 reduces to √τ2ds2 M′ H = τ 2 2 2 gIJdzId¯zJ + 1 2 gIJw′Iw′J + 1 2 gIJ y′ I −ReFIKw′Ky′ J −ReFJLw′L +τ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ d′Zγ′′Y′ γ′ +v(−,1) γγ′′ d′ ¯Zγ′′Y′ γ′ i −2 X γ,γ′∈Γrig (vM −1)γγ′Y′ γ ¯Y′ γ′ + τ 2 2 2 X γ,γ′∈Γrig v(+) γγ′ d′Zγ′d′ ¯Zγ−τ 2 2 X γ,γ′∈Γrig (M−1Q)γγ′ X ˜γ∈Γrig v(+,1) γ˜γ d′Z˜γ X ˜γ′∈Γrig v(−,1) γ′˜γ′ d′ ¯Z˜γ′. C.3 HK structure Now we want to prove that the limiting space M′ H is an HK manifold. For this purpose, it is enough to show that it carries a holomorphic symplectic structure, which in turn can be achieved by constructing a globally defined holomorphic symplectic form on the trivial CP 1 bundle over M′ H, which gets interpretation of the twistor space. Such symplectic form has a representation Ω= it−1ω′ + + ω′ 3 + it ω′ −, (C.22) (C.22) where t is the stereographic coordinate on CP 1. Then the metric (2.32) must be such that ω′ 3 is the K¨ahler form in the complex structure in which ω′ + is holomorphic. where t is the stereographic coordinate on CP 1. Then the metric (2.32) must be such that ω′ 3 is the K¨ahler form in the complex structure in which ω′ + is holomorphic. To find such Ω, note that locally it can always be trivialized by the choice of Darboux coordinates Ω= dηI [i] ∧dµ[i] I , (C.23) (C.23) – 50 – – 50 – where, as usual, the index [i] labels open patches of an atlas on the twistor space. Thus, what we need is to specify a consistent set of Darboux coordinates. We claim that one recovers the metric (2.32) if one identifies these Darboux coordinates away form the poles of CP 1 with the corresponding Darboux coordinates on the twistor space of the initial QK manifold MH, i.e. one takes ηI(t) = ξI(t), µI(t) = ˜ξI(t), (C.24) (C.24) whereas the Darboux coordinates around t = 0 are obtained by applying a holomorphic symplectic transformation (cf. (B.22)) with the generating function given by JHEP01(2018)156 H[+] = τ 2 2 4t2 f 2t τ2 η[+]  + G(η[+], µ), (C.25) (C.25) where the prepotential f(zI) is defined in (2.33). This identification is possible because, under the restriction γ ∈Γrig, the integral equations (B.10) fixing the Darboux coordinates on ZM become a closed system for ξI and ˜ξI. They also involve ξ0 and ξX, but these Darboux coordinates are fixed in terms of the frozen fields and do not receive any quantum corrections (this happens because the components p0 and pX of the magnetic charge are taken to vanish) ξ0 = τ1 + τ2 2 t−1 −t  , ξX = ζX + τ2 2 zXt−1 −¯zXt  . C.3 HK structure (C.26) (C.26) Besides, it is important to note that FI(z) = fI(z) due to the condition on the inter- section numbers (2.8) and the restriction on the charges of worldsheet instantons. This makes it possible to replace (B.10) by X ′ γ(t) = exp " −2πi  Θ′ γ + τ2 2 Z′ γ t−1 −¯Z′ γ t  + 1 4πi X γ′∈Γrig Ωγ′ ⟨γ, γ′⟩ Z ℓγ′ dt′ t′ t + t′ t −t′ log 1 −X ′ γ′(t′)  # , (C.27) X ′ γ(t) = exp " −2πi  Θ′ γ + τ2 2 Z′ γ t−1 −¯Z′ γ t  ( ) (C.27) " + 1 4πi X γ′∈Γrig Ωγ′ ⟨γ, γ′⟩ Z ℓγ′ dt′ t′ t + t′ t −t′ log 1 −X ′ γ′(t′)  # , (C.27) where where X ′ γ = σγ e−2πi(q0ξ0+qXξX+qIηI−pIµI), Θ′ γ = q0τ1 + qXζX + qIζI −pI ˜ζI, Z′ γ = q0 + qXzX + qIzI −pIfI(z). (C.28) (C.28) The resulting system of integral equations coincides with the equations for Darboux coordi- nates on the twistor space of the HK moduli space of a 4d N = 2 gauge theory compactified on a circle [46], which has flavor charges q0 and qX and is characterized by the holomorphic prepotential f(zI). Let us finally show that this twistorial construction indeed leads to the metric (2.32). To this end, we first perform the symplectic transformation generated by (C.25). Although it appears to be similar to the canonical transformation generated by (B.23) which we – 51 – encountered in the computation of the D-instanton corrected HM metric, they are not identical because the prepotential f is not homogeneous in contrast to F. Keeping this difference in mind, computing the expansion coefficients of ηI [+] and µ[+] I around t = 0 and substituting them into encountered in the computation of the D-instanton corrected HM metric, they are not identical because the prepotential f is not homogeneous in contrast to F. C.3 HK structure Keeping this difference in mind, computing the expansion coefficients of ηI [+] and µ[+] I around t = 0 and substituting them into ω′ + = −idηI,0 [+] ∧dµ[+] I,0, ω′ 3 = dηI,0 [+] ∧dµ[+] I,0 + dηI,−1 [+] ∧dµ[+] I,1, (C.29) (C.29) JHEP01(2018)156 which follows from a combination of (C.22) and (C.23), one finds that the basis of (1,0) forms encoding the complex structure J′ 3 consists of dzI and Y′ I (C.20), whereas the K¨ahler form ω′ 3 is given by which follows from a combination of (C.22) and (C.23), one finds that the basis of (1,0) forms encoding the complex structure J′ 3 consists of dzI and Y′ I (C.20), whereas the K¨ahler form ω′ 3 is given by ω′ 3 = dζI ∧d˜ζI + iτ 2 2 2 gIJdzI ∧d¯zJ − 1 128π4 X γ,γ′∈Γrig ΩγΩγ′⟨γ, γ′⟩d′J (1) γ ∧d′J (1) γ′ − 1 8π2 X γ∈Γrig Ωγ h (qIdζI −pId˜ζI) ∧d′J (1) γ + τ2d′Zγ ∧d′J (1,+) γ i . (C.30) (C.30) Next we observe that the last term in (C.30) can be rewritten as Next we observe that the last term in (C.30) can be rewritten as τ2 16π2 X γ∈Γrig Ωγ  d′J (1,+) γ ∧d′Zγ −d′J (1,−) γ ∧d′ ¯Zγ  = iτ2 2 X γ,γ′∈Γrig  d′Θγ ∧  v(+,1) γγ′ d′Zγ′ −v(−,1) γγ′ d′ ¯Zγ′  + τ2v(+) γγ′ d′Zγ′ ∧d′ ¯Zγ  . (C.31) γ∈Γrig = iτ2 2 X γ,γ′∈Γrig  d′Θγ ∧  v(+,1) γγ′ d′Zγ′ −v(−,1) γγ′ d′ ¯Zγ′  + τ2v(+) γγ′ d′Zγ′ ∧d′ ¯Zγ  . (C.31) (C.31) Besides, one has Besides, one has Besides, one has Besides, one has dζI ∧d˜ζI − 1 128π4 X γ,γ′∈Γrig ΩγΩγ′⟨γ,γ′⟩d′J (1) γ ∧d′J (1) γ′ = i 2 gIJY′ I ∧¯Y′ J −2i X γ,γ′∈Γrig (vM−1)γγ′Y′ γ∧¯Y′ γ′ (C.32) −τ2 X γ,γ′∈Γrig M−1 γγ′  C′ γ− i 16π2 X ˜γ∈Γrig Ω˜γ⟨γ,˜γ⟩d′J (1) ˜γ  ∧ X ˜γ′∈Γrig  v(+,1) γ′˜γ′ d′Z˜γ′+v(−,1) γ′˜γ′ d′ ¯Z˜γ′  , ∧¯Y′ J −2i X γ,γ′∈Γrig (vM−1)γγ′Y′ γ∧¯Y′ γ′ (C.32) (C.32) 2 X γ,γ′∈Γrig −τ2 X γ,γ′∈Γrig M−1 γγ′  C′ γ− i 16π2 X ˜γ∈Γrig Ω˜γ⟨γ,˜γ⟩d′J (1) ˜γ  ∧ X ˜γ′∈Γrig  v(+,1) γ′˜γ′ d′Z˜γ′+v(−,1) γ′˜γ′ d′ ¯Z˜γ′  , where where C′ γ = −2gIJ qI −Re FIKpK  d˜ζJ −Re FJLdζL −1 2 gIJ pI dζJ. C.3 HK structure (C.33) C′ γ = −2gIJ qI −Re FIKpK  d˜ζJ −Re FJLdζL −1 2 gIJ pI dζJ. (C.33) (C.33) – 52 – Finally, we need also the following identity Finally, we need also the following identity Finally, we need also the following identity i X γ,γ′,γ′′∈Γrig M−1 γγ′  v(+,1) γγ′′ d′Zγ′′ ∧¯Y′ γ′ −v(−,1) γγ′′ d′ ¯Zγ′′ ∧Y′ γ′  = − X γ,γ′,˜γ′∈Γrig M−1 γγ′  C′ γ − i 16π2 X ˜γ∈Γrig Ω˜γ⟨γ, ˜γ′⟩d′J (1) ˜γ  ∧  v(+,1) γ′˜γ′ d′Z˜γ′ + v(−,1) γ′˜γ′ d′ ¯Z˜γ′  −i 2 X γ,γ′∈Γrig  v(+,1) γγ′ d′Zγ′ −v(−,1) γγ′ d′ ¯Zγ′  ∧ " d′Θγ −i 2 X γ,γ′∈Γrig  v(+,1) γγ′ d′Zγ′ −v(−,1) γγ′ d′ ¯Zγ′  ∧ " d′Θγ (C.34) (C.34) JHEP01(2018)156 JHEP01(2018)156 −τ2 X γ′′∈Γrig (M−1Q)γγ′′  X ˜γ∈Γrig  v(+,1) γ′′˜γ d′Z˜γ + v(−,1) γ′′˜γ d′ ¯Z˜γ    # , which allows express terms with d′Θγ and C′ γ in terms of (1,0)-forms and their conjugate. Collecting all these relations together and suing them in (C.30), one arrives at the following expression for the K¨ahler form which allows express terms with d′Θγ and C′ γ in terms of (1,0)-forms and their conjugate. Collecting all these relations together and suing them in (C.30), one arrives at the following expression for the K¨ahler form ω′ 3 = iτ 2 2 2 gIJdzI ∧d¯zJ + i 2 gIJY′ I ∧¯Y′ J −2i X γ,γ′∈Γrig (vM −1)γγ′Y′ γ∧¯Y′ γ′ +iτ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ d′Zγ′′ ∧¯Y′ γ′ +Y′ γ′ ∧v(−,1) γγ′′ d′ ¯Zγ′′ i (C.35) −iτ 2 2 X γ,γ′∈Γrig (M−1Q)γγ′ X ˜γ∈Γrig v(+,1) γ˜γ d′Z˜γ∧ X ˜γ′∈Γrig v(−,1) γ′˜γ′ d′ ¯Z˜γ′ + iτ 2 2 2 X γ,γ′∈Γrig v(+) γγ′ d′Zγ′ ∧d′ ¯Zγ. ω′ 3 = iτ 2 2 2 gIJdzI ∧d¯zJ + i 2 gIJY′ I ∧¯Y′ J −2i X γ,γ′∈Γrig (vM −1)γγ′Y′ γ∧¯Y′ γ′ +iτ2 X γ,γ′,γ′′∈Γrig M−1 γγ′ h v(+,1) γγ′′ d′Zγ′′ ∧¯Y′ γ′ +Y′ γ′ ∧v(−,1) γγ′′ d′ ¯Zγ′′ i (C.35) (C.35) −iτ 2 2 X γ,γ′∈Γrig (M−1Q)γγ′ X ˜γ∈Γrig v(+,1) γ˜γ d′Z˜γ∧ X ˜γ′∈Γrig v(−,1) γ′˜γ′ d′ ¯Z˜γ′ + iτ 2 2 2 X γ,γ′∈Γrig v(+) γγ′ d′Zγ′ ∧d′ ¯Zγ. It precisely corresponds to the metric (2.32). precisely corresponds to the metric (2.32). It precisely corresponds to the metric (2.32). It precisely corresponds to the metric (2.32). D Torus reduction of 5d gauge theory In this appendix we perform compactification of the five-dimensional action (3.2) on the torus. The spacetime metric gˆµˆν, whose signature in our conventions is (−, +, +, +, +), is taken to be as in (3.11) where coordinates x3 and x4 parametrize the torus directions. The periodicity of holonomies under large gauge transformations is set to be 2π, which makes natural to define the variables ϑI 1 = 1 2π I S1 3 AI 3 dx3, ϑI 2 = 1 2π I S1 4 AI 4 dx4 (D.1) (D.1) with period 1. Introducing ϑI τ ≡ϑI 1 −τϑI 2 (D.2) (D.2) and assuming the independence of all fields on the torus coordinates, one finds that the kinetic term for vectors and the Chern-Simons term give, respectively, Z T 2 dx3dx4√̺ FIJF I ˆµˆνF J ˆµˆν = VFIJF I µνF Jµν + 8π2 τ2 FIJ∂µϑI τ∂µ ¯ϑJ τ , (D.3) Z T 2 dx3dx4FIJKǫˆµˆνˆλˆρˆσAI ˆµF J ˆνˆλF K ˆρˆσ = 16π2FIJKǫµνλ F I µν ϑJ 2 ∂λϑK 1 −ϑJ 1 ∂λϑK 2  −2AI µ∂νϑJ 1 ∂λϑK 2  . (D.3) – 53 – – 53 – Note that the CS term does not produce the factor of volume because the integrand does not contain the factor √̺. As a result, integrating by parts and using the Bianchi identity ǫµνλ∂µF I νλ ≡0, the reduced action can be brought to the following form Note that the CS term does not produce the factor of volume because the integrand does not contain the factor √̺. As a result, integrating by parts and using the Bianchi identity ǫµνλ∂µF I νλ ≡0, the reduced action can be brought to the following form S5→3d bos = − Z d3x  FIJ  V 8π F I µνF Jµν + π τ2 ∂µϑI τ∂µ ¯ϑJ τ + V 4π ∂µϕI ∂µϕJ  + π 2 FIJKǫµνλF I µν ϑJ 2 ∂λϑK 1 −ϑJ 1 ∂λϑK 2  . (D.4) (D.4) The action (D.4) contains the vector fields AI µ and therefore describes a coupling of three-dimensional tensor multiplets. It can be turned into a nonlinear σ-model for hypermultiplets by dualizing the vector fields into scalars. 17One should remember that due to our choice of signature the contraction of two Levi-Civita symbols gives ǫµρσǫνρσ = −2δµ ν . D Torus reduction of 5d gauge theory This is done by adding to the action the term Z JHEP01(2018)156 ∆S = π Z d3x λI ǫµνλ∂µF I νλ (D.5) (D.5) such that the variation with respect to the Lagrange multipliers λI induces equations of motion which are simply the Bianchi identity. Integrating this term by parts and varying the total action S5→3d bos + ∆S with respect to F I µν, one finds instead F I µν = −4π2 V FIJǫµνλ  ∂λλJ + 1 2 FJKL ϑK 2 ∂λϑL 1 −ϑK 1 ∂λϑL 2  , (D.6) (D.6) where FIJ is the inverse of FIJ. Substituting this field strength back into the action, one obtains17 a σ-model for complex fields ϑI τ and real fields ϕI and λI: S3d bos = − Z d3x  FIJ  π τ2 ∂µϑI τ∂µ ¯ϑJ τ + V 4π ∂µϕI ∂µϕJ  (D.7) +4π3 V FIJ  ∂µλI + 1 2 FIKL ϑK 2 ∂µϑL 1 −ϑK 1 ∂µϑL 2  ∂µλJ + 1 2 FJMN ϑM 2 ∂µϑN 1 −ϑM 1 ∂µϑN 2  . (D.7) E Toric data Let Y be a smooth compact threefold described by a homogeneous polynomial equation in some coordinate patch of a desingularized ambient four-dimensional toric Fano variety e A. The relevant topological information about Y can be encoded by two sets of data. First, one should specify a reflexive polytope ∆with vertices belonging to a lattice M ≃Z4. We will denote the dual polytope, defined within the dual lattice N ≃Hom(M, Z), by ∆∗. Both of them can be represented by matrices with 4 columns whose rows correspond to points in M (resp. N) defining their vertices. A crucial property of ∆∗is that it contains a single interior point, which is the origin of N, while all other lattice points contained in ∆∗lie on its boundary (including the vertices). Moreover, the boundary lattice points have an important geometric interpretation corresponding to the toric divisors Di of the ambient space. Due to this, it will be convenient to introduce a matrix ¯∆∗whose rows describe all such boundary points. – 54 – However, these polytopes define in general a singular variety. The desingularization of the ambient space is encoded by the second data, a simplicial triangulation of ∆∗by unit-volume simplices with a vertex at the origin, or more precisely a fine, star, regular triangulation. It is specified by describing each four-simplex by a set of 4 toric divisors corresponding to its edges. Thus, it can be encoded by a matrix also with 4 columns and the number of rows equal to the number of simplices used in the triangulation. The entries of the matrix specify rows of ∆∗. As a side remark, let us recall that different triangulations of ∆∗may (or may not) cor- respond to different Calabi-Yau geometries. When more than one triangulation describes the same Calabi-Yau, these represent different chambers of the K¨ahler cone of Y, also known as phases of the geometry. JHEP01(2018)156 Below we specify the toric data for the two Calabi-Yau manifolds used in section 4. We call them by their number in the database [75]. Polytope 337 (geometry 1). E Toric data The polytopes corresponding to this example are ∆=              1 0 0 0 1 2 0 0 0 0 1 0 0 3 1 0 0 0 0 1 0 2 0 1 −1 1 2 −1 3 −2 −2 −2 −2 −2 −2 3 −3 −3 −2 3 3 −3 −2 −3              , ¯∆∗=          −1 0 −1 −1 −1 0 −1 0 −1 0 0 −1 −1 1 −1 −1 0 0 −1 −1 0 1 −1 0 1 −1 2 1 2 −1 2 2          . (E.1) (E.1) They define a singular variety. We are interested in the Calabi-Yau manifold defined by the desingularization corresponding to geometry 1 in the list of [75]. E Toric data Such desingularization admits three phases, described by the following three triangulations T1 =                     0 1 2 3 0 1 2 6 0 1 3 4 0 1 4 6 0 2 3 4 0 2 4 6 1 2 3 6 1 3 4 5 1 3 5 6 1 4 5 7 1 4 6 7 1 5 6 7 2 3 4 6 3 4 5 6 4 5 6 7                     , T2 =                      0 1 2 3 0 1 2 6 0 1 3 5 0 1 4 5 0 1 4 6 0 2 3 4 0 2 4 6 0 3 4 5 1 2 3 6 1 3 5 6 1 4 5 7 1 4 6 7 1 5 6 7 2 3 4 6 3 4 5 6 4 5 6 7                      , T3 =                        0 1 2 3 0 1 2 6 0 1 3 5 0 1 4 5 0 1 4 7 0 1 6 7 0 2 3 4 0 2 4 6 0 3 4 5 0 4 6 7 1 2 3 6 1 3 5 6 1 4 5 7 1 5 6 7 2 3 4 6 3 4 5 6 4 5 6 7                        . E Toric data (E.2) T1 =                     0 1 2 3 0 1 2 6 0 1 3 4 0 1 4 6 0 2 3 4 0 2 4 6 1 2 3 6 1 3 4 5 1 3 5 6 1 4 5 7 1 4 6 7 1 5 6 7 2 3 4 6 3 4 5 6 4 5 6 7                     , T2 =                      0 1 2 3 0 1 2 6 0 1 3 5 0 1 4 5 0 1 4 6 0 2 3 4 0 2 4 6 0 3 4 5 1 2 3 6 1 3 5 6 1 4 5 7 1 4 6 7 1 5 6 7 2 3 4 6 3 4 5 6 4 5 6 7                      , T3 =                        0 1 2 3 0 1 2 6 0 1 3 5 0 1 4 5 0 1 4 7 0 1 6 7 0 2 3 4 0 2 4 6 0 3 4 5 0 4 6 7 1 2 3 6 1 3 5 6 1 4 5 7 1 5 6 7 2 3 4 6 3 4 5 6 4 5 6 7                        . (E.2) (E.2) We recall that the i-th row of a triangulation T has components Tij = (nj)j=1...4, encoding the 4-simplex delimited by the four toric divisors {Dnj+1}j=1...4 where Dn is the divisor specified by the n-th row of ¯∆⋆. E Toric data We recall that the i-th row of a triangulation T has components Tij = (nj)j=1...4, encoding the 4-simplex delimited by the four toric divisors {Dnj+1}j=1...4 where Dn is the divisor specified by the n-th row of ¯∆⋆. – 55 – – 55 – Polytope 1439 (geometry 2). In this example the two polytopes are encoded by the matrices   Polytope 1439 (geometry 2). In this example the two polytopes are encoded by the matrices   ∆=       1 0 0 0 0 1 0 0 2 3 4 0 2 3 0 4 −6 −5 4 −8 −6 −5 −8 4       , ¯∆∗=          −1 −1 1 1 −1 −1 1 2 −1 −1 2 1 −1 −1 3 3 −1 3 −2 −2 1 −1 0 0 −1 −1 2 2 0 1 −1 −1          . (E.3) (E.3) They again define a singular manifold. This time we take the desingularization corre- sponding to geometry 2 in the classification of [75], which is described by the following triangulation matrix   They again define a singular manifold. This time we take the desingularization corre- sponding to geometry 2 in the classification of [75], which is described by the following triangulation matrix   JHEP01(2018)156 T =                      0 1 4 6 0 1 4 7 0 1 5 6 0 1 5 7 0 2 4 6 0 2 4 7 0 2 5 6 0 2 5 7 1 3 4 6 1 3 4 7 1 3 5 6 1 3 5 7 2 3 4 6 2 3 4 7 2 3 5 6 2 3 5 7                      . (E.4) (E.4) Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] S. Kachru, A. Klemm, W. Lerche, P. Mayr and C. Vafa, Nonperturbative results on the point particle limit of N = 2 heterotic string compactifications, Nucl. Phys. B 459 (1996) 537 [hep-th/9508155] [INSPIRE]. [2] A. Klemm, W. Lerche, P. Mayr, C. Vafa and N.P. Warner, Selfdual strings and N = 2 supersymmetric field theory, Nucl. Phys. 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How ozone boosts biofilter performance
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How ozone boosts biofilter performance Marvin Yeung  Prakit Saingam  Yang Xu  Jinying Xi Marvin Yeung  Prakit Saingam  Yang Xu  Jinying Xi Video Byte Keywords: Microbiome, biofilters, biofilter microbiome, ozonation, ozone, biofiltration systems, gaseous volatile organic compounds, biodiversity, bacteria, industrial filtration Posted Date: February 25th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-276037/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/2 Abstract Ozone is a strong disinfectant known to kill bacteria and viruses. But when pumped into biofiltration systems at low doses ozone seems to have the opposite effect, boosting microbe activity and thus filtration performance all while relieving clogging issues by reducing overall biomass. To find out how ozone improves biofilter performance, researchers examined two toluene-treating biofilters at the microbial level. One served as the control, while the other was exposed to a low dose of ozone. The microbiome of the exposed filter consistently showed 30% higher biodiversity than the control filter with an increased capacity to break down amino and carboxylic acids. In addition, ozonation led to a higher abundance of stress-tolerant and biofilm-forming species causing a shift in the degradation pathways of certain compounds. The tighter microbial co-occurrence network observed for the filter exposed to ozone suggests a more stable microbial community robust to shocks a highly desirable feature in full-scale biofilters. These findings offer valuable insight into how and why low concentrations of ozone improve biofilter performance and could help engineers build better filters for high-value industrial applications. Page 2/2 Page 2/2
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Original Reaction Sequence of Pb(Yb<sub>1/2</sub>Nb<sub>1/2</sub>)O<sub>3</sub>-PbTiO<sub>3</sub>: Consequences on Dielectric Properties and Chemical Order
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University of Dundee Original Reaction Sequence of Pb(Yb1/2Nb1/2)O3-PbTiO3 Cochard, Charlotte; Karolak, Fabienne; Bogicevic, Christine; Guedes, Orland; Janolin, Pierre- Eymeric Published in: Advances in Materials Science and Engineering DOI: University of Dundee Original Reaction Sequence of Pb(Yb1/2Nb1/2)O3-PbTiO3 Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Link to publication in Discovery Research Portal Citation for published version (APA): Cochard, C., Karolak, F., Bogicevic, C., Guedes, O., & Janolin, P.-E. (2015). Original Reaction Sequence of Pb(Yb1/2Nb1/2)O3-PbTiO3: Consequences on dielectric properties and chemical order. Advances in Materials Science and Engineering, 2015, Article 408101. https://doi.org/10.1155/2015/408101 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights i h d Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Download date: 24. Oct. 2024 Hindawi Publishing Corporation Advances in Materials Science and Engineering Volume 2015, Article ID 408101, 6 pages http://dx.doi.org/10.1155/2015/408101 Hindawi Publishing Corporation Advances in Materials Science and Engineering Volume 2015, Article ID 408101, 6 pages http://dx.doi.org/10.1155/2015/408101 1Laboratoire SPMS, Universit´e Paris Saclay, CentraleSup´elec, CNRS; Grande Voie des Vignes, 92295 Chatenay-Malabry, France 2´Etudes et Productions Schlumberger, 1 rue Henri Becquerel, 92140 Clamart, France 1Laboratoire SPMS, Universit´e Paris Saclay, CentraleSup´elec, CNRS; Grande Voie des Vignes, 92295 Chatenay-Malabry, France 2´Etudes et Productions Schlumberger, 1 rue Henri Becquerel, 92140 Clamart, France Correspondence should be addressed to Pierre-Eymeric Janolin; pierre-eymeric.janolin@centralesupelec.fr Received 20 September 2015; Revised 4 December 2015; Accepted 8 December 2015 Academic Editor: Wenbin Yi Copyright © 2015 Charlotte Cochard et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The solid solution [Pb(Yb1/2Nb1/2)O3]1−x-[PbTiO3]x was synthesized with 𝑥≤60%, using several high-temperature techniques as well as room-temperature mechanosynthesis. The high-temperature synthesis reveals a reaction path involving the synthesis first of the end-members before the solid solution. The density and dielectric constant measured on the ceramics prepared from these powders indicate the crucial role of the synthesis technique in the subsequent properties. Mechanosynthesis results in ceramics with higher density and dielectric constant. Identical optimized sintering conditions were then applied to all investigated compositions and the resulting dielectric properties and chemical orders were compared. All polar orders (antiferroelectricity, ferroelectricity, and relaxor behavior) were evidenced. The 1 : 1 chemical order on the B-site of Pb(Yb1/2Nb1/2)O3 results in the formation of a double perovskite Pb2YbNbO6, and the superstructures in the X-ray diagrams signing the existence of this order persist up to 30% PbTiO3. The underlying mechanism for substitution of Yb or Nb by Ti is presented. 1. Introduction synthesis route has been designed for Pb(BB󸀠)O3 perovskites, the so-called “B-site oxide mixing route” [7, 8] involving the reaction of all oxides but PbO. Depending on the valence of the B-site elements, this leads to the synthesis of a columbite through B2+O2−+B󸀠 2 5+O5 2−→BB󸀠 2O6 [9] or of a wolframite through B2 3+O3 2−+ B󸀠 2 5+O5 2−→2BB󸀠O4 [10] or of a rutile through B4+O2 2−+B󸀠4+O2 2−→BB󸀠O4 [11]. In a second step, this columbite, wolframite, or rutile is mixed with PbO to get Pb(B,B󸀠)O3. For Pb(B,B󸀠)O3-PbTiO3 solid solution, either TiO2 is added to the other B-site oxides and then PbO is added to the product of this reaction [7, 8] or TiO2 + PbO is added to the wolframite/columbite/rutile [1–4]. Solid solution between ferroelectric PbTiO3 and perovskites with two elements on the B site (of the general formula A(BB󸀠)O3) have attracted considerable interest as they lead to high-performance functional materials such as Pb(Mg1/3,Nb2/3)O3-PbTiO3 [1], Pb(Zn1/3,Nb2/3)O3-PbTiO3 [2], Pb(Sc1/2,Nb1/2)O3-PbTiO3 [3], and Pb(In1/2,Nb1/2)O3- PbTiO3 [4], which are of both fundamental and commercial interests. Pb(Yb1/2Nb1/2)O3-PbTiO3 is one of such materi- als. It exhibits superior high-temperature high-piezoelectric properties, especially at the so-called “morphotropic” phase boundary with a Curie temperature of 370∘C (645 K) and a piezoelectric coefficient 𝑑33 ∼470–510 pC/N [5, 6] which makes it desirable for high-temperature actuators and sen- sors.h In the case of Pb(B1/2B󸀠 1/2)O3 ferroelectric perovskites, the order on the B site has a dramatic influence on the properties of these materials. Generally speaking, disorder on the B site of the perovskite (i.e., random occupancy by any of the two elements) leads to a relaxor behavior whereas an ordered alternating occupancy of the B site along the [111] The synthesis of these A(BB󸀠)O3 compounds is delicate as pyrochlore phases may easily be formed when all oxides are reacting and are detrimental to the properties. 3. Results and Discussion Starting with the conventional solid-state technique, after 7 h at 950∘C, PYN-PT is formed (see Figure 1(a)). However, for shorter calcination times (4 h), extra diffraction peaks related to Pb(Yb1/2Nb1/2)O3 and PbTiO3 also appear in Figure 1(b). This suggests that the reaction sequence is actually PbO + TiO2 + YbNbO4 →Pb(Yb1/2Nb1/2)O3 + PbTiO3 →PYN- PT. It is highly unusual that the reaction sequence of such a solid solution involves forming first the end-members. In order to test whether it is actually possible to synthesize the solid solution from its end-members, we synthesized Pb(Yb1/2Nb1/2)O3 and PbTiO3 separately, before mixing them and calcining them for 950∘C 4 h. We indeed obtained PYN-PT, as can be seen in Figure 1(c). The pyrochlore impu- rity phase can be eliminated and pure PYN-PT synthesized under optimized synthesis conditions (namely, at 800∘C for 12 h); see Figure 1(d). q 2 4 For the solid-state technique, we investigated various calcination temperatures and times (800–1000∘C and 2– 12 h) to get the pure perovskite phase. The reactive sintering technique, as its name indicates, enables carrying out both the synthesis and the sintering of the ceramics from a pellet composed of the reactants [11]. For all compositions inves- tigated, the optimized conditions for simultaneous synthesis and sintering were 950 to 1050∘C for 4 h. For the mechanosyn- thesis, we used a Retsch mill PM 100, and the pure perovskite phase was obtained after 9 h at 450 rpm. This process takes place at room temperature as the thermal energy of the other techniques is replaced here by mechanical energy.hf The X-ray diffraction was carried out on a Bruker D2 phaser to check the crystallinity and purity of the products and on an 18 kW high-precision diffractometer equipped with a rotating Cu anode for the superstructures detec- tion. The X-ray diagrams were either compared with the corresponding JCPDS file (for PbTiO3) or to the literature [16] (for Pb(Yb1/2Nb1/2)O3, as no JCPDS file is available) to confirm the perovskite phase and its purity. Scanning Electronic Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX) (Leo Gemini 5130) were performed on Pyrochlore phases have been shown to form at moderate temperatures (600–700∘C) in Pb(BB󸀠)O3 and Pb(BB󸀠)O3- PbTiO3 solid solutions, even using the B-site mixing route [7, 9, 18, 19], and may still be present in the final product. 1. Introduction An alternative Advances in Materials Science and Engineering 2 Advances in Materials Science and Engineering Intensity (a.u.) 20 25 30 35 40 45 50 55 60 2𝜃(∘) (a) (b) (c) (d) (e) PYN PT Parasitic ⟨022⟩ ⟨021⟩ ⟨002⟩ ⟨111⟩ ⟨001⟩ ⟨011⟩ Figure 1: X-ray diagram of PYN-PT powder (for 𝑥= 60%) obtained by various techniques: at 950∘C by solid-state reaction of PbO + TiO2 + YbNbO4 (a) for 7 h and (b) for shorter calcination time (4 h) revealing the presence of PYN and PT and (c) by solid-state reaction of Pb(Yb1/2Nb1/2)O3 + PbTiO3 leading to PYN-PT after 4 h. Pure PYN-PT can be synthesized by solid-state reaction at 800∘C for 12 h (d) as well as by mechanosynthesis (e) of PbO + TiO2 + YbNbO4. Intensity (a.u.) 20 25 30 35 40 45 50 55 60 2𝜃(∘) (a) (b) (c) (d) (e) ⟨022⟩ ⟨021⟩ ⟨002⟩ ⟨111⟩ ⟨001⟩ ⟨011⟩ direction by the two elements leads generally to antiferro- electric behavior [12, 13]. In the latter case, the structure may be described as a “double perovskite” of general formula Pb2BB󸀠O6. This order doubles the crystallographic unit cell along the [111] direction, giving rise to superstructure X- ray reflections, and was shown to be driven more by the sum of the ionization energies of the two B-site elements rather than by size or charge difference [14]. In the case of Pb(Yb1/2Nb1/2)O3 (PYN), the sum of the ionization energies is large (75 eV) leading to a 1 : 1 chemical order on the B site which could only be broken by adding lithium to the compound [15]. In this work we investigate three techniques to synthe- size [Pb(Yb1/2Nb1/2)O3]1−x-[PbTiO3]x (PYN-PT) with 𝑥≤ 60% (Ti-richer compositions were considered analogous to PbTiO3) and compared the properties of the ceramics made out of these powders. The investigated techniques are conventional solid-state mixing, reactive sintering, and mechanosynthesis. An original intermediate reaction step involving the formation of the two end-members is evi- denced. The chemical order on the B-site, reminiscent of the one of PYN, is shown to persist in the powders obtained from mechanosynthesis up to composition with 30% PbTiO3. 1. Introduction Figure 1: X-ray diagram of PYN-PT powder (for 𝑥= 60%) obtained by various techniques: at 950∘C by solid-state reaction of PbO + TiO2 + YbNbO4 (a) for 7 h and (b) for shorter calcination time (4 h) revealing the presence of PYN and PT and (c) by solid-state reaction of Pb(Yb1/2Nb1/2)O3 + PbTiO3 leading to PYN-PT after 4 h. Pure PYN-PT can be synthesized by solid-state reaction at 800∘C for 12 h (d) as well as by mechanosynthesis (e) of PbO + TiO2 + YbNbO4. 2. Experimental Procedure Commercial-grade oxides PbO (Interchim), Yb2O3 (Rhˆone- Poulenc), Nb2O5, and TiO2 (both Alfa Aesar) with purity greater than 99.5% were used as starting materials. Raw materials were mixed in absolute ethanol and subsequently dried in a dry-off oven at 80∘C for 24 h before calcination. For every synthesis technique used, the first step consisted in obtaining the YbNbO4 wolframite from the calcination of Yb2O3 and Nb2O5 at 1200∘C for 4 h. For the synthesis of the solid solution, and still for every synthesis techniques, we followed the conventional route, mixing stoichiometric quantities of PbO, TiO2, and YbNbO4. fractured surfaces to investigate the purity and microstruc- ture of the samples. The average grain sizes were determined using the line interception method on SEM images [17]. The relative density was calculated using geometrical dimension or the Archimedes’ method. Dielectric measurements were carried out using an Agilent 4294A impedance analyzer on ceramics with gold-sputtered electrodes. 3. Results and Discussion 14000 12000 10000 8000 6000 4000 2000 0 𝜖󳰀 350 400 450 500 550 600 650 700 750 800 Temperature (K) 40 35 30 25 20 15 10 5 0 Tan 𝛿 1.3 wt% PbO 3wt% PbO Mechanosynthesis Solid-state reaction + PVA Solid-state reaction + Solid-state reaction + t In order to prevent the formation of pyrochlore phases, alternative high-pressure and/or high-temperature tech- niques have been developed, for example, hot isostatic press. They, however, require heavy equipment. The last technique used to synthesize PYN-PT, mechanosynthesis, was shown to enable the synthesis of pure perovskite phase without such heavy equipment. This room-temperature technique, also known as ball-milling, was first applied to metallic alloys in the late 70s and to ferroelectric oxides 20 years later [8, 20, 21]. The strain and the grain size reduction taking place during the mechanosynthesis lead to broad and asymmetric peaks in the diffraction patterns making the identification of minority phases in presence difficult. The pure perovskite phase was considered to be obtained when subsequent milling did not change the diffraction pattern. The corresponding X- ray diagram is reported in Figure 1(e) where the large width and asymmetric profile of the peaks associated with the mechanical strain developed in the nanograins can be clearly observed. The difference with the high-temperature synthesis techniques here is that the grain size of the reactants is much smaller (∼20 nm versus microsized grains) and therefore present a much higher reactivity. Temperature (K) Figure 2: Real part of the dielectric constant 𝜖󸀠(line) and losses tan 𝛿 (dotted line) of PYN-PT with 60% PbTiO3 measured at 1 kHz and as a function of temperature on ceramics obtained by various synthesis techniques: +PVA, +1 wt% PbO, and +3 wt% PbO designate the binder used with powders obtained by the solid-state technique. All samples were sintered at 950∘C. p g y Powders obtained by the solid-state or mechanosynthesis techniques have been sintered (at 950∘C for 4 h) and the absence of pyrochlore after sintering was confirmed by X-ray diffraction (not shown). The density and dielectric constant at room temperature of the resulting ceramics are compared with the ceramics obtained by reactive sintering in Table 1. The dielectric constants reported in Table 1 have been normalized by the density of the ceramic to enable a meaningful comparison. 3. Results and Discussion In PYN- PT, a pyrochlore phase may also be formed but then the end- members, PYN and PT, are formed before reacting together Advances in Materials Science and Engineering 3 to form the PYN-PT solid solution at higher temperature. With optimized synthesis parameters it is possible to obtain pyrochlore-free PYN-PT samples.h Table 1: Relative density (𝑑) and normalized dielectric constant (𝜖󸀠/𝑑) at room temperature and 1 kHz measured on PYN-PT ceramics with 60% PbTiO3 obtained by the various investigated synthesis techniques. For the solid-state technique, PVA or PbO were used as binders. The powder of pure perovskite obtained from solid-state synthesis must be shaped and sintered in order to get the final ceramic. As the reaction temperature and the sintering temperature were the same, a more practical solution to prepare ceramics was investigated: reactive sintering. In this case, the raw reactants are prepressed into a so-called “green” pellet and both the calcination and sintering occur simultaneously. Following this route we observed the same reaction sequence that starting from PbO + YbNbO4 + TiO2 leads to the formation of PYN and PbTiO3 and only afterwards of PYN-PT. 𝑑 𝜖󸀠/𝑑 Reactive sintering 60% 302 Solid-state reaction + PVA 81% 652 Solid-state reaction + 1.3 wt% PbO 72% 771 Solid-state reaction + 3 wt% PbO 86% 613 Mechanosynthesis 91% 953 𝑑 𝜖󸀠/𝑑 Reactive sintering 60% 302 Solid-state reaction + PVA 81% 652 Solid-state reaction + 1.3 wt% PbO 72% 771 Solid-state reaction + 3 wt% PbO 86% 613 Mechanosynthesis 91% 953 14000 12000 10000 8000 6000 4000 2000 0 𝜖󳰀 350 400 450 500 550 600 650 700 750 800 Temperature (K) 40 35 30 25 20 15 10 5 0 Tan 𝛿 1.3 wt% PbO 3wt% PbO Mechanosynthesis Solid-state reaction + PVA Solid-state reaction + Solid-state reaction + Figure 2: Real part of the dielectric constant 𝜖󸀠(line) and losses tan 𝛿 (dotted line) of PYN-PT with 60% PbTiO3 measured at 1 kHz and as a function of temperature on ceramics obtained by various synthesis techniques: +PVA, +1 wt% PbO, and +3 wt% PbO designate the binder used with powders obtained by the solid-state technique. All samples were sintered at 950∘C. 3. Results and Discussion Because the ceramics prepared from reactive sintering have an extremely low density and therefore open pores, their dielectric constants value cannot be simply compared to the ones of ceramics prepared by the other processing methods. However, it clearly appears that processing ceramics by reactive sintering leads to ceramics of poor quality that are unsuitable for potential applications. The ceramics obtained from mechanosynthesis are the densest and exhibit the highest dielectric constant of all investigated synthesis techniques. A binder was necessary to shape the powder obtained by the solid-state technique into pellets before sintering. In the case of the first binder used, PVA, SEM, and EDX analyses confirmed the absence of carbonated residues after sintering. Addition of PbO instead of PVA was also considered as high-temperature synthesis may lead to Pb evaporation. PbO may also act as a liquid sintering aid. The results are contrasted: if the ceramics density increases with PbO excess compared to reactive sintering, adding 3 wt% excess PbO leads to a normalized dielectric constant lower than with a 1.3 wt% excess PbO. This is because the excess PbO overcompensates for Pb loss during sintering and remains as PbO in the ceramics. Its low dielectric constant (∼20) therefore contributes to lowering the ceramic dielectric constant.hl The influence of the synthesis technique on dielectric properties is presented in Figure 2. Mechanosynthesis leads to the highest dielectric constant at the para-ferroelectric phase transition (𝑇𝐶∼530∘C) with 𝜖󸀠 max > 12 000. The ceramics obtained from solid-state technique exhibit slightly lower 𝜖󸀠 max, and, as mentioned earlier, excess of PbO decreases 𝜖󸀠 max. From SEM, we found that, even after sintering, the grain size of PYN-PT obtained by mechanosynthesis remains smaller (∼1 𝜇m) than with other techniques (≥5 𝜇m). 4 Advances in Materials Science and Engineering 800∘C-4h 1000∘C-4h 900∘C-2h 900∘C-6h 1𝜇m 1𝜇m 1𝜇m 1𝜇m Time Temperature Figure 3: SEM images of PYN-PT 60% ceramics obtained after sintering at various temperatures and times. 1000∘C-4h 900∘C-6h 1𝜇m 1𝜇m rature 800∘C-4h 1𝜇m 800∘C-4h Time Time Figure 3: SEM images of PYN-PT 60% ceramics obtained after sintering at various temperatures and times. Figure 3: SEM images of PYN-PT 60% ceramics obtained after sintering at various temperatures and times. 3. Results and Discussion 25000 20000 15000 10000 5000 0 𝜖󳰀 900∘C-2h 900∘C-4h 900∘C-6h 800∘C-4h 1000∘C-4h Temperature (K) 350 400 450 500 550 600 650 700 750 800 Figure 4: Real part of the dielectric constant (𝜖󸀠) measured at 1 kHz from RT to 530∘C of PYN-PT 60% ceramic obtained from various sintering temperatures and times. 25000 20000 15000 10000 5000 0 𝜖󳰀 350 400 450 500 550 600 650 700 750 800 The grain size of ceramics obtained from mechanosynthesis is close to the optimum grain size of Pb-based perovskites giving rise to the best properties. This smaller grain size, combined with strain effects [22], also contributes to lowering the transition temperature by ∼10∘C. The grain size of ceramics obtained from mechanosynthesis is close to the optimum grain size of Pb-based perovskites giving rise to the best properties. This smaller grain size, combined with strain effects [22], also contributes to lowering the transition temperature by ∼10∘C. Sintering conditions for ceramics obtained by mechano- synthesis were then further optimized with the objective to determine sintering parameters valid over the entire com- positional range investigated here (from 0 to 60% PbTiO3). The SEM investigation reveals that, as expected, the grain size increases with increasing sintering temperature and time. Taking the example of PYN-PT 0.60 (Figure 3), the grain size increases from 0.086 𝜇m to 1.27 𝜇m when increasing the sintering temperature from 800 to 1000∘C and keeping the sintering time constant at 4 h. A similar increase is observed when increasing the sintering time (from 0.25 𝜇m at 900∘C for 2 h to 0.78 𝜇m at 900∘C for 6 h). Together with the decrease in the number and size of porosities, this larger grain size leads to an increase of the dielectric constant [1, 4] (Figure 4). At the smallest grain size (0.086 𝜇m), the dielectric peak at the Curie temperature is broad but still existent indicating the persistence of the ferroelectric properties down to this grain size. Comparable increase of grain size with sintering temperature/time was observed for other compositions within the PYN-PT solid solution.h Temperature (K) Figure 4: Real part of the dielectric constant (𝜖󸀠) measured at 1 kHz from RT to 530∘C of PYN-PT 60% ceramic obtained from various sintering temperatures and times. [25, 26]), relaxor (for 25, 30, and 40% PbTiO3 content) and ferroelectric compositions (50 and 60% PbTiO3 content) can be synthesized by this method. 3. Results and Discussion Our study focuses on the synthesis of the ceramics, and the complete description of The evolution of the dielectric constant with temperature and the room-temperature diffractograms for selected com- positions is presented in Figures 5 and 6, in order to show that antiferroelectric (pure PYN [23, 24] and PYN-5% PT Advances in Materials Science and Engineering 5 20000 0 15000 10000 5000 𝜖󳰀 300 400 500 600 700 800 Temperature (K) 25% 30% 40% 50% 60% 5% PYN ×5 ×5 Figure 5: Real part of the dielectric constant (𝜖󸀠) measured at 1 kHz from RT to 530∘C on PYN-PT ceramics exhibiting antiferroelectric (pure PYN and PYN-5% PT), relaxor (for 25, 30, and 40% PbTiO3 content), or ferroelectric (50 and 60% PbTiO3 content) behaviors. All samples were sintered between 950∘C and 1050∘C. 20000 0 15000 10000 5000 𝜖󳰀 300 400 500 600 700 800 Temperature (K) 25% 30% 40% 50% 60% 5% PYN ×5 ×5 idea here. It stems from the strong stability of the Yb/Nb order on the B site of PYN which has been evidenced as an intermediate step in the solid-state synthesis technique. Start- ing from pure PYN, we mentioned before that the chemical formula of this double perovskite may be written Pb2YbNbO6 in order to reflect the chemical order existing on the B site along the [111] crystallographic direction. When PbTiO3 is added, Titanium atoms substitute equally each B site (i.e., Yb or Nb), in order to preserve electroneutrality. This is coherent with the intermediate reaction step observed with high- temperature techniques where the end-members are formed before the solid solution. In this scheme, Titanium disturbs a preexisting order rather than inserting itself in-between Yb/Nb chains; that is, along the [111] direction, the alterna- tion of B-cations would be [Yb-Nb]n-Ti-[Yb-Nb]n-Ti. This situation can be safely rejected since it would lead to a new chemical order and the superstructure reflections would be significantly shifted in the X-ray diagram with respect to their position in PYN. The composition [Pb(Yb1/2Nb1/2)O3]1−x- [PbTiO3]x may then be written Pb2(Yb1−xTix)(Nb1−xTix)O6 to reflect the underlying 1 : 1 chemical order giving rise to the superstructures reported in Figure 6. It is only when there are, on average, more Titanium atoms on the B sites than Ytterbium or Niobium atoms (i.e., for compositions with PbTiO3 content over 33%) that the chemical order is broken and the corresponding superstructures disappear from the X-ray diagram. Conflict of Interests Figure 6 exhibits superstructure reflections (marked with a square) indicating that the chemical B-site order of PYN persists for PYN-PT compositions with PbTiO3 content up to 30% with no indication of Ti segregation (broadening of reflections, lowering of intensity, etc.). This chemical order reflects an equally probable substitution of Ytterbium and Niobium by Titanium. The details of the microstructural model explaining the origin of these superstructures will be published elsewhere but let us give nevertheless the main The authors declare that there is no conflict of interests regarding the publication of this paper. 4. Conclusion In conclusion we have shown that the end-members of the solid solution Pb(Yb1/2Nb1/2)O3-PbTiO3 are formed first before the solid solution is obtained in high-temperature synthesis techniques, using the B-site oxides mixing route. Mechanosynthesis was shown to provide a room-temperature alternative to these techniques leading to ceramics with smaller grains but higher dielectric properties and density. Sintering conditions were optimized for all powders with PbTiO3 content up to 60% and lead to ceramics exhibiting the full diversity of ferroelectric behaviors. From a structural point of view, the so-obtained ceramics exhibit a signature of B-site order reminiscent of the PYN one for compositions up to 30%, that is up to when there is an equal number of Titanium, Ytterbium, and Niobium on the B site of the perovskite. 15 20 25 Figure 6: X-ray diagrams for PYN-PT ceramics obtained by mechanosynthesis for PbTiO3 content between 0 (PYN) and 60%. Squares (◼) indicate superstructure reflections due to chemical order on the B site. Triangles (△) mark superstructures due to antiparallel displacements. All samples were sintered between 950∘C and 1050∘C. their properties together with the type of ferroelectric order they derivate from shall be presented elsewhere.l 3. Results and Discussion Such disordered compositions (with Ti content larger than 33%) may be then written Pb(Yb1−x/2,Nb1−x/2,Tix)O3 and the structure of PYN-PT solid solution is described as a simple perovskite. Figure 5: Real part of the dielectric constant (𝜖󸀠) measured at 1 kHz from RT to 530∘C on PYN-PT ceramics exhibiting antiferroelectric (pure PYN and PYN-5% PT), relaxor (for 25, 30, and 40% PbTiO3 content), or ferroelectric (50 and 60% PbTiO3 content) behaviors. All samples were sintered between 950∘C and 1050∘C. 60% 50% 40% 30% 25% 20% 16% 5% PYN 10 15 20 25 ⟨001⟩ Figure 6: X-ray diagrams for PYN-PT ceramics obtained by mechanosynthesis for PbTiO3 content between 0 (PYN) and 60%. Squares (◼) indicate superstructure reflections due to chemical order on the B site. Triangles (△) mark superstructures due to antiparallel displacements. All samples were sintered between 950∘C and 1050∘C. 60% 50% 40% 30% 25% 20% 16% 5% PYN 10 15 20 25 ⟨001⟩ References [16] W. K. Choo, H. J. Kim, J. H. Yang et al., “Crystal struc- ture and B-site ordering in antiferroelectric Pb(Mg1/2W1/2)O3, Pb(Co1/2W1/2)O3 and Pb(Yb1/2Nb1/2)O3,” Japanese Journal of Applied Physics, vol. 32, no. 9, pp. 4249–4253, 1993. [1] J. Carreaud, P. Gemeiner, J. M. Kiat et al., “Size-driven relaxation and polar states in PbMg1/3Nb2/3O3-based system,” Physical Review B—Condensed Matter and Materials Physics, vol. 72, no. 17, Article ID 174115, pp. 4–9, 2005. [17] American Society for Testing and Materials, “Standard test methods for determining average grain size,” ASTM E112-13, ASTM International, West Conshohocken, Pa, USA, 2013. [2] M. A. Hentati, H. Dammak, H. Khemakhem, and M. P. Thi, “Dielectric properties and phase transitions of [001], [110], and [111] oriented Pb(Zn1/3Nb2/3)O3-6%PbTiO3 single crystals,” Journal of Applied Physics, vol. 113, no. 24, Article ID 244104, 2013. [18] V. V. Bhat, M. V. Radhika Rao, and A. M. Umarji, “Dilatometric approach for the determination of the solid state reaction- onset of the lead based relaxor ferroelectric system,” Materials Research Bulletin, vol. 38, no. 6, pp. 1081–1090, 2003. [3] R. Haumont, B. Dkhil, J. M. Kiat, A. Al-Barakaty, H. Dammak, and L. Bellaiche, “Cationic-competition-induced monoclinic phase in high piezoelectric (PbSc1/2Nb1/2O3)1−𝑥−(PbTiO3)𝑥 compounds,” Physical Review B, vol. 68, no. 1, Article ID 014114, 2003. [19] Y. Zupei, Z. Shaorong, Q. Shaobo, C. Bin, and T. Changsheng, “Reaction mechanisms of PMN-PT powder prepared by molten salt synthesis,” Ferroelectrics, vol. 265, no. 1, pp. 225–232, 2002. [20] J. Xue, D. Wan, S.-E. Lee, and J. Wang, “Mechanochemical synthesis of lead zirconate titanate from mixed oxides,” Journal of the American Ceramic Society, vol. 82, no. 7, pp. 1687–1692, 1999. [4] K. Alilat, M. P. Thi, H. Dammak, C. Bogicevic, A. Albareda, and M. Doisy, “Grain size effect on electromechanical properties and non-linear response of dense nano and microstructured PIN-PT ceramics,” Journal of the European Ceramic Society, vol. 30, no. 9, pp. 1919–1924, 2010. [21] J. Xue, J. Wang, and D. Wan, “Nanosized barium titanate powder by mechanical activation,” Journal of the American Ceramic Society, vol. 83, no. 1, pp. 232–234, 2000. [5] J. B. Lim, S. Zhang, and T. R. Shrout, “Relaxor behavior of piezo- electric Pb(Yb1/2Nb1/2)O3-PbTiO3 ceramics sintered at low temperature,” Journal of Electroceramics, vol. 26, no. 1–4, pp. 68– 73, 2011. [22] M. Alguer´o, J. Ricote, T. Hungr´ıa, and A. Castro, “High-sensitiv- ity piezoelectric, low-tolerance-factor perovskites by mechano- synthesis,” Chemistry of Materials, vol. 19, no. 20, pp. References 4982–4990, 2007. [6] T. Yamamoto and S. Ohashi, “Dielectric and piezoelectric prop- erties of Pb(Yb1/2Nb1/2)O3-PbTiO3 solid solution system,” Japanese Journal of Applied Physics, vol. 34, no. 9, pp. 5349–5353, 1995. [23] V. Demidova, E. Gagarina, T. Ivanova, and V. Sakhnenko, “Atomic structure and phase transitions in antiferroelectric PbYb0.5Nb0.5O3,” Ferroelectrics, vol. 159, pp. 191–196, 1994.h [24] J. R. Kwon and W. K. Choo, “The antiferroelectric crystal structure of the highly ordered complex perovskite Pb(Yb1/2Nb1/2)O3,” Journal of Physics: Condensed Matter, vol. 3, no. 13, pp. 2147–2155, 1991. [7] M. Orita, H. Satoh, K. Aizawa, and K. Ametani, “Preparation of ferroelectric relaxor Pb(Zn1/2Nb2/3)O3-Pb(Mg1/3Nb2/3)O3- PbTiO3 by two-step calcination method,” Japanese Journal of Applied Physics, vol. 31, no. 9, part 1, pp. 3261–3264, 1992. [25] S.-J. Ahn, J.-J. Kim, J.-H. Kim, and W.-K. Choo, “Nondegenerate hard-mode Raman studies of phase transition in antiferro- electric lead ytterbium niobate-based perovskite compound,” Journal of Raman Spectroscopy, vol. 37, no. 1–3, pp. 202–207, 2006. [8] R. M. V. Rao, A. Halliyal, and A. M. Umarji, “Perovskite phase formation in the relaxor system [Pb(Fe1/2Nb1/2)O3]1−𝑥- [Pb(Zn1/3Nb2/3)O3]𝑥,” Journal of the American Ceramic Society, vol. 79, no. 1, pp. 257–260, 1996. [9] S. L. Swartz and T. R. Shrout, “Fabrication of perovskite lead magnesium niobate,” Materials Research Bulletin, vol. 17, no. 10, pp. 1245–1250, 1982. [26] H. Lim, H. J. Kim, and W. K. Choo, “X-ray and dielectric studies of the phase transitions in Pb(Yb1/2Nb1/2)O3-PbTiO3 ceramics,” Japanese Journal of Applied Physics B, vol. 34, no. 9, pp. 5449–5452, 1995. [10] P. Groves, “Fabrication and characterisation of ferroelectric perovskite lead indium niobate,” Ferroelectrics, vol. 65, no. 1, pp. 67–77, 2011. [11] T. R. Shrout, P. Papet, S. Kim, and G.-S. Lee, “Conventionally prepared submicrometer lead-based perovskite powders by reactive calcination,” Journal of the American Ceramic Society, vol. 73, no. 7, pp. 1862–1867, 1990. [12] C. Cochard, Pb(Yb1/2Nb1/2)O3-PbTiO3: a model solid solution for the study of polar orders [Ph.D. thesis], ´Ecole Centrale Paris, Chˆatenay-Malabry, France, 2015. [13] V. A. Isupov, “Ferroelectric and antiferroelectric perovskites PbB󸀠0.5B󸀠󸀠0.5O3,” Ferroelectrics, vol. 289, no. 1, pp. 131–195, 2003. [14] A. A. Bokov, N. P. Protsenko, and Z.-G. Ye, “Relationship between ionicity, ionic radii and order/disorder in complex perovskites,” Journal of Physics and Chemistry of Solids, vol. 61, no. 9, pp. 1519–1527, 2000. [15] A. A. Bokov, V. Y. Shonov, I. P. Rayevsky, E. S. Gagarina, and M. F. Acknowledgment The authors acknowledge the financial support of the ANRT under Contract no. 2011/1687. Advances in Materials Science and Engineering 6 References Kupriyanov, “Compositional ordering and phase transitions in Pb(Yb0.5Nb0.5)O3,” Journal of Physics: Condensed Matter, vol. 5, no. 31, pp. 5491–5504, 1993. References Submit your manuscripts at http://www.hindawi.com Scientifica Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Corrosion International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Polymer Science International Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Ceramics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Composites Journal of Nanoparticles Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Biomaterials Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Nanoscience Journal of Textiles Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Nanotechnology Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Crystallography Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Coatings Journal of Advances in Materials Science and Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Smart Materials Research Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Metallurgy Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 BioMed Research International Materials Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Nanomaterials Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Nanomaterials Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Composites Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Metallurgy Journal of Nanoparticles Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com als Scientifica Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Ceramics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Biomaterials Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Nanoscience Journal of Textiles Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Crystallography Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Coatings Journal of Advances in Materials Science and Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Nanomat Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Nanomaterials Scientifica Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Ceramics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Biomaterials Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Nanoscience Journal of Textiles Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Crystallography Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Coatings Journal of Advances in Materials Science and Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Nanomat Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Nanomaterials Advances in Materials Science and Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Materials Science and Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Scientifica Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Advances in Materials Science and Engineering Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Scientifica Hindawi Publishing Corporation
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Detecting clinically significant events through automated language analysis: Quo imus?
NPJ schizophrenia
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REFERENCES 1. Bedi, G. et al. Automated analysis of free speech predicts psychosis onset in high-risk youths. npj Schizophr. 1, 15030 (2015). 1. Bedi, G. et al. Automated analysis of free speech predicts psychosis onset in high-risk youths. npj Schizophr. 1, 15030 (2015). 1. Bedi, G. et al. Automated analysis of free speech predicts psychosis onset in high-risk youths. npj Schizophr. 1, 15030 (2015). 2. Elvevåg, B., Foltz, P. W., Weinberger, D. R. & Goldberg, T. E. Quantifying incoherence in speech: an automated methodology and novel application to schizophrenia. Schizophr. Res. 93, 304–316 (2007). To realize the potential of the transformative next steps, we must routinely and systematically strive to obtain larger data sets containing multiple language samples from participants collected over time. This will allow quantifying the joint time course of the disease(s) and changes in language. Increased sample size further improves the methodologies, permitting moving beyond the less- reliable cross-validation to the use of the gold-standard for validating ML results, which is a ‘hold-out’ data set. In such an approach all modeling is conducted blind to the hold-out set, and when modeling is completed, the model is run on the held-out set to measure expected performance in the larger population, thereby ensuring generalization while lowering the risk of overfitting. At least as importantly, realistically sized data sets allow the application of larger combinations of more sophisticated NLP/ML techniques that move beyond the often used simple word-count features. This permits deeper characterization of more important aspects of language, such as semantic structures, discourse organization, as well as acoustic characteristics.6 1 3. Elvevåg, B., Foltz, P. W., Rosenstein, M. & DeLisi, L. E. An automated method to analyze language use in patients with schizophrenia and their first-degree relatives. J. Neurolinguistics 23, 270–284 (2010). 3. Elvevåg, B., Foltz, P. W., Rosenstein, M. & DeLisi, L. E. An automated method to analyze language use in patients with schizophrenia and their first-degree relatives. J. Neurolinguistics 23, 270–284 (2010). 4. Rosenstein, M., Foltz, P. W., DeLisi, L. E. & Elvevåg, B. Language as a biomarker in those at high-risk for psychosis. Schizophr. Res. 165, 249–250 (2015). 4. Rosenstein, M., Foltz, P. W., DeLisi, L. E. & Elvevåg, B. Language as a biomarker in those at high-risk for psychosis. Schizophr. Res. 165, 249–250 (2015). 5. Nicodemus, K. K. et al. Category fluency, latent semantic analysis and schizophrenia: a candidate gene approach. Cortex 55, 182–191 (2014). 5. www.nature.com/npjschz All rights reserved 2334-265X/16 BRIEF COMMUNICATION OPEN Detecting clinically significant events through automated language analysis: Quo imus? This result follows closely our own and others observations of the value of these techniques in, for example, discriminating patients with schizo- phrenia from controls,2 discriminating schizophrenia probands, first-degree relatives and unrelated healthy controls,3 differentiat- ing those at high risk of psychosis from unrelated putatively healthy participants4 and in a candidate gene study linking language in general to underlying neurobiology,5 all quite encouraging outcomes. Our disappointment is not directly with the Bedi et al.1 paper itself, but that we as a field are, after this long proving period, still at the ‘promising’ stage. This inertia arises from two primary factors. The first is owing to the use of small, often second-hand data sets produced for other studies, which severely constrains the NLP techniques that can be applied and the generality of the obtained results. The second is that the methodologies applied must become sufficiently assimilated into the field to be used effectively in analyses so as to provide valid, reliable measures of the constructs of interest. This understanding permits better linking of the appropriate features of language to the underlying etiologies of interest. 1Institute of Cognitive Science, University of Colorado, Boulder, CO, USA; 2Pearson, Boulder, CO, USA and 3Psychiatry Research Group, Department of Clinical Medicine, University of Tromsø, Norway; and Norwegian Centre for Integrated Care and Telemedicine, University Hospital of North Norway, Tromsø, Norway. Correspondence: P Foltz (peter.foltz@colorado.edu) Received 28 September 2015; accepted 5 October 2015 The authors declare no conflict of interest. The authors declare no conflict of interest. BRIEF COMMUNICATION OPEN Detecting clinically significant events through automated language analysis: Quo imus? Peter W Foltz1,2, Mark Rosenstein2 and Brita Elvevåg3 npj Schizophrenia (2016) 2, Article number: 15054; doi:10.1038/npjschz.2015.54; published online 6 January 2016 hrenia (2016) 2, Article number: 15054; doi:10.1038/npjschz.2015.54; published online 6 January 2016 Schizophrenia (2016) 2, Article number: 15054; doi:10.1038/npjschz.2015.54; published online 6 January 2 is that of a truly high-dimensional language-feature space with the potential to align with the aspirational goals of the NIMH Research Domain Criteria by employing language to locate and pinpoint those with severe mental illness at coordinates within this space. Once localized, the features that define the resulting hypothesized clusters can potentially be calibrated for use in early detection, continuously evaluating treatment and providing links to the biology underlying these diseases, simultaneously superseding our existing diagnostic categories. But this vision is only achievable with purpose-designed studies containing sufficiently large populations with a mix of both healthy participants and individuals sampled across multiple categories of diagnostic groups. Our field must become versed in the use of more powerful applications of NLP/ML techniques and offer more reproducible methodologies. These results, taken with others, are sufficiently encouraging so that it is now time for us to move beyond ‘promising’. We found the recent paper by Bedi et al.1 simultaneously exciting, heartening and, sadly, a bit discouraging. It shows that modern, statistical natural language processing (NLP) and machine- learning (ML) techniques can potentially be useful as a component of diagnosis, here predicting who among those at risk will eventually transition to full-blown psychosis. This result follows closely our own and others observations of the value of these techniques in, for example, discriminating patients with schizo- phrenia from controls,2 discriminating schizophrenia probands, first-degree relatives and unrelated healthy controls,3 differentiat- ing those at high risk of psychosis from unrelated putatively healthy participants4 and in a candidate gene study linking language in general to underlying neurobiology,5 all quite encouraging outcomes. We found the recent paper by Bedi et al.1 simultaneously exciting, heartening and, sadly, a bit discouraging. It shows that modern, statistical natural language processing (NLP) and machine- learning (ML) techniques can potentially be useful as a component of diagnosis, here predicting who among those at risk will eventually transition to full-blown psychosis. © 2016 Schizophrenia International Research Society/Nature Publishing Group REFERENCES Nicodemus, K. K. et al. Category fluency, latent semantic analysis and schizophrenia: a candidate gene approach. Cortex 55, 182–191 (2014). 6. Cohen, A. S., Hong, L. S. & Guevara, A. Understanding emotional expression using prosodic analysis of natural speech: refining the methodology. J. Behav. Ther. Exp. Psychiatry 41, 150–157 (2010). 6. Cohen, A. S., Hong, L. S. & Guevara, A. Understanding emotional expression using prosodic analysis of natural speech: refining the methodology. J. Behav. Ther. Exp. Psychiatry 41, 150–157 (2010). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/ by/4.0/ From our perspective, Figure 3 from Bedi et al.1 is a beautiful, low-dimensional, small, incremental step toward our vision, which © 2016 Schizophrenia International Research Society/Nature Publishing Group
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Quantum dot single-photon sources with ultra-low multi-photon probability
npj quantum information
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ARTICLE OPEN Quantum dot single-photon sources with ultra-low multi- photon probability Lukas Hanschke1, Kevin A. Fischer2, Stefan Appel1, Daniil Lukin2, Jakob Wierzbowski1, Shuo Sun2, Rahul Trivedi2, Jelena Vučković2, Jonathan J. Finley1 and Kai Müller 1 High-quality sources of single photons are of paramount importance for quantum communication, sensing, and metrology. To these ends, resonantly excited two-level systems based on self-assembled quantum dots have recently generated widespread interest. Nevertheless, we have recently shown that for resonantly excited two-level systems, emission of a photon during the presence of the excitation laser pulse and subsequent re-excitation results in a degradation of the obtainable single-photon purity. Here, we demonstrate that generating single photons from self-assembled quantum dots with a scheme based on two-photon excitation of the biexciton strongly suppresses the re-excitation. Specifically, the pulse-length dependence of the multi-photon error rate reveals a quadratic dependence in contrast to the linear dependence of resonantly excited two-level systems, improving the obtainable multi-photon error rate by several orders of magnitude for short pulses. We support our experiments with a new theoretical framework and simulation methodology to understand few-photon sources. npj Quantum Information (2018) 4:43 ; doi:10.1038/s41534-018-0092-0 Received: 27 February 2018 Revised: 20 August 2018 Accepted: 23 August 2018 1Walter Schottky Institut and Physik Department, Technische Universität München, 85748 Garching, Germany and 2E. L. Ginzton Laboratory, Stanford University, Stanford, CA 94305, USA Correspondence: Kai Müller (kai.mueller@wsi.tum.de) These authors contributed equally: Lukas Hanschke, Kevin A. Fischer. www.nature.com/npjqi INTRODUCTION We first provide detailed experimental results supporting that the bi-excitonic system is a superior single-photon source over a 2LS due to re-excitation. Second, we provide a new theoretical analysis for photon sources, using the biexcitonic system as an example. Two-level systems (2LS) provided by excitonic transitions in self- assembled quantum dots (QDs) are commonly used on-demand sources for high-quality single photons.1,2 Crucially, resonant excitation enables nearly transform-limited linewidth3–5 and high photon indistinguishability.6 Combined with nanoresonators, single-photon sources with high emission rates and collection efficiency have been demonstrated7–14 and are now being incorporated into quantum information processors. For example, a solitary high-quality QD source was recently used in exciting demonstrations to create a train of single photons, which were temporally multiplexed to the input of a Boson Sampler.15,16 Boson sampling with this source, to date, has provided one of the best experimental validations of optical quantum computing. The quality of the experimental data in this, and future optical quantum information processors, ultimately relies on the ability of the source to emit precisely one photon when triggered by a laser pulse. However, it has recently been shown (from our work17–19 and others20) that resonant excitation of a 2LS provides a fundamental limitation to the error rate of the single-photon source and hence the information processor. This results from the emission of a photon during the presence of the excitation pulse which leads to re-excitation and multi-photon emission. Two-level systems (2LS) provided by excitonic transitions in self- assembled quantum dots (QDs) are commonly used on-demand sources for high-quality single photons.1,2 Crucially, resonant excitation enables nearly transform-limited linewidth3–5 and high photon indistinguishability.6 Combined with nanoresonators, single-photon sources with high emission rates and collection efficiency have been demonstrated7–14 and are now being incorporated into quantum information processors. For example, a solitary high-quality QD source was recently used in exciting demonstrations to create a train of single photons, which were temporally multiplexed to the input of a Boson Sampler.15,16 Published in partnership with The University of New South Wales Experimental results Dashed line represents Poissonian statistics of driving laser 0.01 0.1 1 10 100 10-5 10-4 10-3 10-2 10-1 100 -60 -30 0 30 60 0 200 400 600 800 Pulse length (1/γ) b g(2)[0] a Intensity (counts) Time delay (ns) Fig. 2 Measured degree of second-order coherence. a Example of a measurement for two-photon excitation and filtering on the 2X emission using a pulse length of 3 ps. b Measured values of g(2)[0] as a function of the pulse length for a resonantly driven two-level system (black) and two-photon excitation of 2X (red). Dashed line represents Poissonian statistics of driving laser 2 0.2 0.3 0.4 914 912 910 H V b X X- Η V 2X X Gate voltage (V) Wavelength (nm) 0 X- e- a Eb -60 -30 0 30 60 0 200 400 600 800 a Intensity (counts) Time delay (ns) b a ) b 0.01 0.1 1 10 100 10-5 10-4 10-3 10-2 10-1 100 Pulse length (1/γ) b g(2)[0] y ( ) b g ( ) 910 911 912 913 914 915 0 5 10 Laser 2X X c Intensity (arb.u.) Wavelength (nm) c Fig. 2 Measured degree of second-order coherence. a Example of a measurement for two-photon excitation and filtering on the 2X emission using a pulse length of 3 ps. b Measured values of g(2)[0] as a function of the pulse length for a resonantly driven two-level system (black) and two-photon excitation of 2X (red). Dashed line represents Poissonian statistics of driving laser 0 1 2 5π 4π 3π 2π π d Intensity (arb. u.) Excitation power (arb. units) d rates. As discussed above, for the resonantly excited 2LS, g(2)[0] is limited by re-excitation that is enabled by emission of a photon during the presence of the pulse. In contrast, for a two-photon excitation of 2X, re-excitation is strongly suppressed. Because the laser is far detuned from the 2X transition by Eb/2 re-excitation following the excitation of 2X can only occur after the cascade 2X j i ! X j i ! 0j i has returned the system to the ground state. Si h i i b bili d d h l l h Excitation power (arb. units) Fig. 1 Two-photon excitation scheme. a Gate voltage dependent photoluminescence. b Schematic illustration of resonant excitation of a 2LS and two-photon excitation of 2X. Solid arrows represent laser drive, while dotted arrows represent spontaneous emission. Experimental results c Example spectrum for two-photon excitation of 2X at 912.34 nm. d Rabi oscillations between 0j i and 2X j i, measured from 2X luminescence Since the re-excitation probability depends on the pulse length T, we performed measurements of g(2)[0] for different values of T. The results are presented as red datapoints in Fig. 2b. For comparison, the values obtained for a resonantly driven 2LS formed by the X−transition of the same QD are presented in Fig. 2b as black datapoints (reproduced from ref. 19). Thereby, the pulse lengths are normalized to γX and γx, respectively. Note, for the 2LS data and very short pulses, g(2)[0] was corrected for an imperfect suppression of the excitation laser which can be quantified by electrically detuning the X−transition.19 For both cases g(2)[0] increases with pulse length and asymptotically approaches the classical limit of 1 for long pulses. Crucially, for all measured pulse lengths, the values obtained from the two- photon excitation scheme are significantly lower than the resonantly excited 2LS. The improvement in g(2)[0] amounts to an improvement of several orders of magnitude for sufficiently short pulses. A power law fit in the short pulse regime (not shown) results coefficients of 0.73 ± 0.07 for the resonantly driven 2LS and 1.89 ± 0.40 for the two-photon excitation scheme. This indicates a scaling behavior of approximately g(2)[0] ∝Tγ for the resonantly driven 2LS and g(2)[0] ∝ðTγÞ2 for the two-photon excitation scheme, which will be confirmed in theoretical considerations below. Slight deviations of the experimental data from this behavior can be attributed to changes in the pulse shape when increasing the pulse length. the emission intensity on the excitation power is presented in Fig. 1d for exciting with 3 ps long pulses and reveals clean Rabi oscillations. Time-resolved measurements reveal lifetimes of 260 ps for the emission from X and 173 ps for the emission from 2X. To generate single photons from this system, the emission has simply to be frequency filtered to the 2X or X transition. The single-photon purity is quantified by the measured degree of second-order coherence g(2)[0] = 〈n(n −1)〉/〈n〉2 where n is the number of photons per pulse. We performed measurements of g(2)[0] for two-photon excitation and frequency filtered detection on 2X using a standard Hanbury-Brown and Twiss (HBT) setup. An example measurement for 3 ps long excitation pulses of area π is presented in Fig. 2a. Experimental results The obtained values for g(2)[0] are so low that fitting the data with a series of peaks does not yield a value. Therefore, we integrate the data over an interval that contains the complete peaks (2.6 ns) and compare the integrated counts around zero time delay to the average intensity of the peaks away from zero delay. After subtracting a constant dark count background we obtain a value of g(2)[0] = 9.4 × 10−5 ± 1.9 × 10−3. The measured value of g(2)[0] is lower than values obtained for a resonantly driven 2LS.17 While the upper limit is dominated by the error which results from the dark counts of the avalanche photodiodes used here, very recently Schweickert et al.27 have reported a value of g(2)[0] = (7.5 ± 1.6) × 10−5 using the same scheme but superconducting detectors with negligible dark count npj Quantum Information (2018) 43 Published in partnership with The University of New South Wales Experimental results b Measured values of g(2)[0] as a function of the pulse length for a resonantly driven two-level system (black) and two-photon excitation of 2X (red). Dashed line represents Poissonian statistics of driving laser rates. As discussed above, for the resonantly excited 2LS, g(2)[0] is limited by re-excitation that is enabled by emission of a photon during the presence of the pulse. In contrast, for a two-photon excitation of 2X, re-excitation is strongly suppressed. Because the laser is far detuned from the 2X transition by Eb/2 re-excitation following the excitation of 2X can only occur after the cascade 2X j i ! X j i ! 0j i has returned the system to the ground state. Since the re-excitation probability depends on the pulse length T, we performed measurements of g(2)[0] for different values of T. The results are presented as red datapoints in Fig. 2b. For comparison, the values obtained for a resonantly driven 2LS f d b h f h d 0 1 2 0.2 0.3 0.4 914 912 910 H V b X X- Η V 2X X Gate voltage (V) Wavelength (nm) 0 X- e- a 5π 4π 3π 2π π d Eb Intensity (arb. u.) Excitation power (arb. units) 910 911 912 913 914 915 0 5 10 Laser 2X X c Intensity (arb.u.) Wavelength (nm) Fig. 1 Two-photon excitation scheme. a Gate voltage dependent photoluminescence. b Schematic illustration of resonant excitation of a 2LS and two-photon excitation of 2X. Solid arrows represent laser drive, while dotted arrows represent spontaneous emission. c Example spectrum for two-photon excitation of 2X at 912.34 nm. d Rabi oscillations between 0j i and 2X j i, measured from 2X luminescence 0.01 0.1 1 10 100 10-5 10-4 10-3 10-2 10-1 100 -60 -30 0 30 60 0 200 400 600 800 Pulse length (1/γ) b g(2)[0] a Intensity (counts) Time delay (ns) Fig. 2 Measured degree of second-order coherence. a Example of a measurement for two-photon excitation and filtering on the 2X emission using a pulse length of 3 ps. b Measured values of g(2)[0] as a function of the pulse length for a resonantly driven two-level system (black) and two-photon excitation of 2X (red). Experimental results a Example of a measurement for two-photon excitation and filtering on the 2X emission using a pulse length of 3 ps. b Measured values of g(2)[0] as a function of the pulse length for a resonantly driven two-level system (black) and two-photon excitation of 2X (red). Dashed line represents Poissonian statistics of driving laser the emission intensity on the excitation power is presented in Fig. 1d for exciting with 3 ps long pulses and reveals clean Rabi oscillations. Time-resolved measurements reveal lifetimes of 260 ps for the emission from X and 173 ps for the emission from 2X. To generate single photons from this system, the emission has simply to be frequency filtered to the 2X or X transition. The single-photon purity is quantified by the measured degree of second-order coherence g(2)[0] = 〈n(n −1)〉/〈n〉2 where n is the number of photons per pulse. We performed measurements of g(2)[0] for two-photon excitation and frequency filtered detection on 2X using a standard Hanbury-Brown and Twiss (HBT) setup. An example measurement for 3 ps long excitation pulses of area π is presented in Fig. 2a. The obtained values for g(2)[0] are so low that fitting the data with a series of peaks does not yield a value. Therefore, we integrate the data over an interval that contains the complete peaks (2.6 ns) and compare the integrated counts around zero time delay to the average intensity of the peaks away from zero delay. After subtracting a constant dark count background we obtain a value of g(2)[0] = 9.4 × 10−5 ± 1.9 × 10−3. The measured value of g(2)[0] is lower than values obtained for a resonantly driven 2LS 17 While the upper limit is dominated by the rates. As discussed above, for the resonantly excited 2LS, g(2)[0] is limited by re-excitation that is enabled by emission of a photon during the presence of the pulse. In contrast, for a two-photon excitation of 2X, re-excitation is strongly suppressed. Because the laser is far detuned from the 2X transition by Eb/2 re-excitation following the excitation of 2X can only occur after the cascade 2X j i ! X j i ! 0j i has returned the system to the ground state. Since the re-excitation probability depends on the pulse length T, we performed measurements of g(2)[0] for different values of T. The results are presented as red datapoints in Fig. 2b. Experimental results For comparison, the values obtained for a resonantly driven 2LS formed by the X−transition of the same QD are presented in Fig. 2b as black datapoints (reproduced from ref. 19). Thereby, the pulse lengths are normalized to γX and γx, respectively. Note, for the 2LS data and very short pulses, g(2)[0] was corrected for an imperfect suppression of the excitation laser which can be quantified by electrically detuning the X−transition.19 For both cases g(2)[0] increases with pulse length and asymptotically approaches the classical limit of 1 for long pulses. Crucially, for all measured pulse lengths, the values obtained from the two- photon excitation scheme are significantly lower than the resonantly excited 2LS. The improvement in g(2)[0] amounts to an improvement of several orders of magnitude for sufficiently short pulses. A power law fit in the short pulse regime (not shown) results coefficients of 0.73 ± 0.07 for the resonantly driven 2LS and 1.89 ± 0.40 for the two-photon excitation scheme. This indicates a scaling behavior of approximately g(2)[0] ∝Tγ for the resonantly driven 2LS and g(2)[0] ∝ðTγÞ2 for the two-photon excitation scheme, which will be confirmed in theoretical considerations below. Slight deviations of the experimental data from this behavior can be attributed to changes in the pulse shape when increasing the pulse length. 0 1 2 0.2 0.3 0.4 914 912 910 H V b X X- Η V 2X X Gate voltage (V) Wavelength (nm) 0 X- e- a 5π 4π 3π 2π π d Eb Intensity (arb. u.) Excitation power (arb. units) 910 911 912 913 914 915 0 5 10 Laser 2X X c Intensity (arb.u.) Wavelength (nm) Fig. 1 Two-photon excitation scheme. a Gate voltage dependent photoluminescence. b Schematic illustration of resonant excitation of a 2LS and two-photon excitation of 2X. Solid arrows represent laser drive, while dotted arrows represent spontaneous emission. c Example spectrum for two-photon excitation of 2X at 912.34 nm. d Rabi oscillations between 0j i and 2X j i, measured from 2X luminescence 0.01 0.1 1 10 100 10-5 10-4 10-3 10-2 10-1 100 -60 -30 0 30 60 0 200 400 600 800 Pulse length (1/γ) b g(2)[0] a Intensity (counts) Time delay (ns) Fig. 2 Measured degree of second-order coherence. a Example of a measurement for two-photon excitation and filtering on the 2X emission using a pulse length of 3 ps. Experimental results The sample consists of InGaAs QDs of low areal density (<1 μm−2) embedded in the intrinsic region of an n-i Schottky diode. The QDs are grown at a distance of 35 nm from the n-doped region which allows control of the charge occupancy of the QDs. A field- dependent photoluminescence measurement is presented in Fig. 1a and confirms clear charge stability plateaus for the neutral exciton transitions X and emission from a negatively charged trion (X−). The latter can be used as a true 2LS with emission rate γX (Fig. 1b—left) while the former is part of a four-level system given by the biexciton 2X, exciton X and ground state 0 (Fig. 1b—right). Due to anisotropy in QD shape, the exchange interaction results in two X levels where one couples 2X and 0 with horizontal polarization and the other with vertical polarization.21,22 Depend- ing on the specific type of QD, the two X levels are non- degenerate with a fine structure splitting of 0–100 μeV. This system is well-known for the generation of entangled photon pairs.23–25 Due to the Coulomb interaction, the energy of 2X is detuned from twice the X energy by the binding energy Eb. Therefore, 2X can be excited via a two-photon process where the laser energy is detuned from X by Eb/2.24,26 The emission rates of this system are γ2x and γx. A typical spectrum for two-photon excitation of 2X is presented in Fig. 1c and confirms identical intensities for 2X and X emission as expected. The dependence of In this letter, we investigate an alternative scheme which is based on a four-level system, given by the biexciton-exciton ladder in a QD, and demonstrate that it facilitates significantly higher single-photon purity than a resonantly driven 2LS due to dramatically reduced re-excitation. At the same time, it maintains a simple implementation and high single-photon generation rates. Moreover, it enables an even higher brightness since it eliminates the need for polarization suppression of the excitation laser. Published in partnership with The University of New South Wales Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. 0.01 0.1 1 10 100 10-5 10-4 10-3 10-2 10-1 100 -60 -30 0 30 60 0 200 400 600 800 Pulse length (1/γ) b g(2)[0] a Intensity (counts) Time delay (ns) Fig. 2 Measured degree of second-order coherence. Theoretical results b, c Simulated photocount distribution Pn, i.e., the probability for n different detections to occur, either from the emission of b a 2LS or c a 3LS filtered on either the 2X or X′ transition frequency A3LSðtÞ ¼ Z t 0 dt0 ðμ  Eðt0ÞÞ2 hEb (4) (4) and We can then calculate the pulse-wise second-order coherences gð2Þ k ½0 from the integrated versions of the correlators17 We can then calculate the pulse-wise second-order coherences gð2Þ k ½0 from the integrated versions of the correlators17 H3LSðtÞ ¼ μ  EðtÞ ð Þ2 2Eb 0j i 2X h j þ 2X j i 0 h j ð Þ (5) (5) Gð2Þ k t1; t2 ð Þ ¼ tr J Lk ½ V t2; t1 ð ÞJ Lk ½ V t1; 0 ð Þρð0Þ ½ : (10) (10) Gð2Þ k t1; t2 ð Þ ¼ tr J Lk ½ V t2; t1 ð ÞJ Lk ½ V t1; 0 ð Þρð0Þ ½  where σ 2 0j i X0 h j; X0 j i 2X h j f g are the system’s dipole operators. The operator 0j i 2X h j only appears after adiabatic elimination of the intermediate state. These coherences were calculated for A = π pulses (we took γ ¼ γx ¼ γ2x=2), driving both the 2LS (Fig. 3a—black) and 3LS (Fig. 3a—red), and they very closely match the experimental results of Fig. 2b. Small differences between experiment and theory result from experimental inaccuracies, such as the error in determining the pulse area of π, inaccuracies in the pulse shape, as well as drifts and fluctuations in power over the duration of the measurements.19 We also note that we performed all quantum simulations with the Quantum Toolbox in Python (QuTiP).29 The dynamics of the systems under spontaneous emission into Markovian reservoirs are captured in the density operator for the systems, whose evolutions can be written in terms of a Liouvillian as ρ t1 ð Þ ¼ V t1; t0 ð Þρ t0 ð Þ ¼ T exp R t1 t0dtLðtÞ h i ρ t0 ð Þ; (6) (6) where T←is the chronological operator which orders the infinitesimal products in Eq. (6). The Liouvillian is a superoperator defined by The final goal is to compare these results to the theoretical photocount distribution Pn, from which the photodetectors sample. Theoretical results Next, we gain insight into the behavior through a theoretical study of the emission from an ideal 2LS and 2X system. First, consider an ideal 2LS,19,28 with a ground state e j i and an excited state X j i. Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. 3 10-5 10-4 10-3 10-2 10-1 100 0.01 0.1 1 10 100 10-5 10-4 10-3 10-2 10-1 100 10-5 10-4 10-3 10-2 10-1 100 P0 P1 P2 Pn b P0 P1 P2 Pulse length (1/γ) Pn c 2LS 3LS a g(2)[0] Fig. 3 Probabilities for different photodetection events. a Simulated values of g(2)[0] as a function of the pulse length for a resonantly driven two-level system (black) and two-photon excitation of 2X (red). Dashed line represents Poissonian statistics of driving laser. b, c Simulated photocount distribution Pn, i.e., the probability for n different detections to occur, either from the emission of b a 2LS or c a 3LS filtered on either the 2X or X′ transition frequency 10-5 10-4 10-3 10-2 10-1 100 10-5 10-4 10-3 10-2 10-1 100 P0 P1 P2 Pn b 2LS 3LS a g(2)[0] 10-5 10-4 10-3 10-2 10-1 100 2LS 3LS a g(2)[0] Suppose the system is driven by an optical pulse starting at t = 0, resonant with the e j i $ X j i transition and where the rotating wave approximation holds. As a function of the interacted pulse area a A2LSðtÞ ¼ Z t 0 dt0μ  Eðt0Þ=h; (1) (1) where E(t′) is the envelope of the pulse’s electric field and μ the system’s electric dipole moment, the system undergoes coherent oscillations between its ground e j i and excited X j i states. If the system is initially prepared in the ground state, as is typical in cryogenic experiments, the probability of the system being in the excited state PXðAðtÞÞ shows Rabi oscillations that are nearly sinusoidal where E(t′) is the envelope of the pulse’s electric field and μ the system’s electric dipole moment, the system undergoes coherent oscillations between its ground e j i and excited X j i states. Theoretical results If the system is initially prepared in the ground state, as is typical in cryogenic experiments, the probability of the system being in the excited state PXðAðtÞÞ shows Rabi oscillations that are nearly sinusoidal 10-5 10-4 10-3 10-2 10-1 100 P0 P1 P2 Pn b (2) PXðAðtÞÞ  sin2ðAðtÞ=2Þ; (2) PXðAðtÞÞ  sin2ðAðtÞ=2Þ; for excitation by a short pulse relative to the spontaneous emission time of the 2LS. The Rabi oscillations are captured by the Hamiltonian (in a reference frame rotating at the laser frequency) 0.01 0.1 1 10 100 10-5 10-4 10-3 10-2 10-1 100 P0 P1 P2 Pulse length (1/γ) Pn c c H2LSðtÞ ¼ μ  EðtÞ 2 e j i X h j þ X j i e h j ð Þ; (3) (3) where σ ¼ e j i X h j is the system’s dipole operator. j ih j Second, to model the 2X system we will actually use only a three-level system (3LS) with levels labeled as 0j i, X0 j i and 2X j i. Although there is strictly no transformation that makes these systems equivalent, if the polarization of photons emitted is disregarded in the photon counting procedure, then the behavior of the 3LS mirrors that of the 2X system. Since a two-photon transition excites the system 0j i $ 2X j i via the intermediate state X0 j i, the system undergoes Rabi oscillations that scale linearly with the pulse power rather than the field. Hence, Fig. 3 Probabilities for different photodetection events. a Simulated values of g(2)[0] as a function of the pulse length for a resonantly driven two-level system (black) and two-photon excitation of 2X (red). Dashed line represents Poissonian statistics of driving laser. npj Quantum Information (2018) 43 Published in partnership with The University of New South Wales reservoir is approximately Then, the photocount distribution into the k-th channel is given by In the short pulse regime P2  γ2 2XγX R T 0 R T t1 R T t0 1dt1dt0 1dt2 sin At1 2T  2sin Aðt2t10Þ 2T 2 þγ2XγX R T 0 R T t1 dt1dt0 1 sin At1 2T  2sin A Tt0 1 ð Þ 2T 2  OðT4Þ þ O T2 ð Þ: (21) (21) Pn ¼ Z 1 0 dtn Z tn 0 dtn1    Z t2 0 dt1pn t1; t2; ¼ ; tn ð Þ: (15) (15) Since the leading order of the second integral is lowest, and for an A = π pulse, the 3LS has Note there is only one possible way to count zero photons emitted: P2  γ2XγX R T 0 R T t1 dt1dt0 1 sin At1 2T  2sin A Tt0 1 ð Þ 2T 2  γ2XγXT2 π28 8π2 (22) P0 ¼ lim t!1 tr½Kðt; 0Þρð0Þ: (16) (22) (16) To our knowledge, this is the first use of such a model to extract photocount distributions for photon sources. For a single-photon source, driven with area A = π, only Pn for n < 3 are significant— we calculate these probabilities in Fig. 3 for (b) the 2LS and (c) the 3LS filtered on either transition frequency. For the single-photon sources, g(2)[0] ≈2P2/(P1 + 2P2)2, which scales linearly for the 2LS and quadratically for the 3LS with pulse length. Then, the error rate for the single-photon source is directly accessible as P2 ≈ g(2)[0]/2. or g(2)[0] ≈γ2xγxT2(π2 −8)/4π2. This quadratic scaling matches exactly with the linear region of the theoretical results in Fig. 3 and the experimentally observed results in Fig. 2. In summary, we have quantified the re-excitation process in resonantly driven 2LS and two-photon excitation in 3LS. We have demonstrated that the 3LS, and hence the 2X system, dramatically suppresses re-excitation resulting in orders of magnitude better single-photon source operation. reservoir is approximately p t1; t2; t0 1    γ2X sin At1 2T  2γXeγX t0 1t1 ð Þγ2X sin A t2t0 1 ð Þ 2T 2 if 0<t1<t0 1<t2<T γ2X sin At1 2T  2γXeγX t0 1t1 ð Þ γ2Xsin A Tt0 1 ð Þ 2T 2eγ2X t2T ð Þ if 0<t1<t0 1<T<t2 0 otherwise 8 > > < > > : (18) ρðtÞ ¼ P 1 n¼0 R t 0 dtn R tn 0 dtn1    R t2 0 dt1K t; tn ð ÞJ Lk ½  ´ K tn; tn1 ð ÞJ Lk ½     K t2; t1 ð ÞJ Lk ½ K t1; 0 ð Þρð0Þ: (12) (12) (18) This summation is over different numbers of photon emissions into the k-th reservoir, such that the probability density for a sequence of n emissions at times t1, t2, …, tn < t over the interval [0, t] is given by  γ2X sin At1 2T  2 γXγ2X sin A t2t0 1 ð Þ 2T 2 if 0<t1<t0 1<t2<T γ2X sin At1 2T  2 γX γ2Xsin A Tt0 1 ð Þ 2T 2eγ2X t2T ð Þ if 0<t1<t0 1<T<t2 0 otherwise 8 > > < > > : (19) pn t1; t2; ¼ ; tn; ½0; t ð Þ ¼ tr K t; tn ð ÞJ Lk ½ K tn; tn1 ð ÞJ Lk ½  ½    K t2; t1 ð ÞJ Lk ½ K t1; 0 ð Þρð0Þ: (13) (13) (13) (19) where t0 1 is the time of emission of the first photon at the X transition frequency. Hence, the two-photon error rate at the 2X frequency is We drop the time label by taking the limit pn t1; t2; ¼ ; tn ð Þ  lim t!1 pn t1; t2; ¼ ; tn; ½0; t ð Þ; (14) (14) P2 ¼ Z 1 0 Z 1 0 Z 1 0 dt1dt0 1dt2p t1; t2; t0 1   : (20) (20) which corresponds to the case where the system has entirely decayed after excitation by the laser pulse. In practice, we just integrate for a few spontaneous emission lifetimes after the pulse ends. reservoir is approximately While our experiments are a proof-of-principle with a sample structure that does not provide a high collection efficiency, the technique is directly applicable to QDs embedded in nanophotonic structures where a very efficient detection of emitted photons is possible.34–37 Beyond superior single-photon purity, the 2X scheme has the advantage over a resonantly driven 2LS that no cross-polarized suppression of the excitation laser is necessary because the driving laser and emission are far detuned such that the laser can easily be spectrally filtered out. Thus, it is easier to implement, as misalignment or optical imperfections do not pose an additional possibility for multi-photon errors. For example, very short laser pulses typically degrade cross-polarized suppression due to the increased spectral width of the laser, a wavelength sensitivity of the suppression and less efficient driving. Moreover, it does not reduce the source brightness which would be the case in a resonantly driven 2LS where laser and signal are orthogonally polarized but need to couple to the same transition. To obtain high photon indistinguishability experimentally the emission has still to be filtered to one polarization, however Purcell enhancing just one polarization with a nanoresonator would transform the four-level system to an effective three-level system with a source brightness near unity. Therefore, we expect this scheme combined with appropriate nanoresonators be an excellent candidate for a single-photon source in future quantum information processors. Published in partnership with The University of New South Wales unraveled with respect to n emissions into the k-th reservoir as unraveled with respect to n emissions into the k-th reservoir as unraveled with respect to n emissions into the k-th reservoir as reservoir is approximately Theoretical results The ideal 2LS emits an entirely pure photonic state into the reservoir,30 whereas the 3LS cascade is known to emit an entangled state between the reservoirs.17,23 From the perspective of a single reservoir, i.e., tracing over the other reservoir, this means the state could be highly mixed and hence our previous techniques would not apply easily.17,19,30 Instead, we will use the Mandel photon counting formula, as connected to the system state by Carmichael.31–33 To do this, we first define a new superoperator LðtÞρðtÞ ¼ i HðtÞ; ρðtÞ ½  þ X k D Lk ½ ρðtÞ; (7) (7) with the Dissipator defined as with the Dissipator defined as D½LρðtÞ ¼ J ½LρðtÞ  1 2 LyL; ρðtÞ   (8) D½LρðtÞ ¼ J ½LρðtÞ  1 2 LyL; ρðtÞ   (8) and the recycling (or emission) superoperator and the recycling (or emission) superoperator J ½LρðtÞ ¼ LρðtÞLy: (9) J ½LρðtÞ ¼ LρðtÞLy: (9) K t1; t0 ð Þ ¼ T exp Z t1 t0 dt LðtÞ  J Lk ½  ð Þ   ; (11) Finally, Lk are the loss operators defined by the system operators σk and their coupling rates to the reservoirs γk, i.e., Lk ¼ ffiffiffiffiffi γk p σk. Even though the 2X system physically emits into the same reservoirs for 2X j i ! X j i and X j i ! 0j i, they are at such different frequencies Eb  γ; γX; γ2X ð Þ they can be considered to emit into separate Markovian reservoirs. (11) which can be thought of as an unnormalized map that evolves the density matrix conditioned on no photon emissions into the k-th reservoir. Then, the total density matrix evolution can be s npj Quantum Information (2018) 43 Published in partnership with The University of New South Wales Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. 4 Published in partnership with The University of New South Wales REFERENCES 1. Michler, P. (ed.) Quantum Dots for Quantum Information Technologies (Springer, Cham, 2017). 2. Senellart, P., Solomon, G. & White, A. High-performance semiconductor quantum- dot single-photon sources. Nat. Nanotechnol. 12, 1026–1039 (2017). 3. Prechtel, J. H. et al. Frequency-stabilized source of single photons from a solid- state qubit. Phys. Rev. X 3, 041006 (2013). 4. Hansom, J. et al. Frequency stabilization of the zero-phonon line of a quantum dot via phonon-assisted active feedback. Appl. Phys. Lett. 105, 172107 (2014). 5. Kuhlmann, A. V. et al. Transform-limited single photons from a single quantum dot. Nat. Commun. 6, 8204 (2015). 6. He, Y.-M. et al. On-demand semiconductor single-photon source with near-unity indistinguishability. Nat. Nano 8, 213–217 (2013). Second-order autocorrelation measurements were performed using a HBT set-up consisting of one 50:50 beamsplitter and two single-photon avalanche diodes. The measured count rate for exciting 2X with a pulse of area π was 9 kcps and the dark count rates of the detectors are 251 ± 16 cps and 95 ± 10 cps. The detected photons were correlated with a TimeHarp200 time-counting module. The time-bin width was 60 ps. The integration time for Fig. 2a was 11.05 h and for Fig. 2b between 7.52 h and 12.27 h. In the pulse-wise form gð2Þ½0 ¼ N0 N1 where N0 is the integrated area of the center peak and and N1 is the average area of the side peaks. We used 16 side peaks for the averaging, which is the largest number that we can record with our correlation electronics and the used bin-width of 60 ps. Note, that no long-term decay of the side peaks was observed, indicating the absence of any blinking and consistent with the fact that we use electronically stabilized devices. The error in N0 and N1 is given by ffiffiffiffiffiffi N0 p and ffiffiffiffiffiffi N1 p =4, where the factor of 4 results from the fact that the integrated area of 16 peaks was used to calculate N1. The error in g(2)[0] can then be calculated using quadratic propagation. 7. Somaschi, N. et al. Near-optimal single-photon sources in the solid state. Nat. Photon 10, 340–345 (2016). 8. Unsleber, S. et al. Highly indistinguishable on-demand resonance fluorescence photons from a deterministic quantum dot micropillar device with 74% extrac- tion efficiency. Opt. Express 24, 8539–8546 (2016). 9. Ding, X. et al. REFERENCES On-demand single photons with high extraction efficiency and near-unity indistinguishability from a resonantly driven quantum dot in a micropillar. Phys. Rev. Lett. 116, 020401 (2016). 10. He, Y.-M. et al. Deterministic implementation of a bright, on-demand single photon source with near-unity indistinguishability via quantum dot imaging. Optica 4, 802 (2016). 11. Müller, K. et al. Self-homodyne-enabled generation of indistinguishable photons. Optica 3, 931 (2016). 12. Wang, H. et al. Near-transform-limited single photons from an efficient solid-state quantum emitter. Phys. Rev. Lett. 116, 213601 (2016). 13. Loredo, J. C. et al. Scalable performance in solid-state single-photon sources. Optica 3, 433 (2016). 14. Iles-Smith, J., McCutcheon, D. P. S., Nazir, A. & Mørk, J. Phonon scattering inhibits simultaneous near-unity efficiency and indistinguishability in semiconductor single-photon sources. Nat. Photonics 11, 521–526 (2017). The measured correlations have a constant background that results from dark counts of the detectors. To correct for the background, we first calculate the dark counts per time bin nBG by averaging a large number of bins between the peaks. The background corrected value of g(2)[0] is then given by gð2Þ corr½0 ¼ N0;corr N1;corr where N0,corr = N0 −NBG and N1,corr = N1 −NBG. Note, that the error still results from ffiffiffiffiffiffi N0 p and not ffiffiffiffiffiffiffiffiffiffiffiffi N0;corr p , highlighting the importance of detectors with low dark counts for the characterization single-photon sources with ultra-low multi-photon error rates. single-photon sources. Nat. Photonics 11, 521–526 (2017). 15. Loredo, J. C. et al. Boson sampling with single-photon fock states from a bright solid-state source. Phys. Rev. Lett. 118, 130503 (2017). 16. Wang, H. et al. High-efficiency multiphoton boson sampling. Nat. Photonics 11, 361–365 (2017). 17. Fischer, K. A., Müller, K., Lagoudakis, K. G. & Vuckovic, J. Dynamical modeling of pulsed two-photon interference. New J. Phys. 18, 113053 (2016). 18. Fischer, K. A. et al. Signatures of two-photon pulses from a quantum two-level system. Nat. Phys. 13, 649–654 (2017). METHODS The sample investigated is grown by molecular beam epitaxy (MBE). It consists of a layer of InGaAs QDs with low areal density (<1 μm−2), embedded within the intrinsic region of a Schottky photodiode formed from an n-doped layer below the QDs and a semitransparent titanium gold front contact. The distance between the doped layer and the QDs is 35 nm, which enables control over the charge status of the dot. A weak planar microcavity with an optical thickness of one wavelength is formed from a buried 18-pair GaAs/AlAs distributive Bragg reflector (DBR) and the semitransparent top contact, which enhances the in-coupling and out- coupling of light. We gratefully acknowledge financial support from the DFG via the Nanosystems Initiative Munich, the BMBF via Q.Com (Project No. 16KIS0110), BaCaTeC, the International Graduate School of Science and Engineering (IGSSE) of TUM and the National Science Foundation (Division of Materials Research Grant No. 1503759). D.L. acknowledges support from the Fong Stanford Graduate Fellowship and the National Defense Science and Engineering Graduate Fellowship. J.W. acknowledges support from the PhD programme ExQM of the Elite Network of Bavaria. J.V. gratefully acknowledges support from the TUM Institute of Advanced Study. R.T. acknowledges support from the Kailath Stanford Graduate Fellowship. K.M. acknowledges support from the Bavarian Academy of Sciences and Humanities. All optical measurements were performed at 4.2 K in a liquid helium dipstick set-up. For excitation and detection, a microscope objective with a numerical aperture of NA = 0.68 was used. Cross-polarized measurements were performed using a polarizing beam splitter. To further enhance the extinction ratio, additional thin film linear polarizers were placed in the excitation/detection pathways and a single-mode fibre was used to spatially filter the detection signal. Furthermore, a quarter-wave plate was placed between the beamsplitter and the microscope objective to correct for birefringence of the optics and the sample itself. DISCUSSION As we have shown, the bi-excitonic and effective 3-level systems are superior single-photon sources to a 2LS. We briefly provide an approximate analysis which yields strong insight into the fundamental reason behind this behavior. Previously, we derived an analytic estimate for the two-photon error rate P2 and hence g(2)[0], for short pulses resonantly driving a 2LS.19 Keeping only terms to first order in γT, where T is the pulse length, results for short pulses are P2  γT 8 and gð2Þ½0  γT 4 . Here, we further derive an analytic estimate for the 3LS. For the 3LS, the only way for two emissions to occur at the 2X transition frequency is through the cycle of transitions 0j i ! 2X j i ! X0 j i ! 0j i ! 2X j i: (17) (17) As in the 2LS, the first emission 2X j i ! X0 j i contributes a factor of γ2Xsin At 2T  2 to the emission probability density, under the approximation of a square driving pulse. After the first emission, the system is almost exclusively in X0 j i so X0 j i ! 0j i contributes a factor of γXeγXt. Finally, 0j i ! 2X j i again provides another γ2Xsin At 2T  2. Hence, the two-photon emission density into the first npj Quantum Information (2018) 43 npj Quantum Information (2018) 43 Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. 5 ADDITIONAL INFORMATION For Fig. 1c, d, a weak laser background (due to an imperfect suppression of the excitation laser) was subtracted. This linearly increasing background was directly measured through electrically tuning the quantum dot out of resonance, and typically amounted to less than 10% of the signal by 5π pulse area. Competing interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. p The 3 ps to 80 ps-long excitation pulses were derived from a fs-pulsed titanium sapphire laser (Coherent Mira 900) through pulse shaping. For the 3 ps to 25 ps long pulses, a 4f pulse shaper with a focal length of 1 m and an 1800 lmm−1 grating was used. For the 80 ps long pulses a spectrometer-like filter with a focal length of 1 m and an 1800 lmm−1 grating was used. Longer pulses were obtained through modulating a continuous wave laser. For the modulation, a fibre-coupled and EOM- controlled lithium niobate Mach-Zehnder (MZ) interferometer with a bandwidth of 10 GHz (Photline NIR-MX-LN-10) was used. Such modulators allow control of the output intensity through a DC bias and a radiofrequency input. The radiofrequency pulses were generated by a 3.35 GHz pulse-pattern generator (Agilent 81133A). To obtain a high extinction ratio, the temperature of the modulator was stabilized and precisely controlled (1 mK) using a Peltier element, thermistor, and TEC controller. This enabled a static extinction ratio >45 db. AUTHOR CONTRIBUTIONS L.H., S.A., J.W., and K.M. performed the experiments. K.A.F., D.L., S.S., and R.T. performed the theoretical work and modelling. J.V. and J.J.F. provided expertise. K.A. L.H., S.A., J.W., and K.M. performed the experiments. K.A.F., D.L., S.S., and R.T. performed the theoretical work and modelling. J.V. and J.J.F. provided expertise. K.A. F. and K.M. conceived the idea. All authors participated in the discussion and understanding of the results. L.H. and K.A.F. contributed equally. L.H., S.A., J.W., and K.M. performed the experiments. K.A.F., D.L., S.S., and R.T. performed the theoretical work and modelling. J.V. and J.J.F. provided expertise. K.A. F. and K.M. conceived the idea. All authors participated in the discussion and understanding of the results. L.H. and K.A.F. contributed equally. performed the theoretical work and modelling. J.V. and J.J.F. provided expertise. K.A. F. and K.M. conceived the idea. All authors participated in the discussion and understanding of the results. L.H. and K.A.F. contributed equally. npj Quantum Information (2018) 43 Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. L Hanschke et al. L Hanschke et al. 6 21. Bayer, M. et al. Fine structure of neutral and charged excitons in self-assembled In (Ga)As/(Al)GaAs quantum dots. Phys. Rev. B 65, 195315 (2002). 21. Bayer, M. et al. Fine structure of neutral and charged excitons in self-assembled In (Ga)As/(Al)GaAs quantum dots. Phys. Rev. B 65, 195315 (2002). 33. Gardiner, C. & Zoller, P. The Quantum World of Ultra-Cold Atoms and Light Book II: The Physics of Quantum-Optical Devices, 1–524 (World Scientific, London, 2015). 33. Gardiner, C. & Zoller, P. The Quantum World of Ultra-Cold Atoms and Light Book II: The Physics of Quantum-Optical Devices, 1–524 (World Scientific, London, 2015). 22. Finley, J. J. et al. Fine structure of charged and neutral excitons in InAs-AlGaAs quantum dots. Phys. Rev. B 66, 153316 (2002). 34. Gazzano, O. et al. Bright solid-state sources of indistinguishable single photons. Nat. Commun. 4, 1425 (2013). 23. Akopian, N. et al. Entangled photon pairs from semiconductor quantum dots. Phys. Rev. Lett. 96, 130501 (2006). 35. Claudon, J. et al. A highly efficient single-photon source based on a quantum dot in a photonic nanowire. Nat. Photonics 4, 174–177 (2010). 24. Müller, M. et al. On-demand generation of indistinguishable polarization- entangled photon pairs. Nat. Photonics 8, 224–228 (2014). 36. Gschrey, M. et al. Highly indistinguishable photons from deterministic quantum- dot microlenses utilizing three-dimensional in situ electron-beam lithography. Nat. Commun. 6, 7662 (2015). 25. Huber, D. et al. Highly indistinguishable and strongly entangled photons from symmetric GaAs quantum dots. Nat. Commun. 8, 15506 (2017). 37. Sapienza, L. et al. Nanoscale optical positioning of single quantum dots for bright and pure single-photon emission. Nature Commun 6, 7833 (2015). 26. Ardelt, P. L. et al. Dissipative preparation of the exciton and biexciton in self- assembled quantum dots on picosecond time scales. Phys. Rev. B 90, 241404 (2014). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Published in partnership with The University of New South Wales npj Quantum Information (2018) 43 Published in partnership with The University of New South Wales DATA AVAILABILITY 19. Fischer, K. A. et al. Pulsed rabi oscillations in quantum two-level systems: beyond the area theorem. Quantum Sci. Technol. 3, 014006 (2017). The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request. the area theorem. Quantum Sci. Technol. 3, 014006 (2017). 20. Dada, A. C. et al. Indistinguishable single photons with flexible electronic trig- gering. Optica 3, 493–498 (2016). Published in partnership with The University of New South Wales npj Quantum Information (2018) 43 npj Quantum Information (2018) 43 Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. Quantum dot single-photon sources with ultra-low multi-photon. . . L Hanschke et al. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. 27. Schweickert, L. et al. On-demand generation of background-free single photons from a solid-state source. Appl. Phys. Lett. 112, 093106 (2018). 28. Shore, B. W. Manipulating quantum structures using laser pulses. (Cambridge University Press, Cambridge, 2011). 29. Johansson, J. R., Nation, P. D. & Nori, F. Qutip 2: A python framework for the dynamics of open quantum systems. Comput. Phys. Commun. 184, 1234–1240 (2013). 30. Fischer, K. A. et al. Scattering into one-dimensional waveguides from a coherently-driven quantum-optical system. Quantum 2, 69 (2018). 31. Carmichael, H. An Open Systems Approach to Quantum Optics: Lectures Presented at the Université Libre de Bruxelles, October 28 to November 4, 1991, Vol. 18 (Springer Science & Business Media, Berlin, 2009). 32. Wiseman, H. M. & Milburn, G. J. Quantum Measurement and Control (Cambridge university press, Cambridge, 2009). 32. Wiseman, H. M. & Milburn, G. J. Quantum Measurement and Control (Cambridge university press, Cambridge, 2009). © The Author(s) 2018 npj Quantum Information (2018) 43 npj Quantum Information (2018) 43
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Biofertilzers with natural phosphate, sulphur and Acidithiobacillus in a siol with low available-P
Scientia Agrícola
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767 767 BIOFERTILIZERS WITH NATURAL PHOSPHATE, SULPHUR AND Acidithiobacillus IN A SOIL WITH LOW AVAILABLE-P Newton Pereira Stamford1*; Patrícia Ribeiro dos Santos1; Adriana Muniz Mendes Freire de Moura1; Carolina Etiene de Rosália e Silva Santos1; Ana Dolores Santiago de Freitas1 Newton Pereira Stamford1*; Patrícia Ribeiro dos Santos1; Adriana Muniz Mendes Freire de Moura1; Carolina Etiene de Rosália e Silva Santos1; Ana Dolores Santiago de Freitas1 1UFRPE - Depto. de Agronomia, R. Dom Manoel de Medeiros, s/n°. - 52171-900 - Dois Irmãos, PE - Brasi *Corresponding author <newtonps@fastmodem.com.br> ABSTRACT: The production of mineral fertilizers is a expensive process, since it requires high energy consumption, and cannot be produced by small farmers. Laboratory assays were conducted to produce P- biofertilizers from natural phosphate (B5, B10, B15, B20), applying sulphur at different rates (5; 10; 15 and 20%) inoculated with Acidithiobacillus (S*) and testing increasing periods of incubation. A greenhouse experiment was carried out to evaluate the effect of the biofertilizers in a soil with low available P (Typic Fragiudult) from the “Zona da Mata” of Pernambuco State, grown with yam bean (Pachyrhizus erosus) in two consecutive harvests. The treatments were: Natural Phosphate (NP); biofertilizers produced in laboratory (B5, B10, B15, B20) with sulphur and Acidithiobacillus (NP+S*); natural phosphate with sulphur (20%) without Acidithiobacillus (NP+S); triple super phosphate (TSP) and a control without phosphorus. Plants were inoculated with a mixture of rhizobia strains (NFB 747 and NFB 748) or did not receive rhizobia inoculation. In bioassays pH and available P in the biofertilizers were analyzed. In the greenhouse experiment shoot dry matter, total N and total P in shoots, soil pH and available P were determined. Higher rates of available P were obtained in biofertilizers with sulphur and Acidithiobacillus (NP+S*) and in triple super phosphate (TSP), and biofertilizers with sulphur and Acidithiobacillus (FN+S*) and triple super phosphate (TSP) increased plant parameters. Native rhizobia were as effective as the strains applied in inoculation. After the two harvests soil presented lower pH values and higher rates of available P when the biofertilizers B15 and B20 with sulphur and Acidithiobacillus were applied. Key words: Pachyrhizus erosus, P uptake, phosphorus fertilization, yam bean BIOFERTILIZANTES COM FOSFATO NATURAL, ENXOFRE E Acidithiobacillus EM SOLO COM BAIXO P DISPONÍVEL RESUMO: A produção de biofertilizantes é um processo prático e econômico que reduz o consumo de energia e a sua aplicação visa aumentar o fósforo (P) disponível no solo. Biofertilizantes fosfatados (B5, B10, B15, B20) produzidos em laboratório usando fosfato natural (FN) e enxofre em diferentes concentrações (5; 10; 15 e 20%), inoculado com Acidithiobacillus (S*) e enxofre (20%) sem Acidithiobacillus (S), com incubação por 30, 45 e 60 dias, foi utilizado em um experimento em vasos com solo da Zona da Mata de Pernambuco (Argissolo Amarelo), com baixo nível de P disponível, cultivado com jacatupé (Pachyrhizus erosus), por dois períodos consecutivos. Os tratamentos fosfatados foram: fosfato natural (FN); biofertilizantes (B5, B10, B15, B20), com Acidithiobacillus (FN+S*) e B20, sem Acidithiobacillus (FN+S); superfosfato triplo (ST); e um controle sem fósforo (P0). As plantas foram inoculadas com a mistura das estirpes de rizóbio NFB 747 e 748, e sem inoculação. Nos ensaios em laboratório analisou-se o pH e o P extraído dos biofertilizantes, em cada período de incubação, e nos experimentos em vasos com solo determinou-se a biomassa seca, o N e o P total acumulado da parte aérea, o pH e o P extraído do solo. O P disponível foi mais elevado para os biofertilizantes com Acidithiobacillus (FN+S*) e para o superfosfato triplo. A fertilização fosfatada, condicionou resposta positiva dos biofertilizantes (FN+S*) e do superfosfato triplo. Os rizóbios nativos foram tão eficientes quanto os do inoculante. Após os dois cultivos o P disponível foi maior com os biofertilizantes B15 e B20 com Acidithiobacillus. Palavras-chave: Pachyrhizus erosus, jacatupé, fertilização fosfatada, solo ácido MATERIAL AND METHODS Assays using plate dishes and plastic trays (50 cm x 30 cm x 5 cm) for biofertilizers production using natu- ral phosphate (Gafsa) with addition of sulphur at different rates (5, 10, 15 and 20%) inoculated with Acidithiobacillus (S*) were conducted in laboratory. Bacterial cultures were grown in 125 Erlenmeyer flasks using medium 9K (Garcia Jr, 1991) for 5 days at 180 rpm in a horizontal shaker at 28-30°C. Inoculation was applied at a rate of 1 mL g-1 of sulphur. Biofertilizer with natural phosphate and sulphur (20%) without Acidithiobacillus inoculation (S) was also produced. During the incubation period (30, 45 and 60 days), boiled, filtered water was added to keep samples at 80% of saturation, monitored by daily weighing. The basic materials for production of phospho- rus fertilizers is phosphoric rocks. The most commonly used is apatite a no-restorable resource. The production of P-soluble fertilizers, such as super phosphates and thermo phosphates, requires higher energy consumption, specific strategies, and conduction of researches for the establishment of efficient and economic use of natural phosphates (Goedert & Sousa, 1986). The immediate uti- lization of phosphoric rock in the natural form is very re- stricted because of the low solubility, turning a more com- mon practice perennial crops, mixed with soluble fertil- izers, maintaining a slow nutrient release and uptake (Oliveira et al., 1977). The biofertilizers were produced in trays using natural phosphate (2 kg) plus elemental sulphur at the same rates (5; 10; 15 and 20%) as for plate dishes, and incubated for 60 days. At the end of the incubation peri- ods pH and available P (Mehlich 1) were determined in Petri dishes and in trays, according to EMBRAPA (1997) methodology. The biofertilizers produced in trays were applied in the greenhouse experiment carried out in pots (8 dm3), grown with yam bean legume (Pachyrhizus erosus (L) Urban). Plants were harvested in two consecu- tive crops after 90 days of planting. Studies on the isolation and selection of micro- organisms with ability to promote higher solubilization of phosphoric rocks have been carried out in many works, especially because of the possibility of interaction with microorganisms involved in biological nitrogen fixation (Nahas, 1999). MATERIAL AND METHODS The bacteria involved in the process of biological nitrogen fixation may interact with microorgan- isms that realize phosphoric rock solubilization, especially with the bacteria from the genus Acidithiobacillus, which react with sulphur producing sulphuric acid (Garcia Jr., 1992) and promote intense phosphoric rock solubilization with higher availability of phosphorus in soil, and that, in turn, is favorable to the symbiotic process and plant growth (Santos, 2002). A Typic Fragiudult with low available P was used. The soil was collected in the District of Carpina, Zona da Mata region located in Pernambuco State, North- east of Brazil. Soil samples (0-30 cm layer) were sieved (5 mm), mixed and placed in plastic pots. Results from soil analyses (eight replications) are pH (H2O 1:2.5) 5.9; exchangeable cations (mmolc kg-1) Ca2+ 14.0, Mg2+ 12.8, K 1.7; P (Mehlich 1) 2.7 mg kg-1; total N 0.6 g kg-1; Or- ganic C 8.2 g kg-1; dp 2.66 kg dm-3; dg 1.45 kg dm-3; sand, silt and clay contents 760; 100 and 140 g kg-1, respectively. It is necessary to evaluate and compare the effects of the application of sulphur inoculated with Acidithiobacillus in plant growth and in soil reaction to P soluble fertilizers and natural phosphate, because the sulphuric acid produced in the biological reaction could act in the natural phosphate solubilization and in the soil reaction reducing soil pH, and that could hamper plant growth (Stamford et al., 2002). The pot experiment was arranged in a completely randomized factorial design with three replications (Silva & Silva, 1982). P treatments were: natural phosphate (NP) in the commercial status; biofertilizers produced in labo- ratory (B5, B10, B15, and B20) using natural phosphate with elemental sulphur in the rates 5, 10, 15 and 20% inocu- lated with Acidithiobacillus (NP+S*); natural phosphate with sulphur 20% without Acidithiobacillus (NP+S); triple super phosphate (TSP); and control no P fertiliza- tion (P0). P fertilization was applied following the maxi- mum recommendation for yam bean according to Stam- ford et al. (1999), equivalent to 100 kg P ha-1. Fertilizers were applied 10 cm below and 10 cm side way, based on results obtained by Stamford et al. (1990). INTRODUCTION grains (4 t ha-1), with high percentage of edible oil and protein similar to soybean (Sorensen, 1996). The botani- cal and genetic characteristics of yam bean have been studied by the International Yam bean Program in Den- Yam bean (Pachyrhizus erosus L. Urban) is a le- gume that produce high yields of tubers (60 t ha -1) and Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 768 Stamford et al. mark and researches with rhizobia effectiveness and re- sponse to fertilizers are realized in Brazil by the Nucleus of Biologic Nitrogen Fixation in the Tropics (NFBNT) at the Federal Rural University of Pernambuco. effect of rhizobia inoculation with strains selected for ef- fective N2 fixation on yam bean and the interaction be- tween inoculation with rhizobia strains and phosphorus treatments was also evaluated. Strains of rhizobia previously screened for yam bean submitted to higher temperature conditions (Stam- ford et al., 1995; 1999) were very effective, and in some Brazilian soils, nitrogen fixation by these selected strains may be sufficient to supply nitrogen for good yields (Cruz et al., 1997). To optimize the biological nitrogen fixation may be necessary nutrient application, especially phos- phorus, because of the numerous functions exercised by this element in the symbiotic process (Burity et al., 2000). Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 MATERIAL AND METHODS This study was carried out to evaluate the effect of biofertilizers produced from natural phosphate (Gafsa) and sulphur inoculated with Acidithiobacillus in a soil with low level of available phosphorus, comparing to P soluble fertilizer (triple super phosphate) and natural phosphate on nodules and shoot biomass, total N and to- tal P accumulation on shoot, soil pH and available P. The Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Biofertilizers with natural S e Acidithiobacillus 769 Rhizobia treatments were performed with or without inoculation. Seeds of yam bean were inoculated with strains NFB 747 and NFB 748, selected for effi- cient nitrogen fixation on yam bean in previous experi- ments carried out in acid and high temperature condi- tions (Stamford et al., 1995; 1999). Inoculant was pre- pared in YM medium using 125 mL Erlenmeyers main- tained in a horizontal shaker at 100 gpm, for 5 days, at 28-30oC (containing greater than 108 g-1 in liquid cul- ture). A mixture of both strains was used for inocula- tion (1:1) and applied 1 mL per plant. In the consecu- tive growth plants were reinoculated with rhizobia fol- lowing the same process described for the first crop. The software SANEST (Zonta et al., 1982) was used for statistical analysis of data and treatments means were compared by the Tukey test (P = 0.05) (Silva & Silva, 1982). Laboratory assays The content of available phosphorus in the as- says for biofertilizers production in Petri dishes, col- lected in the different incubation periods showed liberation of P from biofertilizers, increasing three times the results of natural phosphate, and no differences were observed between the periods of incubation (Table 1). The results obtained in trays incubated for 60 days were inferior to available P in assays with Petri dishes, although the biofertilizers produced from natu- ral phosphate and sulphur inoculated with Acidithiobacillus showed potential in phosphorus avail- ability (Table 1). Lime was not applied because of the adequate values of soil pH (5.9) and exchangeable cations (28 mmolc kg-1) for tropical legumes. To reduce the influ- ence of humidity changes on available P in soil, as re- ported by Ruiz et al. (1990), water was applied twice a day (8h00 and 17h00), to keep moisture at 80% of field capacity. In the materials used as P sources, before ap- plication of the P treatments, available P (Mehlich 1) following the EMBRAPA (1997) methodology was ana- lyzed. Biofertilizers with sulphur and Acidithiobacillus (NP+S*) and soluble fertilizer (triple super phosphate) did not show difference, and biofertilizer with sulphur with- out Acidithiobacillus (NP+S) are not different of natural phosphate without sulphur (FN). In general, biofertilizers with sulphur and Acidithiobacillus decreased pH values, and showed a clear effect of H2SO4 produced by the mi- crobiological reaction carried out by Acidithiobacillus bacteria. In the first experiment shoots were harvested 90 days after seeds emergence (DAE), and two leaves per plant were not harvested to promote new growth of yam bean plants, aiming to estimate the residual power of P fertilizers in two consecutive crops. In the later experi- ment, plants were harvested 90 days after the first har- vest. Nodules and shoot dry matter; total N and total P in shoots; soil pH and available P (Mehlich 1) were de- termined. Total N was analyzed by Kjeldhal semimicro method (Kjeltec 1030), and total P in shoots determined following Malavolta et al. (1989). Soil pH (water 1.0:2.5) and available P (Mehlich 1) were determined according to EMBRAPA (1997). Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Acidithiobacillus. 2Values followed by different letters are different (P = 0.05), using the Tukey test. Upper case letters compare data in rows and lower letters in columns. Greenhouse experiment Shoot dry matter of yam bean in the two har- vests were not different under fertilization with either natural phosphate with sulphur and Acidithiobacillus (NP+S*) or triple super phosphate (TSP), which showed higher dry biomass of shoots compared to the others P treatments (Table 2). Biofertilizer with sulphur without Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 P-sources Periods of incubation in Petri dishes in trays 30 days 45 days 60 days 60 days -------------------- Available P - Mehlich 1 (%) -------------------- pH Natural phosphate (NP) 1.9 bA 1.9 bA 2.0 bA 1.1 c 5.4 a Biofertilizer 20 (NP+S) - - - 1.6 c 5.2 a Biofertilizer 5 (NP+S*) 6.5 aA 7.6 aA 7.7 aA 4.7 b 4.2 b Biofertilizer 10 (NP+S*) 6.1 aA 6.5 aA 6.5 aA 5.5 ab 4.1 b Biofertilizer 15 (NP+S*) 5.8 aB 5.8 aB 7.7 aA 6.0 a 4.1 b Biofertilizer 20 (NP+S*) 6.1 aA 6.5 aA 7.0 aA 6.1 a 4.1 b Triple super phosphate (TSP) - - - 6.0 a 5.0 b C.V. (%) 8.93 7.56 6.78 Table 1 - Available P (Mehlich 1) and pH of biofertilizers produced in laboratory assays and triple super phosphate. 1(NP+S*) = natural phosphate plus sulphur inoculated with Acidithiobacillus. (P+S) = natural phosphate plus sulphur without Acidithiobacillus. 2Values followed by different letters are different (P = 0.05), using the Tukey test. Upper case letters compare data in rows and lower letters in columns. ch 1) and pH of biofertilizers produced in laboratory assays and triple super phosphate. Table 1 - Available P (Mehlich 1) and pH of biofertilizers produced in laboratory assays and triple Table 1 - Available P (Mehlich 1) and pH of biofertilizers produced in laborat 8.93 Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 770 Stamford et al. Stamford et al. Acidithiobacillus (NP+S) yielded greater yam bean shoot dry matter than the natural phosphate (NP) and control treatment without P fertilizer (P0). grew poorer and accumulated lower amount of total nitrogen, although natural phosphate (NP) and biofertilizer with sulphur without Acidithiobacillus (NP+S) produced no significant response to total nitrogen accumulation, in comparison to control treatment without application of phosphorus (Table 3). Overall, in treatments with rhizobia inoculation, there was not effect of P application, except when the biofertilizer B15 with Acidithiobacillus (NP+S*) was applied. 0 Dry biomass of plants did not vary with and with- out rhizobia inoculation. 1(NP+S*) = natural phosphate plus sulphur inoculated with Acidithiobacillus. (P+S) = natural phosphate plus sulphur without Acidithiobacillus. 2Values followed by different letters are different (P = 0.05), using the Tukey test. Upper case letters compare data in rows and lower letters in columns. Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Greenhouse experiment Although rhizobia strains led to better results compared to uninoculated plants, in the two consecutive harvests there was no effect of rhizobia inoculation. Phosphorus application affected total nitrogen yields in yam bean shoot dry matter in the first harvest, and in the absence of phosphorus (P0), yam bean 15 In the consecutive harvest, in general, there was effect of phosphorus application compared to the treat- P-sources Harvest 1 Harvest 2 Inoculated Uninoculated Means Inoculated Uninoculated Means -------------------------------- Shoot dry biomass, g planta-1 -------------------------------- Control with no P (P 0) 2.39 cA 2.39 cA 2.39 b 2.24 bA 1.98 bA 2.11 b Natural phosphate (NP) 2.94 bcA 2.58 bcA 2.76 b 3.58 abA 2.34 abB 2.96 b Biofertilizer 20 (NP+S) 3.56 aA 3.17 aA 3.37 a 3.44 abA 3.18 aA 3.31 ab Biofertilizer 5 (NP+S*) 2.69 cA 2.62 bcA 2.66 b 2.99 bA 2.93 abA 2.96 b Biofertilizer 10 (NP+S*) 3.42 abA 3.33 aA 3.37 a 4.37 aA 3.51 aA 3.94 a Biofertilizer 15 (NP+S*) 3.84 aA 3.33 aA 3.58 a 4.08 aA 3.35 aA 3.72 a Biofertilizer 20 (NP+S*) 3.45 abA 3.16 aA 3.31 a 4.12 aA 2.79 abA 3.43 ab Triple super phosphate 3.69 aA 3.03 abA 3.36 a 4.12 aA 3.03 aA 3.57 ab Means 3.25 A 2.96 B 3.62 A 2.88 B C.V. (%) 7.47 3.44 Table 2 - Effects of P treatments on in shoot dry biomass of yam bean inoculated and uninoculated with rhizobia grown in a soil with low available P in two consecutive harvests. 1(NP+S*) = natural phosphate plus sulphur inoculated with Acidithiobacillus. (P+S) = natural phosphate plus sulphur without Acidithiobacillus. 2Values followed by different letters are different (P = 0.05), using the Tukey test. Upper case letters compare data in rows and lower letters in columns Table 2 - Effects of P treatments on in shoot dry biomass of yam bean inoculated and uninoculated with rhizobia grown in a soil with low available P in two consecutive harvests. Table 2 - Effects of P treatments on in shoot dry biomass of yam bean inoculated and uninoculated soil with low available P in two consecutive harvests. ents on in shoot dry biomass of yam bean inoculated and uninoculated with rhizobia grown in a able P in two consecutive harvests. 1(NP+S*) = natural phosphate plus sulphur inoculated with Acidithiobacillus. (P+S) = natural phosphate plus sulphur without Acidithiobacillus. 1(NP+S*) = natural phosphate plus sulphur inoculated with Acidithiobacillus. (P+S) = natural phosphate plus sulphur without Acidithiobacillus. 2Values followed by different letters are different (P = 0.05), using the Tukey test. Upper case letters compare data in rows and lower l tt i l n.4, p.767-773, Oct./Dec. 2003 ientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Greenhouse experiment 2Values followed by different letters are different (P = 0.05), using the Tukey test. Upper case letters compare data in rows and lower letters in columns. P-sources Harvest 1 Harvest 2 Inoculated Uninoculated Means Inoculated Uninoculated Means --------------------------- Total N in shoot dry biomass, mg planta-1 -------------------------- Control with no P (P 0) 48 bA 42 bA 45 b 68 bA 60 bA 64 b Natural phosphate (NP) 72 abA 62 abA 67 ab 135 aA 80 abB 107 a Biofertilizer 20 (NP+S) 79 abA 87 aA 83 a 118 aA 115 aA 116 a Biofertilizer 5 (NP+S*) 66 abA 65 abA 66 ab 133 aA 79 abA 106 a Biofertilizer 10 (NP+S*) 81 abA 83 aA 82 a 132 aA 112 aA 122 a Biofertilizer 15 (NP+S*) 96 aA 82 aA 89 a 132 aA 104 aB 118 a Biofertilizer 20 (NP+S*) 81 abA 88 aA 85 a 131 aA 103 aA 117 a Triple super phosphate 84 abA 84 aA 84 a 137 aA 107 aA 122 a Means 76 A 74 A 123 A 95 B C.V. (%) 16.93 16.27 Table 3 - Effects of P treatments on total N in shoot dry biomass of yam bean inoculated and uninoculated with rhizobia grown in a soil with low available P in two consecutive harvests. Table 3 - Effects of P treatments on total N in shoot dry biomass of yam bean inoculated and uninoculated with rhizobia grown in a soil with low available P in two consecutive harvests. Table 3 - Effects of P treatments on total N in shoot dry biomass of yam bean inoculated and uninoculated with rhizobia grown in a soil with low available P in two consecutive harvests. Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Biofertilizers with natural S e Acidithiobacillus 771 in (Table 5). Soil pH decreased by application of biofertilizers with sulphur and Acidithiobacillus (FN+S*), especially when applied biofertilizers with higher levels of sulphur, indicating the effect of sulphuric acid formed during sulphur oxidation promoted by Acidithiobacillus. Application of B20 biofertilizer with sulphur and without Acidithiobacillus decreases soil pH. This effect probably resulted from sulphuric acid produced by native Acidithiobacillus which during the long experimental pe- riod (180 days) could produce sufficient sulphur oxida- tion. ment without phosphorus. Acidithiobacillus. 2Values followed by different letters are different (P = 0.05), using the Tukey test. Upper case letters compare data in rows and lower letters in columns. Greenhouse experiment However, plants without rhizo- bia inoculation showed not response to natural phosphate and biofertilizers with sulphur without Acidithiobacillus inoculation (NP+S) compared to control treatment (P0). In the two consecutive harvests, the best results of total nitrogen accumulation in shoots were obtained for plants inoculated with specific rhizobia strains, but the effect of rhizobia inoculation was significant only in the consecu- tive harvest. Total P accumulation in dry matter shoots of yam bean produced significant response when the phospho- rus sources were applied, especially with application of biofertilizers with sulphur and Acidithiobacillus (NP+S*) and triple super phosphate (TSP). In the first harvest, natural phosphate (NP) and biofertilizer without Acidithiobacillus (NP+S) were not different to control treatment without phosphorus application (Table 4). In the later harvest, biofertilizers with sulphur and without Acidithiobacillus (NP+S) led to greater total phosphorus accumulation, probably because of the presence of native Acidithiobacillus bacteria which, in the second harvest, produced sulphuric acid sufficient to promote phospho- rus solubilization of natural phosphate. In the biofertilizers, the sulphur bacteria Acidithiobacillus elicits the reaction of sulphur with wa- ter and oxygen, forming higher amounts of sulphuric acid (Garcia Jr., 1992) at varying the rates, as related to the different amounts of elemental sulphur applied. The sulphuric acid produced reacted with the natural phos- phate increasing the available P and lowered pH, accord- ing to the amount of sulphur in the different biofertilizers and depending on the period of incubation, with consis- tent results. The effects of the P treatments on shoot biomass compared with applying natural phosphate (NP) in the commercial status and the control treatment without ap- plying P are conclusive. Similar results were reported by Santos (2002), evaluating the effect of P biofertilizers and soluble fertilizer (triple super phosphate) on mimosa tree legume grown in an acid soil. Klepker & Anghinoni (1995) studying the effect of phosphorus application in maize, reported greater response of soluble fertilizers compared with natural phosphates. In the two consecutive harvests it was not ob- served response to rhizobia inoculation in total phospho- rus in shoot dry matter of yam bean, although in the later harvest, significant response of rhizobia inoculation was observed when biofertilizers (B15 and B20) with sulphur and Acidithiobacillus (NP+S*), B20 with sulphur without Acidithiobacillus (NP+S) and natural phosphate (NP) were applied. Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Greenhouse experiment Table 5 - pH and available P in soil after the two harvests of yam bean in a soil with low available P. Table 5 - pH and available P in soil after the two harvests of yam bean in a soil with low available P. in the biofertilizer without Acidithiobacillus, thus it seems that 90 days after the first harvest the soil bacteria may produce sulphuric acid and could act in P solubilization increasing available P in soil. Brasil & Muraoka (1997) observed a significant correlation between available P in soil and yield of plant biomass using Amazon soils, un- der natural phosphates and soluble fertilizers. Santos (2002), with the same soil used in this work, showed the positive effect of natural phosphate on available P in soil and growth of mimosa tree legume. The results are not so evident because of the use of natural phosphate pelleting the sulphur inoculated with Acidithiobacillus. Probably the reaction for sulphuric acid production was lowered due to the low input of air and water to react with the sulphur into the natural phosphate. The soil pH, determined after the two consecu- tive harvests (180 days) was affected by the P treatments, especially when the biofertilizers produced with higher rates of sulphur and inoculated with Acidithiobacillus were applied but growth was not affected by the reduc- tion in soil pH. The results are in accordance with data obtained by Ernani et al. (2001), with lime application in an acid soil with low available P. Probably the reduc- tion in soil pH observed in the treatments with sulphur and Acidithiobacillus increased the available P and this parameter was considered by He et al. (1996) as a factor of greater effect in solubility of natural phosphates. Also Stamford et al. (2002, 2003), using sulphur inoculated with Acidithiobacillus in amendment of saline and sodic soils observed reduction in soil pH occurring until the total consumption of the added sulphur, promoting soil acidification varying from initial pH 8.2 up to pH 4.5 applying 1.8 t ha-1 of sulphur. Cruz et al. (1997) using different sources of nitrogen and phosphorus fertilizers on yam bean grown in a low available P soil. These authors suggested that for yam bean inoculated with specific and efficient rhizobia strains, it is not necessary nitrogen application for sat- isfactory yields. ACKNOWLEDGEMENTS To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), to Fundação de Apoio e Tecnologia do Estado de Pernambuco (FACEPE) and to the Program in Agronomy (Soil Science Post Gradu- ated Courses) of University Federal Rural of Pernambuco (UFRPE), for financial support and fellowships. Greenhouse experiment The use of natural phosphates and liming is not an adequate and practical technology for heavy acid soils (Vasconcelos et al., 1986). In limed soils, Ernani et al. (2001) did not observe difference using various methods of P fertilizer application, and the best yields were ob- tained with soluble sources of phosphorus, in compari- son to the natural phosphate. Application of P caused a marked increase in to- tal N and total P in plant shoots, and best results were obtained when biofertilizers with natural phosphate with sulphur and Acidithiobacillus and triple super phosphate were used. The positive impact of the biofertilizers pro- duced with natural phosphate plus sulphur inoculated with Acidithiobacillus on total N and total P accumulated in shoots of yam bean holds great promise for improving input from these products as an alternative for partial or total substitution of soluble fertilizers. Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Greenhouse experiment Results of soil pH and available P (Mehlich 1), determined at the end of the later harvest, are presented Results of rhizobia inoculation were not as ef- ficient as the results of the experiments carried out by Table 4 - Effects of P treatments on total P in shoot dry biomass of yam bean inoculated and uninoculated with rhizobia grown in a soil with low available P in two consecutive harvests. grown in a soil with low available P in two consecutive harvests. 1(NP+S*) natural phosphate plus sulphur inoculated with Acidithiobacillus (P+S) natural phosphate plus sulphur without P-sources Harvest 1 Harvest 2 Inoculated Uninoculated Means Inoculated Uninoculated Means ------------------------- Total P in shoot dry biomass, mg planta-1 ------------------------- Control with no P (P 0) 0.55 cA 0.60 bA 0.57 d 0.51 bA 0.57 bA 0.54 c Natural phosphate (NP) 0.72 bcA 0.67 abA 0.69 cd 0.93 aA 0.56 bB 0.74 bc Biofertilizer 20 (NP+S) 0.99 abA 0.91 abA 0.95 ab 0.89 abA 0.77 abA 0.83 ab Biofertilizer 5 (NP+S*) 0.81 abcA 0.68 abA 0.75 bcd 1.20 aA 0.69 abB 0.94 ab Biofertilizer 10 (NP+S*) 1.06 abcA 0.98 aA 0.89 abc 1.14 aA 1.02 aA 1.08 a Biofertilizer 15 (NP+S*) 1.02 aA 0.90 abA 0.98 a 0.83 abA 0.84 abA 0.84 ab Biofertilizer 20 (NP+S*) 1.01 abA 0.96 aA 0.99 a 1.10 aA 0.67 abB 0.88 ab Triple super phosphate 0.99 abA 0.91 abA 0.96 ab 1.16 aA 0.66 abB 0.91 ab Means 0.89 A 0.82 A 0.97 A 0.72 B C.V.(%) 14.52 17.37 Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 772 Stamford et al. Table 5 - pH and available P in soil after the two harvests of yam bean in a soil with low available P. P-sources pH (H2O) Available P (Mehlich) ----------- mg kg-1 ----------- Control with no P (P 0) 5.7 ab 5.4 d Natural phosphate (NP) 6.2 a 16.8 c Triple super phosphate 5.2 bc 31.0 b Biofertilizer 20 (NP+S) 4.9 cd 6.2 d Biofertilizer 5 (NP+S*) 4.8 cd 3.8 d Biofertilizer 10 (NP+S*) 4.8 cd 21.1 c Biofertilizer 15 (NP+S*) 4.6 cd 119.6 a Biofertilizer 20 (NP+S*) 4.4 d 109.1 a C.V. (%) 6.56 17.85 1(NP+S*) = natural phosphate plus sulphur inoculated with Acidithiobacillus. (P+S) = natural phosphate plus sulphur without Acidithiobacillus. 2Values followed by different letters are different (P = 0.05), using the Tukey test. REFERENCES Lombardi (1981) observed effect of “Alvorada” natural phosphate applied with sulphur and Acidithiobacillus on P total and growth of a tropical grass. Native bacteria in soil promoted sulphur oxidation as ef- fective as the inoculated bacteria. However, the coeffi- cient of variation obtained in the experiment was so high that was not possible to evaluate the positive effect of the sulphur inoculation with Acidithiobacillus when com- pared with the soil bacteria. BRASIL, E.C.; MURAOKA, T. Extratores de fósforo em solos da Amazônia tratados com fertilizantes fosfatados. Revista Brasileira de Ciência do Solo, v.21, p.599-606, 1997. BURITY, H.A.; LYRA, M.C.C.P.; SOUZA, E.S. Efetividade da inoculação com rizóbio e fungos micorrízicos arbusculares em mudas de sabiá submetidas a diferentes níveis de fósforo. Pesquisa Agropecuária q g p Brasileira. v.35, p.801-807, 2000. CRUZ, G.N.; STAMFORD, N.P.; SILVA, J.A.A.; CHAMBER-PEREZ, C. Effects of inoculation with Bradyrhizobium and urea application on N2 fixation and growth of yam bean. Tropical Grassland, v.33, p.23-27, 1997. Á EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA. Manual de métodos de análise de solo. 2.ed. Brasília: EMBRAPA, CNLPS, 1997. 212p. In the first harvest the native bacteria present in soil were not effective in the oxidation of sulphur applied Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 Biofertilizers with natural S e Acidithiobacillus 773 SANTOS, K.S. Atuação de fosfato natural com adição de enxofre com Acidithiobacillus na solubilização de fósforo e no desenvolvimento de sabiá (Mimosa caesalpiniaefolia) em solo de tabuleiro. Recife: UFRPE, 2002. 68p. (Dissertação – Mestrado) ERNANI, P.R.; STECKLING, C.; BAYER, C. Características químicas de solo e rendimento de massa seca de milho em função do método de aplicação de fosfatos, em dois níveis de acidez. Revista Brasileira de Ciência do Solo, v.25, p.939-946, 2001. GARCIA JR., O. Isolation and characterization of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans from mineral mines. Revista Brasileira de Microbiologia, v.20, p.1-6, 1991. SILVA, J.A.A.; SILVA J.P. Estatística experimental aplicada à ciência florestal. Recife: (Imprensa da Universidade Federal Rural de Pernambuco; 1982. SORENSEN, M. Yam Bean: Promoting the conservation and use of underutilized and neglected crops. 2. Rome: International Plant Genetic Resource Institute, 1996. 141p. GARCIA JR., O. O enxofre e suas transformações microbianas. In: CARDOSO, E.; SAITO, M.T.; NEVES, M.C.P. Microbiologia do solo. Campinas: SBCS, 1992. p.243-255. GOEDERT, W.J.; SOUSA, D.M.G. Avaliação preliminar de fosfato com acidulação parcial. Revista Brasileira de Ciência do Solo, v.10, p.75- 80, 1986. STAMFORD, N.P.; SANTOS, D.R.; LOPES, A.J.R. REFERENCES Rendimento e teor de nutrientes por cultivares de caupi em Latossolo Vermelho amarelo com diferentes modos de aplicação do fertilizante. Revista Brasileira de Ciência do Solo, v.14, p.57-61, 1990. HE, Z.L.; BALIGAR, V.C.; MARTENS, D.C.; RITCHEY, K.D.; KEMPER, W.D. Factors affecting phosphate rock dissolution in acid soil amended with liming materials and cellulose. Soil Science Society of American Journal, v.60, p.1596-1601, 1996. STAMFORD, N.P.; MEDEIROS, R.; MESQUITA, J.C.P. Avaliação de estirpes de Bradyrhizobium para jacatupé em regime de temperatura STAMFORD, N.P.; MEDEIROS, R.; MESQUITA, J.C.P. Avaliação de estirpes de Bradyrhizobium para jacatupé em regime de temperatura elevada. Revista Brasileira de Ciência do Solo, v.9, p.45-54, 1995. elevada. Revista Brasileira de Ciência do Solo, v.9, p.45-54, 1995. KLEPKER, D.; ANGHINONI, I. Crescimento radicular e aéreo do milho em vasos, em função do nível de fósforo no solo e da localização do adubo fosfatado. Revista Brasileira de Ciência do Solo, v.19, p.403- 408, 1995. STAMFORD, N.P.; SANTOS, C.E.R.S.; MEDEIROS, R.; FREITAS, A.D.S. Efeito da fertilização com fósforo, potássio e magnésio em jacatupé infectado com rizóbio em um Latossolo Álico. Pesquisa Agropecuária Brasileira, v.34, p.1831-1838, 1999. LOMBARDI, M.L.C.O. Dissolução de três fosfatos naturais através da atividade microbiológica do enxofre. Piracicaba, 1981. 62p. Dissertação (Mestrado) – Escola Superior de Agricultura “Luíz de Queiroz”, Universidade de São Paulo. STAMFORD, N.P.; SILVA, J.A.; FREITAS, A.D.S.; ARAÚJO FILHO, J.T. Effect of sulphur inoculated with Acidithiobacillus in a saline soil grown with Leucena and mimosa tree legumes. Bioresource Technology, v.81, p.53-59, 2002. VASCONCELOS, C.A.; SANTOS, H.L.; FRANÇA, G.D.; PITTA, G.V.E.; BAHIA FILHO, A.F.C. Eficiência agronômica de fosfatos naturais para a cultura do sorgo granífero. Revista Brasileira de Ciência do solo, v.10, p.123-128, 1986. MALAVOLTA, E.; VITTI, G.C.; OLIVEIRA, S.A. Avaliação do estado nutricional das plantas. Princípios e aplicação. Piracicaba: POTAFOS, 1989. 201p. NAHAS, E. Solubilização microbiana de fosfatos e de outros elementos. In: SIQUEIRA, J.O; MOREIRA, F.M.S.; LOPES, A.S.; GUILHERME, L.R.G.; FAQUIN, V.; FURTINI NETO, A.E.; CARVALHO, J.G. Interrelação fertilidade, biologia do solo e nutrição de plantas. Lavras: UFLA/DS, 1999. p.467-486. ZONTA, E.P.; MACHADO, A.A.; SILVEIRA JR., P. SANEST - Sistema de análise estatística. Pelotas: Universidade Federal de Pelotas. 1982. 151p. OLIVEIRA, M.C.C. de; LOPES, E.S.; SILVA, M.T.R. de; NAGAI, R. Influência de oxidação microbiológica do enxofre na solubilização de apatita de Araxá. Revista Brasileira de Ciência do Solo, v.1, p.24-28, 1977. RUIZ, H.A.; FERNANDES, B.; NOVAIS, R.F.; ALVAREZ, V.H. Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003 REFERENCES Teor acúmulo e distribuição de fósforo em plantas de soja em relação ao conteúdo de água do solo. Revista Brasileira de Ciência do Solo, v.14, p.181-185, 1990. Received October 10, 2002 Accepted August 28, 2003 Scientia Agricola, v.60, n.4, p.767-773, Oct./Dec. 2003
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Expectations vs. actual behavior of a social robot: An experimental investigation of the effects of a social robot’s interaction skill level and its expected future role on people’s evaluations
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PLOS ONE PLOS ONE RESEARCH ARTICLE Expectations vs. actual behavior of a social robot: An experimental investigation of the effects of a social robot’s interaction skill level and its expected future role on people’s evaluations Aike C. HorstmannID*, Nicole C. Kra¨mer Social Psychology: Media and Communication, University of Duisburg-Essen, Duisburg, Germany Aike C. HorstmannID*, Nicole C. Kra¨mer Aike C. HorstmannID , Nicole C. Kramer Social Psychology: Media and Communication, University of Duisburg-Essen, Duisburg, Germany Social Psychology: Media and Communication, University of Duisburg-Essen, Duisburg, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Horstmann AC, Kra¨mer NC (2020) Expectations vs. actual behavior of a social robot: An experimental investigation of the effects of a social robot’s interaction skill level and its expected future role on people’s evaluations. PLoS ONE 15(8): e0238133. https://doi.org/10.1371/journal. pone.0238133 Editor: Chen Lv, Nanyang Technological University, SINGAPORE Received: May 14, 2020 Accepted: August 9, 2020 Published: August 21, 2020 Copyright: © 2020 Horstmann, Kra¨mer. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Since social robots are taking over roles which are traditionally filled by humans and thus are becoming a growing part of our society, it is crucial to examine what affects people’s evaluation and acceptance of these robots. How a robot is evaluated depends on several factors like the robot’s appearance [1–4], its nonverbal behavior [3, 5], other behavioral aspects (e.g., predict- ability: [6, 7] and cooperativeness: [8]) and media portrayals [9–12]. This study focuses on three aspects: the robot’s behavior, the user’s expectation, and the user’s individual background. Abstract Since social robots are increasingly entering areas of people’s personal lives, it is crucial to examine what affects people’s perceptions and evaluations of these robots. In this study, three potential influences are examined: 1) the robot’s level of interaction skills, 2) the robot’s expected future role as a helpful assistant or a threatening competitor, and 3) peo- ple’s individual background with regard to robots and technology in general. In an experi- mental lab study with a 2x2 between-subjects-design (N = 162), people read a vignette describing the social robot Nao either as assistant or competitor and subsequently inter- acted with Nao, which either displayed high or low interaction skills. Results of a structural equation model show that the robot’s interaction skill level had the strongest effect, with a low level leading to a negative evaluation of the robot’s sociability and competence and sub- sequently a negative general evaluation of the interaction with the robot. A robot which was expected to become a competitor was also evaluated as less sociable than a robot expected to become an assistant. Overall, in case of an actual interaction with a social robot, the robot’s behavior is more decisive for people’s evaluations of it than their expectations or indi- vidual backgrounds. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The Open Access Publication Fund of the University of Duisburg-Essen supported the publication of the article. No additional external funding was received for this study. With regard to behavior, the robot’s level of interaction skills should affect people’s evalua- tions tremendously for several reasons. First of all, behavior in general appears to play a major Competing interests: The authors have declared that no competing interests exist. 1 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE Expectations vs. actual behavior of a social robot role in interactions with non-human entities [13]. Furthermore, interaction skills are essential for a social robot to fulfill its function [14] and according to Davis [15], a technology’s per- ceived usefulness is linked to people’s attitude towards and usage intentions of this technology. When a robot displays low interaction skills, this robot and the interaction with it should be evaluated differently compared to when the robot displays high interaction skills. With regard to expectations, most people have not personally interacted with a social robot yet and thus have to draw information from other sources, which are often mass media reports [11]. Media representations of “good” as well as “bad” fictional robot characters form people’s attitude towards and expectations about social robots and lead to double-minded feelings [10]. Since expectations serve as perceptual filters of the reality [16, 17], whether people expect a social robot to become a helpful assistant or a threatening competitor should affect how people evaluate the robot and the interaction with it. Moreover, when the robot is expected to become a competitor, high interaction skills should be perceived more threatening and thus more neg- atively than low skills. A preliminary analysis of a small part of the data already indicated that the robot’s level of interaction skills as well as people’s expectation regarding the robot’s future role both have an impact on people’s evaluation of this robot as well as on the evaluation of the interaction with the robot t [18]. However, when examining human-robot interaction processes, people’s indi- vidual background needs be taken into account as well. In this work, we additionally look at people’s technical affinity [19], their locus of control when using technologies [20], their previ- ous experiences with real or fictional robots and their negative expectancies regarding robots. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. To get a more comprehensive picture, the focus of this work is to look at these different influ- ences (behavior, expectation, and individual background) in one structural equation model to be able to examine how they relate to each other while affecting how a robot and consequently the interaction with it are evaluated. Summing up, this study examines the main and interac- tion effects of a social robot’s interaction skill level, the robot’s expected future role and users’ personality variables to find out which aspects play what kind of role in people’s perceptions and evaluations of a robot with which they just interacted. PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 A social robot’s level of interaction skills How a social robot behaves while interacting with people, should play a central role for peo- ple’s evaluation of this robot. One reason for this assumption is that behavior in general was found to have a tremendous impact in interactions with non-human entities [13]. According to Rickenberg and Reeves [13], the evaluation of a character “depends on what the character does, what it says, and how it presents itself” (p. 55). Against this background, this study looks at the effects of different behaviors of a social robot, particularly whether this robot displays poor or sophisticated social interaction skills. Since the media’s focus is often predominantly on the success and progress of a technology and neglects problems and setbacks, this leads people to form exorbitantly high expectations about social robots and their skills [11]. In this vein, robots are generally expected to be perfor- mance-oriented, i.e., efficient, reliable, precise, rational, and perfectionist [21–23]. Likewise, research by Kwon et al. [24] showed that people tend to generalize social capabilities for humanoid robots, which also results in very high expectations for these robots. Looking at social robots specifically, people expect them to be able to talk to them, to understand them and to react to them in a sophisticated manner [11]. One of the main characteristics of socially interactive robots is the ability to communicate with high-level dialogue [25]. Even though there are several definitions of social robots, the ability to interact with its environment to some degree (or at least to give the impression to do so) resembles the core definition of a PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 2 / 18 PLOS ONE Expectations vs. actual behavior of a social robot social robot [14]. Without adequate interaction skills, social robots would not be able to fulfill their purpose of interacting with people in a social way and, thus, would not be very useful in their common areas of application like home companionship, nursing care, entertainment, and office/hotel assistance [14]. According to the findings obtained while developing the well- established Technology Acceptance Model, perceived usefulness has a powerful effect on the attitude towards using as well as on the actual use of a technology [15]. If a social robot has low interaction skills, this should reduce its perceived usefulness and consequently lead to detri- mental consequences for the evaluation of this robot. A social robot’s level of interaction skills Research showed that when a robot’s functions fall behind people’s expectations this leads to negative communication outcomes like disappointment, mistrust, and rejection [26]. Thus, a robot displaying low levels of inter- action skills should result in a negative evaluation of the robot’s sociability and competence. How the robot is evaluated should then also affect the general evaluation of the interaction with the robot. Against this background, the following hypotheses were postulated: H1: A robot with a low level of interaction skills leads to a more negative evaluation of a) the robot’s sociability and b) its competence, which consequently leads to c) a more negative general evaluation of the interaction with the robot, compared to a robot with a high inter- action skill level. A social robot’s expected future role In general, there are mainly two prominent prospects for social robots–one which is feared and one which is desired. Since both images are promoted in media, this leads people to have double-minded feelings towards robots [10]. On the one hand, there is this negative view on social robots becoming competitors. People are worried about autonomous robots and loss of control, which is often accompanied by a fear that humans will be either replaced or dominated by robots (known as “Frankenstein Syn- drom”; [27–29]). Mass media, particularly science fiction formats, have a great influence on people’s image of social robots and promote the idea of robots developing their own agenda and revolting against humans [30]. In this vein, a study by Horstmann and Kra¨mer [11] showed that the more “bad” fictional robot characters people know, the more do they fear robots to become superior and a threat to humans. To extend these survey-based findings, this study aims to examine in a systematic-experimental way whether a negative expectation about a robot affects how people evaluate this robot after they interacted with it. It is assumed that interacting with a robot which is expected to learn from humans to become better and more efficient than them and to take away tasks from them is undesirable and that this robot as well as the interaction with it are evaluated poorly. On the other hand, there is also this positive image of social robots functioning as assistants, either in domestic, public, or work environments [11]. This idea of having an electronic help, which makes life easier by carrying out tasks that are unpleasant or strenuous, is very appealing to most people [11, 28, 31]. Thus, people should evaluate a robot which is expected to assist humans with various tasks in the future positively and should also enjoy the interaction with this robot. A social robot’s expected future role In order to examine whether manipulating people’s expectations by framing the robot in a negative light (by describing it as competitor working against humans) or in a positive light (by describing it as assistant working for humans) influences how they evaluate the robot and consequently the interaction with it, the following hypotheses were formulated: H2: When a robot is expected to take over the role of a competitor, this leads to a more nega- tive evaluation of a) the robot’s sociability and b) its competence, which consequently leads PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 3 / 18 PLOS ONE Expectations vs. actual behavior of a social robot to c) a more negative general evaluation of the interaction with the robot, compared to when the robot is expected to become an assistant. A social robot’s expected future role influencing the perception of its interaction skill level It is crucial for the acceptance and successful employment of social robots to find out which influencing variables may interact with each other while affecting the evaluation of a robot. It is known from interpersonal studies, that how a person’s behavior is interpreted is heavily influenced by how desirable or rewarding it is to interact with the person [16]. It is a function of all pre-interactional relationship and communicator characteristics (e.g., personality, repu- tation, and nature of the relationship) and all interactional behaviors (e.g., an amusing com- munication style or positive feedback; [32]). Transferring this to human-robot interaction, how a robot’s behavior is perceived should also be affected by how desirable it is for people to interact with this robot. Since there are ambiguous views on social robots, whether the robot is expected to become a threatening competitor or a helpful assistant in the future should affect how the robot’s level of interaction skills are perceived. More specifically, a social robot’s skills might be perceived differently depending on how people think this robot will use these skills in the future. In case of a robot which is expected to become a competitor working against humans, high interaction skills could be perceived as daunting. In science fiction, robots are often portrayed as highly skilled and extremely intelli- gent, often surpassing humans and threatening to obtain world domination [27, 33–35]. Research has shown that these are scenarios that people are also afraid of in real life [28, 29], especially when they are able to recall a lot of these negative fictional robot characters [11]. Thus, in case of a threatening competitor robot, people would most likely prefer this robot to have low interaction skills. However, if a robot is expected to be a helpful assistant working for humans, people would probably like to have this robot to be equipped with sophisticated inter- action skills. Against this background, this study aims to examine how different levels of a social robot’s interaction skills affect people’s evaluation of the robot and subsequently the interaction with it when the robot is framed as undesirable competitor compared to when it is framed as a desirable assistant. A social robot’s expected future role influencing the perception of its interaction skill level The following is hypothesized: H3: When a robot is described as competitor, a high level of interaction skills leads to a more negative evaluation of a) the robot’s sociability and b) its competence, which consequently leads to c) a more negative general evaluation of the interaction with the robot. PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 Sample Results of an a priori power analysis using Gpower 3.1 software (based on 95% power and a medium effect size of f2 = 0.15; [39, 40]) recommended a sample size of 111 participants. Fur- thermore, our goal was to have at least 40 participants in each condition, resulting in a mini- mum size of 160 participants. In the end, 189 people participated in the lab study, of which 25 had to be excluded because they did not pass the manipulation check making sure people read the vignettes with sufficient attention (see next section). Additionally, one person was removed because of insufficient language skills and one because the vignette was accidently read after instead of before the interaction. Of the remaining 162 participants, 97 reported to be female and 65 to be male. The age ranged from 18 to 42 years with an average of 22.85 years (SD = 3.88). With regard to education, most participants were university students (94.4%) and either possessed university entrance-level qualifications (59.3%) or a university degree (38.9%). Material and methods The laboratory study employed an experimental 2 (assistant vs. competitor expectation) x 2 (high vs. low level of interaction skills) between-subjects design. Participants were randomly assigned to the conditions. The ethics committee of the division of Computer Science and Applied Cognitive Sciences at the Faculty of Engineering of the University of Duisburg-Essen approved the study and written informed consent was obtained. The individual pictured in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details. Users’ individual background In conclusion the following hypotheses are postulated: H4: In addition to the main and interaction effects of the robot’s level of interaction skills and the robot’s expected future role, people’s technological background, their previous experi- ences with robots, and their negative expectancies regarding robots influence people’s eval- uation of a) the robot’s sociability and b) its competence, which consequently affects c) the general evaluation of the interaction with the robot. H4: In addition to the main and interaction effects of the robot’s level of interaction skills and the robot’s expected future role, people’s technological background, their previous experi- ences with robots, and their negative expectancies regarding robots influence people’s eval- uation of a) the robot’s sociability and b) its competence, which consequently affects c) the general evaluation of the interaction with the robot. Users’ individual background In addition to the robot’s behavior and what future role people expect this robot to have, the individual background of the person interacting with the robot should have a great influence on how the robot and the interaction with it are evaluated. First of all, people’s technological background should be taken into account. On the one hand, there is technical affinity, which describes a person’s positive attitude, excitement, and trust toward technology [19]. A high technical affinity resembles a high enthusiasm about new technologies [19], which should also lead to a very positive attitude towards robots in general. On the other hand, there is locus of control when using technology, which describes whether a person feels capable and confident or rather helpless and overwhelmed when handling technological devices [20, 36]. How com- petent a person feels handling technological devices usually relates to his or her actual techno- logical competence and knowledge [20]. Thus, a high technological locus of control should 4 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE Expectations vs. actual behavior of a social robot lead to a more realistic view on what social robots are capable of, as it appeared to be the case in previous research [11]. Moreover, a person’s experiences with real robots but also with robots portrayed in mass media (real and fictional ones) should be considered. Most people have never interacted with a real robot before and thus a heavily used source of information for this still rather unfamiliar technology are media reports about real robots and, even more prevalent, science fiction repre- sentations (cf. [11]). Particularly since these portrayals often lack accuracy and elicit a biased picture of robots, their influences need to be considered. Since negative expectations and fears regarding robots are often discussed in human-robot interaction research (cf. [9, 28, 37, 38]), as well as in literature (cf. [27, 33–35]), their influence should be taken into account as well. Especially the fear of robots gaining consciousness and becoming more intelligent and thus superior to humans represents a common science fiction scenario [30]. Since science fiction is widely spread and easily accessible for everyone, they are believed to have a great impact on people’s attitude towards social robots (e.g., [10, 11, 30]). Thus, the influence of people’s negative expectancies regarding social robots to become supe- rior to and dominate humans is also considered in this study. Experimental setting and procedure First, a cover story was presented explaining that the participants are invited to the lab for a final evaluation of the robot’s improved interaction skills. After written consent was obtained 5 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE Expectations vs. actual behavior of a social robot from the participants, they were asked to read a vignette, where in the first part the robot Nao’s (SoftBanks Robotics) interaction skills were described as very elaborated. In the second part of the vignette, the robot was either described as competitor (Nao’s interaction and communica- tion skills will be superior to human skills and it will take over many tasks which are currently executed by humans, because it will be able to do them in a more efficient, reliable, and safe way) or as assistant (Nao will be very helpful and assist humans with many exhausting and onerous tasks, to make them easier and more pleasant to do). This was followed by a manipu- lation check asking about the robot Nao’s interaction skills (possible answers: high, low, or medium) and its future role (possible answers: assistant, replacement, or no information on future application) as it was described in the vignette. Since only participants were included who passed this check, we are confident that we successfully manipulated people’s expectations with regard to the robot’s future role. For the subsequent interaction, participants sat in front of the robot Nao, which was placed in the middle of a table (see Fig 1). After the main functions of the robot were explained, the experimenter gave the robot a start signal and left the room. The robot then explained that it needed a moment for preparation and will notify the participant when it was ready to start. This gave the experimenter enough time to enter the adjacent room from which the robot was then controlled during the interaction. By using a webcam installed in the lab, the experi- menter was able to see and hear the participant and to let the robot react in accordance with the participant’s answers (wizard of oz design; see [41]). To explain why the participants would be alone with the robot during the interaction, they were told that the robot produces a lot of data for the final evaluation which can only be processed by a high-performance com- puter located a few rooms down the hall. Questionnaires Evaluation of the robot’s sociability and competence. To assess the robot’s perceived sociability, the Social Attractiveness subscale (Interpersonal Attraction Scale [42]; Likert scale ranging from 1 = “strongly disagree” to 5 = “strongly agree”; 5 items; e.g., “I think the robot Nao could be a friend of mine.”; α = 0.80) as well as the Sociability subscale (Source Credibility Scale [43]; semantic differential scale ranging from 1 to 5; 6 items; e.g., “unpleasant–pleasant”; α = 0.84) were adapted to ask about the robot Nao instead of a person. Additionally, the two subscales Perceived Enjoyment (5 items; e.g., “I enjoy the robot talking to me.”; α = 0.87) and Perceived Sociability (4 items; e.g., “I find the robot pleasant to interact with.”; α = 0.84) of the Acceptance of a Social Robot Scale ([44]; 1 = “strongly disagree” to 5 = “strongly agree”) were employed as well. People’s evaluation of the competence of the robot Nao was assessed with the Task Attrac- tiveness subscale (Interpersonal Attraction Scale [42]; Likert scale ranging from 1 = “strongly disagree” to 5 = “strongly agree”; 5 items; e.g., “The robot Nao would be a poor problem solver.”; α = 0.81) and the Competence subscale (Source Credibility Scale [43]; semantic differ- ential scale ranging from 1 to 5; 6 items; e.g., “unintelligent–intelligent”; α = 0.89), after both scales’ items were adjusted to an interaction with a robot instead of with another person. Evaluation of the interaction with the robot. To assess how people generally evaluate the interaction with the robot, adapted versions of the Evaluation subscale ([45]; 4 items; e.g., “I was enjoying the interaction with the robot Nao.”; 1 = „strongly disagree”to 5 = „strongly agree“; α = 0.81) and the Overall Rewardingness scale ([46]; 4 items; e.g., “The opportunity to interact with the robot Nao again is very desirable.”; 1 = „strongly disagree”to 5 = „strongly agree“; α = 0.85) were used. People’s individual background. Experimental setting and procedure The experimenter explained that she wanted to make sure that the data will be transferred and saved correctly, which is why she would like to observe these processes during the interaction. The webcam was justified by explaining that in case of error messages in the data, the devel- opers of Nao’s interaction skills could use the videos to see what went wrong. Participants were also told that the robot would guide them through the interaction and that they should ring a bell when both interaction tasks were completed, which would notify the experimenter. The first interaction task was a figure guessing game. Participants received a list with the names and small pictures of 32 different figures (celebrities as well as comic and movie Fig 1. The experimental set-up. https://doi.org/10.1371/journal.pone.0238133.g001 Fig 1. The experimental set-up. https://doi.org/10.1371/journal.pone.0238133.g001 6 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE Expectations vs. actual behavior of a social robot characters) and were instructed to choose one of them without telling the robot which one they chose. The robot then asked different questions (e.g., “Is it a real person or a fictional fig- ure from a comic or movie?”) to eventually guess the figure. A decision tree was constructed so that the robot was able to name the chosen figure after five questions. After three rounds of this game, the robot introduced the second interaction task: creating a profile about each other. Here, the robot and the participant took turns asking each other personal questions to fill out a given profile template (e.g., age, height, hobbies, and favorite movie). In the high interaction skills condition, participants were able to communicate with the robot in a rather natural and sophisticated way. In the low interaction skills condition, the communication was more restricted (e.g., robot can only understand “Yes” or “No”), the robot deliberately applied some false grammar (e.g., false conjugation of verbs) and misunderstood the participants’ answers several times. After the two interaction tasks were completed, the experimenter came back to the lab and asked the participant to continue with questionnaires on the computer. At the end of the experiment, participants were debriefed and their time and effort were compensated with money or course credits. PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 Questionnaires The participants’ technological background was mea- sured with the Locus of Control when Using Technology Scale (KUT [20]; 8 items; e.g., “I feel so helpless regarding technical devices that I rather keep my hands off of them.”; 1 = “strongly disagree” to 5 = “strongly agree”; α = 0.85) and the Technical Affinity as Handling of and Atti- tude toward Electronic Devices Scale (TA-EG [19]; 19 items; e.g., “I enjoy trying an electronic device.”; 1 = “does not apply at all” to 5 = “applies completely”; α = 0.83). Their experiences with robots were measured by asking participants how often (0 = “never”; 1 = “very rarely” to 5 = “very often”) they have had contact with real robots before. In a similar way, they were asked how often they have watched reports or something similar about real PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 7 / 18 PLOS ONE Expectations vs. actual behavior of a social robot robots and how often they have watched fictional movies or series where robots played an important role before. To assess participants’ negative expectancies regarding robots, 12 items asking about com- mon science fiction scenarios were employed ([11]; e.g., “Robots will try to free themselves from humans.”; 1 = “strongly disagree” to 5 = “strongly agree”; α = 0.88). Demographical background. Participants’ age, sex, educational level, and current employment or training status were assessed. Further questionnaires assessed, but not analyzed for this paper. In addition, the Char- acter subscale of the Source Credibility Scale [43], the Anxiety subscale of the Acceptance of a Social Robot Scale [44], Themes of Relational Communication: Trust, Dominance, and Equal- ity [47], Contact Intentions [6], the General Anxiety subscale of the Frankenstein Syndrome Questionnaire [48] and Knowledge of Fictional Robot Characters [11] were assessed, but not used for the analyses of this paper. Results For an overview of the descriptive values of the main influencing and dependent variables see Table 1. To test all hypotheses (H1 to H4), a structural equation model (SEM) was employed based on the theoretical deliberations outlined before. The process of structural equation modelling comprises several statistical techniques which enable analyses of the relationships between one or more, either continuous or discrete, independent variables and one or more, either continu- ous or discrete, dependent variables [49]. Structural equation modelling basically combines two multivariate techniques: multiple regression analysis and factor analysis [50]. On the one hand, it is possible to simultaneously estimate multiple dependence relationships akin to mul- tiple regression equations and on the other hand, various measures can be incorporated for each concept similar to factor analysis [50]. A SEM has three major advantages over traditional multivariate techniques: measurement errors are explicitly assessed, latent (unobserved) vari- ables can be estimated via manifest (observed) variables and a model can be tested to examine whether a conceptual or theoretical structure fits the data [51]. The aim of the current study is to examine the relative influences of a social robot’s interaction skills (H1), the robot’s ex- pected future role (H2), a combination of the robot’s interaction skills and its expected future role (H3) and people’s individual background (H4) on a) the robot’s perceived competence Table 1. Descriptive statistics of the main influencing and dependent variables. = Source Credibility Scale [43]; IAS = Interpersonal Attraction Scale [42]; ASRS = Acceptance of a Social Robot Scale [44]. Results Robot’s behavior Robot’s expected future role High skills Lows skills Assistant expectation Competitor expectation Total M SD M SD M SD M SD M SD Perceived competence Competence (SCS) 4.03 0.66 3.38 0.80 3.78 0.82 3.63 0.78 3.71 0.80 Task Attractiveness (IAS) 4.11 0.61 3.53 0.72 3.86 0.68 3.78 0.78 3.82 0.73 Perceived sociability Sociability (SCS) 4.32 0.53 3.97 0.65 4.23 0.55 4.06 0.67 4.14 0.61 Social Attractiveness (IAS) 3.24 0.91 2.89 0.93 3.21 0.91 2.93 0.95 3.07 0.94 Perceived Sociability (ASRS) 3.67 0.82 3.17 0.89 3.58 0.79 3.27 0.96 3.03 0.96 Perceived Enjoyment (ASRS) 4.16 0.68 3.74 0.92 4.11 0.77 3.79 0.86 3.95 0.83 Interaction evaluation Evaluation 4.23 0.58 3.61 0.87 4.08 0.72 3.77 0.85 3.92 0.80 Overall Rewardingness 3.95 0.73 3.32 0.89 3.73 0.83 3.54 0.89 3.63 0.87 SCS = Source Credibility Scale [43]; IAS = Interpersonal Attraction Scale [42]; ASRS = Acceptance of a Social Robot Scale [44] PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 8 / 18 PLOS ONE Expectations vs. actual behavior of a social robot and b) its perceived sociability and how these subsequently affect c) the general evaluation of the interaction with the robot. For this SEM, perceived sociability, perceived competence and interaction evaluation as well as technological background and experiences with robots served as latent variables. Interaction skill level, expected future role, skill level x expected role, and negative expectancies regarding robots were included as manifest variables (see Fig 2). The SEM analysis was computed using IBM SPSS AMOS 25.0 for Windows (IBM SPSS sta- tistics, released 2017). The latent variable perceived sociability is made up of the four manifest variables sociability (Source Credibility Scale [43]), social attractiveness (Interpersonal Attrac- tion Scale [42]), perceived enjoyment, and perceived sociability (Acceptance of a Social Robot Scale [44]). The latent variable perceived competence consists of the two manifest variables competence (Source Credibility Scale [43]) and task attractiveness (Interpersonal Attraction Scale [42]). The latent variable interaction evaluation consists of the two manifest variables evaluation and overall rewardingness. The latent variable technological background consists of the two manifest variables locus of control when using technology and technical affinity, while the latent variable experiences with robots consists of the three manifest variables contact with real robots, reception of reports about real robots, and reception of robot science fiction mov- ies/series. 9 / 18 PLOS ONE Table 2. Coefficients of the manifest variables’ loadings on the latent dimensions. Latent dimension Manifest variables β SE p Perceived competence Competence (SC) 0.65 0.07 .008 Task Attractiveness 0.67 0.06 .015 Perceived sociability Sociability (SC) 0.67 0.05 .002 Social Attractiveness 0.72 0.05 .001 Perceived Sociability 0.87 0.02 .001 Perceived Enjoyment 0.90 0.02 .004 Interaction evaluation Evaluation 0.92 0.02 .002 Overall Rewardingness 0.91 0.02 .005 Technological background Technological Locus of Control 0.56 0.11 .002 Technical Affinity 1.05 0.21 .002 Experiences with robots Contact with real robots 0.48 0.09 .003 Reception of robot reports 0.73 0.09 .004 Reception of science fiction 0.70 0.08 .002 Table 2. Coefficients of the manifest variables’ loadings on the latent dimensions. below 0.08 indicate a good fit with the data). For this model, the RMSEA was 0.05, CFI was 0.96, TLI was 0.95, and the SRMR was 0.06, indicating an overall good model fit. Influence of the robot’s level of interaction skills There was a significant direct effect of the robot’s interaction skill level on participants’ evalua- tion of the robot’s sociability (β = .54, SE = 0.08, p = .001) as well as competence (β = .28, SE = 0.08, p = .005), which confirms the assumptions of H1a and H1b. There was a significant indirect effect of the robot’s level of interaction skills on people’s general evaluation of the interaction with the robot via perceived sociability and competence (β = .41, SE = 0.07, p = .001), in support of H1c. In summary, the robot’s behavior in terms of a high or low interaction skill level had a sig- nificant effect on participants’ evaluation of the robot’s sociability and competence, which also significantly affected people’s general evaluation of the interaction with the robot. The robot was evaluated as less sociable and less competent when it displayed a low level of interaction skills (see Table 1 for the descriptive values). Low interaction skills were also found to indi- rectly lead to a less positive general evaluation of the interaction with the robot (see Table 1). Results The standardized model results for the latent dimensions can be found in Table 2. There were no missing data and variables were tested for multivariate normality and multi- collinearity. For the evaluation of the model fit, standard criteria were applied following the suggestions from Hu and Bentler [52, 53]: The root mean square error of approximation (RMSEA; values below 0.08 indicate an acceptable fit), comparative fit indices (CFI/TLI; values above 0.90 indicate a good fit), and the standardized root mean square residual (SRMR; values Fig 2. Structural equation model for the evaluation of the robot’s perceived competence and sociability as well as the general evaluation of the interaction. h //d i /10 13 1/j l 0238133 002 r the evaluation of the robot’s perceived competence and sociability as well as the general evaluation of the interaction. tural equation model for the evaluation of the robot’s perceived competence and sociability as well as the general evaluati Fig 2. Structural equation model for the evaluation of the robot’s perceived competence and sociability as well as https://doi.org/10.1371/journal.pone.0238133.g002 9 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 Expectations vs. actual behavior of a social robot PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 Influence of the robot’s expected future role The expectation of the robot’s future role had a significant direct effect on the evaluation of the robot’s sociability (β = .20, SE = 0.08, p = .011), but not on the evaluation of its competence (β = .12, SE = 0.08, p = .173), which is in accordance with H2a but not with H2b. There was also only a marginally significant indirect effect of the expectation of the robot’s future role on the interaction evaluation via perceived sociability and competence (β = .15, SE = 0.07, p = .067). Therefore, H2c is not fully supported. To sum up, how people evaluated the robot’s sociability was influenced by their expectation regarding the robot’s future role. When the robot was described to aim to become a threaten- ing competitor in the future, this robot was evaluated less sociable than when it was described aiming to become a helpful assistant (see Table 1). The robot’s expected future role did not sig- nificantly affect how competent this robot was perceived. On a marginal significant level, peo- ple’s expectation regarding the robot’s future role indirectly affected their general evaluation of the interaction with the robot. When the robot was expected to become a competitor, the interaction with it was evaluated less positive (see Table 1). 10 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE Expectations vs. actual behavior of a social robot Influence of people’s individual background With regard to individual backgrounds, participants’ technological background (β = -.30, SE = 0.12, p = .001) as well as their experiences with robots (β = .37, SE = 0.13, p = .001) had a sig- nificant direct effect on negative expectancies regarding robots. However, neither negative expec- tancies, nor technological background, nor experiences with robots had a significant direct effect on the evaluation of the robot’s sociability (negative expectancies: β = -.06, SE = 0.08, p = .426; technological background: β = .14, SE = 0.10, p = .108; experiences with robots: β = .03, SE = 0.13, p = .808) or competence (negative expectancies: β = -.10, SE = 0.09, p = .235; technological back- ground: β = .11, SE = 0.11, p = .155; experiences with robots: β = -.07, SE = 0.14, p = .503). Like- wise, there were also no indirect effects (via negative expectancies) of technological background and experiences with robots, neither on the robot’s perceived sociability (technological back- ground: β = .02, SE = 0.03, p = .292; experiences with robots: β = -.02, SE = 0.04, p = .304) nor on its perceived competence (technological background: β = .03, SE = 0.04, p = .141; experiences with robots: β = -.04, SE = 0.04, p = .153). This leads to the rejection of the hypotheses H4a and H4b. With regard to people’s evaluation of the interaction, experiences with robots (β = -.05, SE = 0.11, p = .615) as well as negative expectancies (β = -.08, SE = 0.08, p = .324) had no signif- icant indirect effect on the general evaluation of the interaction. There was a marginally signifi- cant indirect effect of people’s technological background on the general evaluation of the interaction with the robot (β = .15, SE = 0.09, p = .053). Therefore, H4c is not fully supported. In summary, none of the personality variables technological background, experiences with robots, or negative expectancies regarding robots had a significant direct effect on the evalua- tion of the robot’s sociability or its competence. There was also no significant indirect effect of people’s experiences with robots and their negative expectancies regarding robots on the gen- eral evaluation of the interaction with the robot. Combined influence of the robot’s level of interaction skills and its expected future role There was no significant direct effect of the interaction variable (robot’s skill level x robot’s expected future role) on the robot’s perceived sociability (β = .03, SE = 0.08, p = .665) or com- petence (β = -.02, SE = 0.08, p = .807), which leads to a rejection of H3a as well as H3b. With regard to the combined influence of the robot’s expected future role and its displayed level of interaction skills, there was also no significant indirect effect on the general evaluation of the interaction (β = .00, SE = 0.08, p = .980), which leads to the rejection of H3c. Summing up, there was no significant direct interaction effect of the robot’s level of interac- tion skills and the robot’s expected future role on the robot’s perceived sociability and its com- petence, as well as no significant indirect interaction effect on the general evaluation of the interaction with the robot. Influence of people’s individual background However, there was a marginally significant indirect effect of people’s technological background revealing that people with a higher affinity and competence with regard to technological devices appear to indirectly evaluate the interac- tion with the robot more positively. The robot’s level of interaction skills With regard to the robot’s behavior during the interaction, Rickenberg and Reeves [13] were among the first to emphasize the strong effects that the behavior of a non-human interaction partner has on people. They further explain that these effects “are not unilaterally good or bad; they can be either or both” and that “an animated character turns up the volume on social presence, which means that it can accentuate the effects of everything presented” (p. 55). In other words, the behavior of an interaction partner is of crucial importance when it comes to the perception and evaluation of this interaction partner. In accordance with these findings, the results of this study show that when a robot presents low interaction skills during people’s interaction with it, the robot is evaluated as less competent and less sociable compared to when the robot displays high interaction skills. Furthermore, these negative evaluations lead people to also generally evaluate the interaction with the robot less positively. The robot with low interaction skills was probably harder and less comfortable to interact with, which was accentuated by its displayed behavior during the interaction. Another considerable explana- tion is that this robot was also perceived as less useful, since a social robot displaying low inter- action skills defeats its purpose of socially interacting with people [14]. And since perceived usefulness is known to influence the attitude towards as well as the usage intention of a tech- nology [15], this could explain why the competence and the sociability of the robot displaying low interaction skills are evaluated more negatively. These negative evaluations of the robot’s attributes then cause people to also negatively evaluate the interaction with the robot in general. Not many people have interacted with a social robot before and thus often rely on available information sources like mass media, which usually present a rather positively biased view on the skills of social robots [11, 30]. This was shown to lead to heightened expectations [11], which increases the possibility that these expectations are negatively violated when people interact with a real social robot which does not measure up to those high expectations [24, 26]. These violations of expectancies are linked to detrimental communication outcomes in inter- personal settings [32] and were also shown to lead to disappointment, mistrust, and rejection in human-robot interactions [24, 26]. Discussion Since social robots are increasingly entering various areas of people’s daily lives, it is crucial to examine what affects people’s perceptions and evaluations of these robots and their interac- tions with them. Ultimately this will affect people’s acceptance of social robots and their will- ingness to use this technology [15]. For this study, our goal was to examine what influences people’s evaluations of specific attributes of a robot with which they just interacted, but also what subsequently influences people’s more general evaluation of the interaction with the robot. For this purpose we decided to look at three aspects which we assumed to have substan- tial influence: 1) the robot’s displayed level of interactions skills as a behavioral component, 2) what kind of role this robot is expected to have in the future (helpful assistant or threatening competitor), and 3) people’s individual background with regard to technology, experiences with robots, and negative expectancies regarding robots. After setting people’s expectation regarding the robot Nao to either become a threatening competitor or a helpful assistant in the future, they interacted with Nao, which then either displayed a low or high interaction skill level. That people had a personal encounter with a real robot in this study extends insights of studies which are based on surveys only and allows a more realistic situation to examine peo- ple’s evaluation of a robot’s characteristics and the interaction with this robot. Post-hoc statistical power analysis Post hoc power analyses using GPower 3.1 were computed with the actual sample size of 162 participants. The statistical power for this study was 0.24 for detecting a small effect (f2 = .02) and exceeded 0.99 for a medium effect (f2 = .15) as well as for a large effect size (f2 = .35; Cohen, 1988). Thus, more than adequate statistical power was reached for medium to large effect sizes, but less than adequate power for a small effect size. 11 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE Expectations vs. actual behavior of a social robot PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 The robot’s expected future role In addition to the display of different levels of interaction skills, it was examined what effects different expectations of the robot’s future purpose have. The robot which was portrayed as trying to compete with and eventually replace humans was evaluated as less sociable compared to the robot which was expected to become a beneficial assistant. Moreover, when people expected the robot to become a competitor, the negative evaluations of the robot’s attributes also lead them to generally evaluate the interaction with the robot more negatively–at least on a marginally significant level. This confirms and extends previous research on the two different prominent views on social robots [11, 28, 31] by showing that framing a robot as competitor or assistant signifi- cantly influences how sociable this robot is perceived and also partly how an interaction with this robot is evaluated subsequently. A previous study showed that people who know more “bad” fictional robot characters have stronger negative expectancies regarding robots becom- ing the superior life form which will outrace and dominate us humans one day [11]. The cur- rent study extends these survey-based findings in a systematic-experimental way and shows that the fear of a robot becoming a threatening competitor also causes people to evaluate the sociability of the robot they just interacted with more negatively. These findings hold important implications for the future shaping of the perception of social robots. If people have no access to personal experiences or existing categories into which something or someone can be encoded, they have to rely on statements and judgements from third parties [54]. Since social robots are not very common yet, many people base their knowl- edge about this technology on what they learn from others, a main source being mass media [11]. Here, the negative and fearsome view of robots developing their own agenda, revolting against and dominating humans is often promoted [28–30]. However, as this study shows, people’s reliance on third party information can also be used to shape people’s expectations and their subsequent evaluations regarding social robots in a positive way. This could be achieved by explaining and emphasizing the positive features and anticipated usefulness of social robots. What kind of role people expected the robot to have in the future had no effect on how competent the robot was perceived, probably because in both descriptions the robot was described as having high interaction skills. The robot’s expected future role After that, only the actual competence of the social robot, represented by its displayed level of interaction skills, seemed to be able to influence people’s evaluation of the robot’s competence. Also, the indirect influence of the robot’s expected future role on the general evaluation of the robot was rather weak. This can be explained by the circumstance that the robot’s perceived competence had a stronger influence on the interaction evaluation than its perceived sociability. The robot’s expected future role mainly influenced its perceived sociability and not so much its perceived competence. The robot’s level of interaction skills This might have also happened in this setting since peo- ple were told that they will be interacting with a social robot in possession of sophisticated 12 / 18 PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 PLOS ONE Expectations vs. actual behavior of a social robot interaction skills. In case of the robot which then displayed low interaction skills, this could have caused a negative expectancy violation leading to a negative evaluation of this robot and the interaction with it. interaction skills. In case of the robot which then displayed low interaction skills, this could have caused a negative expectancy violation leading to a negative evaluation of this robot and the interaction with it. The robot’s expected future role influencing the perception of its interaction skill level There was no direct interaction effect of the robot’s interaction skill level and the expectation of its future role on the evaluation of the robot’s competence or sociability. Consequently, there was also no indirect interaction effect on the general evaluation of the interaction with the robot. The expectation of the robot to become an assistant or a competitor seems not to PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 13 / 18 PLOS ONE Expectations vs. actual behavior of a social robot have affected how the robot’s level of interaction skills were perceived. An explanation could be that the effect of the robot’s behavior during the interaction was too strong by itself to be influenced by what kind of role people expect this robot to have one day in the future. This is also supported by the effect sizes, which were largest for the robot’s level of interaction skills. The behavior is what people experienced live during their interaction with the robot, while the expected role was probably perceived as being far in the future and thus not as present as the behavior displayed live and in that moment. This explanation also goes in line with the insights by Reeves and Rickenberg [13], who emphasize the extraordinary influence of an agent’s behavior during an interaction on the subsequent evaluation of the agent. Also, the low interaction skill level was probably brought to the fore during the interaction since people usually expect precision, sophisticated abilities, and high efficiency from robots [21, 23]. Since all these aspects were not given in the condition with the poorly skilled social robot, this robot was likely perceived as useless given its purpose of socially interacting with its environment [14]. This perception appears not to be affected by which role the robot is sup- posed to take over in the future, probably because with these poor skills the robot seems unsuitable for any kind of social setting. People’s individual background In this study, we additionally considered various personality variables. The influence of peo- ple’s technological background, their experiences with real and fictional robots, and their neg- ative expectancies regarding robots were taken into account while examining the main effects of the robot’s level of interaction skills and the robot’s expected future role. However, there was no significant effect found caused by these variables. There was a marginally significant indirect effect of people’s technological background on the general evaluation of the interac- tion with the robot. People with a more pronounced technological background, which includes their technical affinity and their locus of control when using technology, evaluated the interaction with the robot better. This can likely be traced back to these people being more enthusiastic about technology in general, which was already found in a previous study to lead to a more positive evaluation of the technology and the interaction with it [7]. An explanation for the overall not significant effects of people’s individual backgrounds could be that the effects of the robot’s expected future role and, even more, the robot’s behavior during the interaction were so strong that people’s technological background, their previous experiences with robots as well as their negative expectancies regarding robots did not make any further notable difference in the evaluation of the robot and the interaction with it. This again is supported by the findings of human-agent interaction studies, such as the one by Rick- enberg and Reeves [13], which suggest that the behavior of an agent plays a pivotal role for the effect this agent has on a potential user. Limitations and future research The study has some limitations regarding the generalizability of the results since most partici- pants were students and thus predominantly young and highly educated. In future studies, the setting should also be moved to the participants´ personal environments and instead of a one- time event, interactions with the robot should take place several times over a longer period of time to examine long-term effects. Moreover, the dependent variable measures were self- reported, an experiment with behavioral measurements could bring further insights, for exam- ple, with regard to the willingness to interact with this robot and robots in general. However, we would like to emphasize that the participants of this study had a real interaction with a robot, which extend insights of studies solely based on surveys and offers a crucial basis for a PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 14 / 18 PLOS ONE Expectations vs. actual behavior of a social robot more realistic evaluation of the robot’s characteristics and the interaction with this robot. In future studies, other pre-existing or interactional characteristics should be considered along with other factors which influence the perception and evaluation of a robot and the interaction with a robot. For instance, the appearance of a robot was found to have a great influence on people’s perception of it [1–4] and might also affect how the robot’s present and future role are perceived. Thus, appearance should also be considered in future studies dealing with the ques- tion which variables may influence the evaluation of a social robot. S1 File. Complete data set. S1 File. Complete data set. (SAV) S1 File. Complete data set. (SAV) Conclusion The current study provides novel insights regarding the effects of a social robot’s level of inter- action skills as a behavioral component, the robot’s expected future role, and people’s individ- ual backgrounds on the evaluation of this robot’s sociability and competence and subsequently on the general evaluation of the interaction with this robot. Since knowledge of “malevolent” fictional robots was found to be related to negative expec- tancies regarding real robots [11], this study systematically examined whether a robot which is expected to become a threatening competitor is evaluated differently after people had a real interaction with it. Results showed indeed that the robot presented as a future competitor is evaluated as less sociable. However, the most central insight from this study confirms previous findings [13] in show- ing that the behavior of the robot in the actual interaction has the most pivotal influence on how the robot and the interaction with it are evaluated. With the largest effect sizes of this study, a social robot displaying low interaction skills leads to a more negative evaluation of the robot’s sociability and competence as well as subsequently of the interaction with the robot. Summing up, whether people expect a robot to become a competitor to them in the future plays an important role in how this robot’s sociability is evaluated. However, the robot’s behav- ior in the actual interaction appears to be the key variable influencing people’s evaluations of the robot and the interaction with it in general. Thus, when people get to the point where they interact with a social robot themselves, the robot’s behavior will be more decisive for how this robot is perceived and evaluated than people’s negative expectations regarding the robot’s future role or prior attitudes and personality traits. PLOS ONE | https://doi.org/10.1371/journal.pone.0238133 August 21, 2020 References 1. Goetz J, Kiesler S, Powers A. Matching robot appearance and behavior to tasks to improve human- robot cooperation. In: Proceedings of the 12th IEEE International Workshop on Robot and Human Inter- active Communication—RO-MAN ’03; 2003 Oct 31-Nov 02; Millbrae, CA. Piscataway, NJ: IEEE; 2003. p. 55–60. https://doi.org/10.1109/ROMAN.2003.1251796 2. Li D, Rau PLP, Li Y. A cross-cultural study: Effect of robot appearance and task. International Journal of Social Robotics. 2010; 2:175–86. https://doi.org/10.1007/s12369-010-0056-9 3. Rosenthal-von der Pu¨tten AM, Kra¨mer NC, Herrmann J. 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https://openalex.org/W4294323840
https://jurnal.iain-bone.ac.id/index.php/attadib/article/download/2439/1207
Indonesian
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PENGUATAN PENDIDIKAN KARAKTER BERBASIS BUDAYA ORGANISASI DI SDIT AL MADANI TAPIN
Atta'dib
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cc-by-sa
3,537
PENGUATAN PENDIDIKAN KARAKTER BERBASIS BUDAYA ORGANISASI DI SDIT AL MADANI TAPIN Sulaiman Jazuli email: sj@staidarululumkandangan.ac.id Prodi Pendidikan Agama Islam STAI Darul Ulum Kandangan Abstract This study aims to investigate the process of strengthening organizational culture-based character education at SDIT Al Madani Tapin. This type of research is qualitative with a narrative analysis approach. Instruments and techniques of data collection by means of observation, interviews, and documentation. The results showed that the main characters based on organizational culture at SDIT Al Madani Tapin are independent, have quranic character, are competitive, and have environmental insight. Meanwhile, in strengthening organizational culture-based character education, SDIT Al Madani uses a parent school procurement strategy, collaborates with parents in monitoring student behavior, and holds a class market once a year. The method used is the method of habituation, the method of reward and punishment, and the method of exemplary. The media used are class stars, shalih- shalihah diaries, and school rules. Meanwhile, in providing evaluations, SDIT Al Madani conducts daily academic assessments, and assesses the character of students. s: Strengthening, Character Education, Organizational Culture-Based Education, Pendahuluan Inti dari pendidikan sebenarnya adalah suatu bimbingan yang diberikan kepada peserta didik agar dapat keluar segala potensi yang terdapat dalam dirinya. Sedangkan karakter menurut Kamus Lengkap Bahasa Indonesia sebagaimana dikutip oleh Helmawati adalah tabiat, sifat-sifat kejiwaan, akhlak, atau budi pekerti yang membedakan seseorang dengan yang lain.(Helmawati, 2017) Menurut Amirulloh nguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 20 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Syarbini setelah menarik benang merah berdasarkan penjelasan para ahli, pengertian karakter tidak berbeda dengan definisi akhlak dalam Islam, yaitu perbuatan yang telah menyatu dalam jiwa seseorang sehingga segala sikap yang muncul darinya secara spontanitas dan tidak perlu dipikirkan lagi.(Syarbini, 2014) Mendidik karakter anak bukan hanya tanggung jawab orang tua semata, tetapi juga merupakan tanggung jawab masyarakat dan sekolah. Ketiga unsur tersebut sebagaimana disebutkan oleh Ki Hajar Dewantara dengan Tri Pusat Pendidikan. Ketiga unsur tersebut harus saling bersinergi dalam menanamkan akhlak atau karakter terhadap anak, sebab membentuk karakter anak tidak semudah membalikkan telapak tangan. Dalam membentuk anak agar memiliki karakter yang unggul pastilah memerlukan proses, perjuangan, kesabaran, ketelitian, dan tanggung jawab dari seluruh unsur Tri Pusat tersebut.(Helmawati, 2017) Selain orang tua sebagai pendidik utama dan paling pertama, seorang guru merupakan pendidik kedua setelah orang tua. Bahkan tidak berlebihan apabila seorang pendidik yang berada di suatu sekolah hendak membentuk anak agar berkarakter kuat, pendidik tersebut juga harus memiliki karakter yang kuat. Hal ini karena seorang pendidik juga merupakan teladan bagi peserta didiknya. Namun untuk tercapainya penenaman karakter anak di lingkungan sekolah, bukan hanya tanggung jawab pendidik tertentu saja, tetapi perlu dukungan dari berbagai pihak yang terdapat di lingkungan sekolah tersebut.(Helmawati, 2017) Walaupun yang bertanggung jawab terhadap pendidikan di sekolah adalah para guru dan pemangku kebijakan, akan tetapi pihak sekolah juga harus bersinergi dengan para orang tua peserta didik. Hal ini senada dengan yang disampaikan oleh lickona sebagaimana dikutip oleh Tutuk Ningsih bahwa agar pembentukan karakter bisa berjalan dengan efektif, maka sudah seharusnya pihak sekolah dengan orang tua saling mendukung dan bekerjasama dalam membentuk karakter peserta didik.(Tutuk, 2015) SDIT Al Madani Tapin memiliki keunikan tersendiri dibanding sekolah-sekolah yang berada di Kabupaten Tapin. METODE Jenis penelitian ini termasuk penelitian lapangan. Pengumpulan data dilakukan pada 11 November 2019. Lokasi penelitian bertempat di SDIT Al Madani Tapin, yang beralamat di Jl. Haur Raya RT/RW: 09/03 Kelurahan Rangda Malingkung, Kabupaten Tapin, Kalimantan Selatan, 71114. Subjek penelitiannya adalah kepala sekolah dan semua pendidik. Jenis penelitiannya adalah kualitatif dengan pendekatan analisis naratif. Instrumen dan Teknik pengumpulan data dengan cara observasi, wawancara dan dokumentasi. Sedangkan Teknik analisis data dilakukan dengan analisis naratif. Pendahuluan Keunikan tersebut dapat dilihat dari komitmen sekolah dalam membangun karakter sebagaimana tertuang dalam visi dan misi yang berbasis budaya organisasi. Selain itu, sekolah ini juga melibatkan orang tua peserta didik dalam melakukan penguatan pendidikan karakter. 21 nguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 21 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Berdasarkan latar belakang tersebut, penulis tertarik untuk mengetahui tentang bagaimana penguatan pendidikan karakter berbasis budaya organisasi di SDIT Al-Madani Tapin Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 HASIL DAN PEMBAHASAN Pendidikan Karakter Berbasis Budaya Sekolah Berdasarkan hasil wawancara, pendidikan karakter utama yang terdapat di SDIT Al Madani Tapin ada 4 (empat), yaitu mandiri, berakhlaq qurani, kompetetif, dan berwawasan lingkungan. Keempat karakter yang paling diutamakan di sekolah tersebut sesuai dengan visi sekolah. Keempat karakter tersebut dapat dirincikan sebagai berikut: 1. Mandiri a. Disiplin b. Terbiasa menabung c. Bertanggungjawab d. Berlatih wirausaha dalam pemanfaatan lingkungan hidup 2. Berakhlaq Qurani a. Mengenal Allah dan Rasul-Nya b. Terbiasa shalat 5 waktu berjamaah. c. Terbiasa shalat sunnah dhuha. d. Terbiasa dzikir pagi, petang, dan sesudah shalat. e. Terbiasa senyum, salam, dan sapa. f. Berprilaku sopan dan santun. g. Berpenampilan rapi dan menutup aurat. 22 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 3. Kompetitif 3. Kompetitif a. Tuntas belajar dengan nilai akademis rata-rata 80. b. Hafal Qur’an juz 28, 29, dan 30. b. Hafal Qur’an juz 28, 29, dan 30. c. Mempersentasikan satu karya hasil belajar bewawasan lingkungan. d. Memiliki keterampilan unggul sesuai minat dan bakat yang dimiliki. e. Terbiasa melibatkan diri secara aktif dalam menyelesaikan masalah dengan memanfaatkan keterampilan diri. f. Bersemangat mengikuti berbagai ajang kompetisi dalam rangka mengembangkan potensi diri. 4. Berwawasan lingkungan a. Menciptakan lingkungan yang bersih, sehat dan menyenangkan. 4. Berwawasan lingkungan a. Menciptakan lingkungan yang bersih, sehat dan menyenangkan. b. Menyediakan sarana dan prasarana penunjang pendidikan yang ramah lingkungan. c. Meningkatkn rasa kepedulian terhadap pelestarian, pencegahan, pencemaran dan pencegahan kerusakan lingkungan a. Menciptakan lingkungan yang bersih, sehat dan menyenangkan. b. Menyediakan sarana dan prasarana penunjang pendidikan yang ramah lingkungan. c. Meningkatkn rasa kepedulian terhadap pelestarian, pencegahan, pencemaran dan pencegahan kerusakan lingkungan. Penguatan Pendidikan Karakter Berbasis Budaya Sekolah a. Metode Pembiasaan. a. Metode Pembiasaan. Penerapan metode ini dapat dilihat dari dibiasakannya peserta didik untuk meletakkan sepatu, alat makan, sajadah, mukena dan bantal pada rak dan tempat yang telah disediakan. Dan para peserta didik dibiasakan untuk melakukan shalat dhuha secara berjama’ah serta dibiasakan membuka kran seperlunya Ketika berwudhu. b. Metode Reward dan Punishment Metode ini digunakan salah satunya dengan pemberian bintang kelas. Ketika peserta didik melakukan hal-hal yang baik, maka akan diberi reward bintang kelas. Ketika bintang kelas terkumpul dan dihitung, maka peserta didik yang memiliki bintang kelas terbanyak akan mendapatkan penghargaan di akhir semester. Akan tetapi, apabila peserta didik melakukan hal yang buruk dan tidak menaati peraturan, maka akan mendapatkan pengurangan poin. Poin yang kurang ini nantinya bisa dibayar dengan dissuruh bersih-bersih, diberi motivasi dan lain sebagainya. b. Metode Reward dan Punishment Metode ini digunakan salah satunya dengan pemberian bintang kelas. Ketika peserta didik melakukan hal-hal yang baik, maka akan diberi reward bintang kelas. Ketika bintang kelas terkumpul dan dihitung, maka peserta didik yang memiliki bintang kelas terbanyak akan mendapatkan penghargaan di akhir semester. Akan tetapi, apabila peserta didik melakukan hal yang buruk dan tidak menaati peraturan, maka akan mendapatkan pengurangan poin. Poin yang kurang ini nantinya bisa dibayar dengan dissuruh bersih-bersih, diberi motivasi dan lain sebagainya. Penguatan Pendidikan Karakter Berbasis Budaya Sekolah 1. Strategi SDIT Al Madani Tapin memiliki strategi dalam penguatan pendidikan karakter berbasis budaya sekolah. Strategi tersebut adalah: a. Bersinergi dengan orang tua peserta didik dalam mendidik. Buktinya dapat dilihat dari keberadaan sekolah orang tua yang pada awalnya diadakan dua bulan sekali. Tetapi sekarang diadakannya setiap empat bulan sekali. Dalam sekolah orang tua ini, pihak sekolah memberikan bekal kepada para orang tua mengenai hal-hal yang berhubungan dengan wawasan, prinsip, dan aplikasi dalam pengasuhan anak dan pemahaman psikologi anak secara praktis. Pada empat bulan pertama berupa pembekalan kemudian empat bulan berikutnya penyerahan raport karakter, begitu seterusnya. b. Bekerjasama dengan para orang tua dalam melakukan pemantauan karakter peserta didik. Para orang tua diberikan buku b. Bekerjasama dengan para orang tua dalam melakukan pemantauan karakter peserta didik. Para orang tua diberikan buku 23 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 centangan karakter sehari-hari. Dengan begini, orang tua tidak lepas tangan terhadap Pendidikan anaknya. Terutama dalam mendidik karakter anak. centangan karakter sehari-hari. Dengan begini, orang tua tidak lepas tangan terhadap Pendidikan anaknya. Terutama dalam mendidik karakter anak. centangan karakter sehari-hari. Dengan begini, orang tua tidak lepas tangan terhadap Pendidikan anaknya. Terutama dalam mendidik karakter anak. c. Mengadakan pasar kelas dalam setahun sekali untuk mendidik kemandirian peserta didik. c. Mengadakan pasar kelas dalam setahun sekali untuk mendidik kemandirian peserta didik. 2. Metode 2. Metode c. Metode Keteladanan c. Metode Keteladanan Metode ini merupakan salah satu metode yang paling berpengaruh dalam mendidik karakter peserta didik. Di SDIT Al Madani, para pendidik dituntut untuk memberikan suri tauladan yang bagus seperti tidak terlambat datang ke sekolah, menggunakan pakaian yang rapi dan sopan, membuang sampah pada tempatnya, menyambut peserta didik yang datang ke sekolah. 3. Media Media yang digunakan oleh SDIT Al Madani Tapin dalam memberikan penguatan Pendidikan karakter adalah: nguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 24 24 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 a. Bintang kelas diberikan kepada peserta didik yang memperlihatkan karakter-karakter yang positif a. Bintang kelas diberikan kepada peserta didik yang memperlihatkan karakter-karakter yang positif b. Diary shalih-shalihah yang wajib diisi oleh orang tua dalam melakukan pemantauan terhadap peserta didik terkait perilaku. c. Melalui sticker yang ditempelkan pada dinding sekolah. d. Aturan-aturan yang berlaku di sekolah 4. Evaluasi d. Aturan-aturan yang berlaku di sekolah 4. Evaluasi a. Dalam mengevaluasi peserta didik dilakukan melalui penilaian harian akademis dan juga penilaian karakter peserta didik. Penilaian karakter tersebut tidak terlepas dari penilaian orang tua dan pembinaan peserta didik. b. SDIT Al Madani memiliki acuan dalam penilaian karakter peserta didik, yaitu bintang kelas, buku diary shalihh-shalih-shalihah, dan raport karakter. Bintang kelas diberikan kepada peserta didik yang melakukan hal baik dan mentaati peraturan. Dalam diary anak shalih-shalihah yang diisi oleh guru dan orang tua terdapat 4 variabel, yaitu mandiri, akhlak qurani, kompetitif, dan berwawasan lingkungan. Sedangkan raport karakter adalah buku pedoman yang digunakan guru sebagai acuan penilaian di sekolah. c. Pemberian bintang prestasi untuk tambahan nilai peserta didik apabila selama di kelas berperilaku baik. c. Pemberian bintang prestasi untuk tambahan nilai peserta didik apabila selama di kelas berperilaku baik. Hasil Penelitian Karakter-karakter utama berbasis budaya organisasi di SDIT Al Madani adalah mandiri, berakhlaq Qurani, kompetitif, dan berwawasan lingkungan. Dalam melakukan penguatan Pendidikan karakter tersebut, SDIT Al Madani menggunakan: 1. Strategi yang digunakan adalah mengadakan sekolah orang tua, melakukan pemantauan terhadap karakter anak Bersama-sama dengan orang tua, dan mengadakan pasar kelas sekali dalam setahun. 2. Metode penguatan Pendidikan karakter di SDIT Al Madani Tapin yaitu dengan metode pembiasaan, metode reward dan punishment, dan metode keteladanan. 3. Media yang digunakan adalah bintang kelas, diary shalih-shalihah, sticker, dan aturan yang berlaku di sekolah. 3. Media yang digunakan adalah bintang kelas, diary shalih-shalihah, sticker, dan aturan yang berlaku di sekolah. 25 Penguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 4. Evaluasi yang dilakukan SDIT Al Madani diantaranya adalah melalui penilaian harian akademis dan juga penilaian karakter peserta didik, acuan dalam penilaian karakter peserta didik, yaitu bintang kelas, buku diary shalihh-shalih-shalihah, dan raport karakter, dan bintang prestasi untuk tambahan nilai peserta didik. 4. Evaluasi yang dilakukan SDIT Al Madani diantaranya adalah melalui penilaian harian akademis dan juga penilaian karakter peserta didik, acuan dalam penilaian karakter peserta didik, yaitu bintang kelas, buku diary shalihh-shalih-shalihah, dan raport karakter, dan bintang prestasi untuk tambahan nilai peserta didik. 2. Penguatan Pendidikan Karakter Berbasis Budaya Organisasi 1. Pendidikan Karakter Karakter dalam Bahasa Yunani disebut dengan charassein yang berarti corak yang telah terukir dan tidak hilang. Sifat karakter tersebut bersifat tetap dan menjadi pembeda antara satu orang dengan yang lainnya.(Darmiatun, 2013) Karakter juga merupakan kualitas moral yang menjadi pembeda antara seseorang dengan seseorang lainnya.(Hapudin, 2019) Dalam Bahasa lain, karakter disebut juga dengan akhlak. Kata akhlak berupakan bentuk jamak dari khuluq. Secara Bahasa dapat diartikan sebagai kebiasaan, watak alami, tabiat, keluhuran budi dan agama. Sedangkan pengertian akhlak secara istilah menurut al-Jurjânî adalah sesuatu yang telah terbentuk dalam diri dan sesuatu tersebut dilakukan tanpa dipikirkan terlebih dulu. Apabila yang ditampilkan itu perbuatan baik, maka disebut perilaku yang baik. Sedangkan apabila yang dinampakkan perbuatan yang buruk, maka disebut perilaku yang buruk.(Al-Kharraz, 2009) Pendidikan karakter merupakan usaha yang tidak hanya mengajarkan mana yang benar dan mana yang salah, tetapi juga bentuk usaha dalam menanamkan kebiasaan-kebiasaan yang baik. Jadi, kunci keberhasilan dalam menanamkan pendidikan karakter harus melibatkan pengetahuan yang baik, perasaan yang baik, dan perilaku yang baik.(Darmiatun, 2013) Selain harus melibatkan tiga hal di atas, agar pendidikan karakter yang diimplementasikan dapat berhasil, maka ketiga elemen yang bertanggungjawab haruslah ikut terlibat. Elemen-elemen tersebut menurut istilah Thomas Lickona adalah keluarga, sekolah, dan komunitas. Sedangkan menurut istilah Ki Hajar Dewantara adalah alam keluarga, alam perguruan, dan alam pemuda. (Yahya, 2019) Berdasarkan pendapat kedua tokoh tersebut, dapat diambil kesimpulan bahwa pendidikan karakter itu bukan semata tanggungjawabnya ada pada lembaga informal saja, tetapi juga lembaga formal maupun nonformal. 26 nguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 26 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Penguatan pendidikan karakter sesungguhnya bertujuan untuk mengarahkan seluruh pemangku kebijakan yang ada di sekolah dalam membentuk pola pikir, sikap, dan tindakan dalam pengelolaan instansi pendidikan. terapat lima nilai karakter utama dalam mengembangkan prioritas gerakan penguatan pendidikan karakter. Lima nilai karakter utama tersebut adalah nilai religius, nasionalis, mandiri, gotong royong, dan integritas. (Akbar et al., 2018) Penguatan pendidikan karakter sesungguhnya bertujuan untuk mengarahkan seluruh pemangku kebijakan yang ada di sekolah dalam membentuk pola pikir, sikap, dan tindakan dalam pengelolaan instansi pendidikan. 1. Pendidikan Karakter terapat lima nilai karakter utama dalam mengembangkan prioritas gerakan penguatan pendidikan karakter. Lima nilai karakter utama tersebut adalah nilai religius, nasionalis, mandiri, gotong royong, dan integritas. (Akbar et al., 2018) Budaya organisasi adalah seperangkat pola dan nilai-nilai yang dipahami dan menjadi jiwa bagi suatu organisasi sekaligus dipraktikkan. Budaya organisasi ini kemudian menjadi sebuah identitas dan keunikan yang telah melekat bagi warga sekolah. (Arifin & Wahyudi, 2018) Pendidikan karakter berbasis budaya organisasi ini bertujuan agar praksis Pendidikan yang yang ikut terlibat di dalamnya seluruh sistem, struktur, dan pelaku pendidikan dapat menciptakan iklim dan lingkungan sekolah serta mengatasi ruang-ruang kelas. Fokus berdasarkan basis budaya sekolah ini terdapat pada pembiasaan dan pembentukan nilai-nilai karakter utama yang menjadi prioritas dalam satuan Lembaga pendidikan. (Roestamy, 2020) Dalam memberikan penguatan Pendidikan karakter berbasis budaya organisasi ini, diperlukan Langkah dalam pelaksanaannya. Langkah pertama yang harus dilaksanakan adalah pemilihan nilai utama Pendidikan karakter. Nilai utama ini yang akan menjadi fokus utama satuan pendidikan dan sekaligus sebagai identitas sekolah. Sehingga segala kegiatan, program dan juga pengembangan karakter yang dilakukan berpusat pada nilai utama tersebut. Langkah kedua adalah dengan cara mendiskripsikan nilai-nilai utama tersebut ke dalam indikator dalam bentuk perilaku yang bisa dilihat dan diverifikasi. Penentuan indikator ini bertujuan agar dapat menumbuhkan nilai-nilai pendukung pada karakter utama yang telah ditetapkan. Dengan kedua langkah 27 nguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 27 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 tersebut, satuan Pendidikan dapat membuat kekhasan, keunikan, dan keunggulan yang menjadi pembeda dengan sekolah lainnya. (Roestamy, 2020) tersebut, satuan Pendidikan dapat membuat kekhasan, keunikan, dan keunggulan yang menjadi pembeda dengan sekolah lainnya. (Roestamy, 2020) Fokus utama dalam memberikan penguatan pendidikan karakter (PPK) berbasis budaya organisasi khususnya sekolah adalah melalui pembiasaan dan pembentukan karakter yang menjadi prioritas suatu instansi pendidikan. Pembiasaan tersebut selalu terhubung dengan semua kegiatan yang ada di sekolah dan tercermin dalam suasana dan lingkungan yang kondusif. (Roestamy, 2020) Fokus utama dalam memberikan penguatan pendidikan karakter (PPK) berbasis budaya organisasi khususnya sekolah adalah melalui pembiasaan dan pembentukan karakter yang menjadi prioritas suatu instansi pendidikan. Pembiasaan tersebut selalu terhubung dengan semua kegiatan yang ada di sekolah dan tercermin dalam suasana dan lingkungan yang kondusif. (Roestamy, 2020) 3. Dalam melakukan penguatan pendidikan karakter berbasis budaya organisasi, diperlukan adanya strategi, metode, media, evaluasi. a. nguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 28 1. Pendidikan Karakter Strategi yang dapat dilakukan dalam memberikan penguatan pendidikan karakter adalah melalui pengintegrasian kegiatan yang dilakukan peserta didik baik di sekolah ataupun di rumah, pengintegrasian mealui program yang dijalankan pihak sekolah, dan juga melalui kelas yang diatur dengan baik.(Sultonurohmah, 2017) b. metode yang dapat dilakukan dalam mendidik karakter peserta didik diantaranya adalah (1) Metode keteladanan yaitu memperlihatkan karakter-karakter yang terpuji di hadapan peserta didik. (2) Metode pembiasaan adalah suatu proses yang dilakukan dalam membiasakan karakter-karakter tertentu terhadap peserta didik untuk mengubah kebiasaan-kebiasaan yang buruk ke arah yang lebih baik. (3) Metode nasihat adalah metode yang dilakukan oleh pendidik dapat berupa pemberian motivasi dan pemberian peringatan kepada peserta didik terhadap karakter yang ditampilkan. (4) Metode Bercerita adalah suatu cara yang dilakukan dalam mendidik karakter melalui cerita-cerita yang dapat diambil manfaatnya. (5) Metode perumpaaan adalah cara yang ditempuh oleh peserta didik dengan memberikan perumpamaan-perumpamaan yanbg telah b. metode yang dapat dilakukan dalam mendidik karakter peserta didik diantaranya adalah (1) Metode keteladanan yaitu memperlihatkan karakter-karakter yang terpuji di hadapan peserta didik. (2) Metode pembiasaan adalah suatu proses yang dilakukan dalam membiasakan karakter-karakter tertentu terhadap peserta didik untuk mengubah kebiasaan-kebiasaan yang buruk ke arah yang lebih baik. (3) Metode nasihat adalah metode yang dilakukan oleh pendidik dapat berupa pemberian motivasi dan pemberian peringatan kepada peserta didik terhadap karakter yang ditampilkan. (4) Metode Bercerita adalah suatu cara yang dilakukan dalam mendidik karakter melalui cerita-cerita yang dapat diambil manfaatnya. (5) Metode perumpaaan adalah cara yang ditempuh oleh peserta didik dengan memberikan perumpamaan-perumpamaan yanbg telah 28 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 terjadi di masa lalu.(Jannah, 2019) (6) Metode reward dan punishment. Reward adalah cara yang dilakukan oleh pendidik melalui apresiasi disebabkan karena perbuatan oeserta didik layak mendapat pujian. Sedangkan punishment adalah sebuah cara yang dilakukan oleh pendidik yang mengandung hukuman sebagai efek jera karena perbuatan buruk yang diperbuat peserta didik.(Rosyid, 2018) c. Media secara umum dapat diartikan sebagai sebuah perantara atau alat berbentuk fisik yang digunakan untuk menyampaikan suatu pesan tertentu kepada penerima pesan.(Maiyena, 2013) d. Evaluasi merupakan alat ukur yang digunakan untuk mengukur keberhasilan suatu pekerjaan. Dalam konteks pendidikan karakter, evaluasi diartikan sebagai alat ukur yang digunakan dalam menentukan tingkat keberhasilan penguatan karakter yang telah ditetapkan oleh lembaga/sekolah. (Zuriani, 2016) DAFTAR PUSTAKA Akbar, S., Sa’dijah, C., Wahyuni, S., Arafik, M., Samawi, A., Saryono, D., Supriyono, Relisa, & Marjanto, D. K. (2018). Penguatan Pendidikan Karakter di Sekolah Dasar. Puslitjakbud. Al-Kharraz, K. ibn J. ibn U. (2009). Mausuah Al-Akhlaq. Arifin, I., & Wahyudi. (2018). Manajemen Pendidikan Karakter Berbasis Budaya Sekolah dan Profesionalisme Guru : Kajian Teori dan Riset (p. 330). Darmiatun, D. dan S. (2013). Implementasi Pendidikan Karakter di Sekolah (Bintoro (ed.); I). Gava Media. Hapudin, M. S. (2019). Manajemen Karakter: Membentuk Karakter Baik Pada Anak (S. R. Zaid (ed.)). Tazkia Press. Helmawati. (2017). Pendidikan Karakter Sehari-Hari (Nita (ed.)). Remaja Rosdakarya. Jannah, M. (2019). Metode Dan Strategi Pembentukan Karakter Religius Yang Diterapkan Di Sdtq-T An Najah Pondok Pesantren Cindai Alus Martapura. Al-Madrasah: Jurnal Pendidikan Madrasah Ibtidaiyah, Penguatan Pendidikan Karakter Berbasis Budaya Organisasi Di Sdit Al Madani Tapin (Sulaiman Jazuli) h. 20-30 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 Atta’dib Jurnal Pendidikan Agama Islam, Prodi Pendidikan Agama Islam Fakultas Tarbiyah IAIN Bone, Vol. 3, No. 1, Juni 2022 4(1), 77–102. Maiyena, S. (2013). Pengembangan Media Poster Berbasis Pendidikan Karakteruntuk Materi Global Warming. Jurnal Materi Dan Pembelajaran Fisika, 3(1). Rosyid, M. Z. (2018). Reward & punishment dalam pendidikan. Literasi Nusantara. Sultonurohmah, N. (2017). Strategi penanaman nilai karakter jujur dan disiplin siswa. AL IBTIDA’: Jurnal Program Studi Pendidikan Guru Madrasah Ibtidaiyah, 5(2), 1–21. Syarbini, A. (2014). Model pendidikan karakter dalam keluarga. Elex Media Komputindo. Tutuk, N. (2015). Implementasi pendidikan karakter. Tutuk, N. (2015). Implementasi pendidikan karakter. Zuriani, Y. (2016). Pelaksanaan Evaluasi Pendidikan Karakter di Sekolah Dasar Islam Terpadu (SDIT) IQRO’IPUH. Annizom, 1(3).
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Size of carotid artery intraplaque hemorrhage and acute ischemic stroke: a cardiovascular magnetic resonance Chinese atherosclerosis risk evaluation study
Journal of cardiovascular magnetic resonance
2,019
cc-by
7,456
(2019) 21:36 (2019) 21:36 Liu et al. Journal of Cardiovascular Magnetic Resonance https://doi.org/10.1186/s12968-019-0548-1 Liu et al. Journal of Cardiovascular Magnetic Resonance https://doi.org/10.1186/s12968-019-0548-1 Size of carotid artery intraplaque hemorrhage and acute ischemic stroke: a cardiovascular magnetic resonance Chinese atherosclerosis risk evaluation study ng Liu1,2†, Maoxue Wang3,4†, Bing Zhang3*†, Wei Wang1, Yun Xu5, Yongjun Han6, Chun Yuan7 and Abstract Background: To determine the usefulness of the size of carotid artery intraplaque hemorrhage (IPH) in discriminating the risk of acute ischemic stroke using cardiovascular magnetic resonance (CMR) vessel wall imaging Methods: Symptomatic patients with carotid atherosclerotic plaque who participated in a cross-sectional, multicenter study of CARE-II (NCT02017756) were included. All patients underwent carotid and brain CMR imaging. Carotid plaque burden and the size of plaque compositions including calcification, lipid-rich necrotic core (LRNC), and IPH were measured. Presence of acute cerebral infarct (ACI) in ipsilateral hemisphere of carotid plaque was determined. The relationship between carotid plaque features and presence of ipsilateral ACI was then analyzed. Results: Of 687 recruited patients (62.7 ± 10.1 years; 69.4% males) with carotid plaque, 28.5% had ACI in ipsilateral hemispheres. Logistic regression revealed that carotid plaque burden was significantly associated with the presence of ACI before and after adjusted for clinical confounding factors. The volume of LRNC, %LRNC volume, volume of IPH, and %IPH volume were significantly associated with ACI before (volume of LRNC: OR = 1.297, p = 0.005; %LRNC volume: OR = 1.119, p = 0.018; volume of IPH: OR = 2.514, p = 0.003; %IPH volume: OR = 2.202, p = 0.003) and after (volume of LRNC: OR = 1.312, p = 0.006; %LRNC volume: OR = 1.90, p = 0.034; volume of IPH: OR = 2.907, p = 0.007; % IPH volume: OR = 2.374, p = 0.004) adjusted for clinical confounding factors. The association between volume of IPH and ACI remained statistically significant after further adjusted for plaque volume (OR = 2.813, p = 0.016) or both plaque volume and volume of LRNC (OR = 4.044, p = 0.024). ns: In symptomatic patients with carotid atherosclerotic plaques, the size of IPH is independently with ipsilateral ACI, suggesting the size of IPH might be a useful indicator for the risk of ACI. Trial registration: Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique Identifier: N tration: Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique Identifier: NCT02017756 Keywords: Carotid artery, Atherosclerosis, Intraplaque hemorrhage, Acute cerebral infarct, Cardiovascular magnetic resonance imaging Keywords: Carotid artery, Atherosclerosis, Intraplaque hemorrhage, Acute cerebral infarct, Cardiovascular magnetic resonance imaging Subject Terms: Magnetic Resonance Imaging (MRI), Ischemic stroke, Atherosclerosis, Stenosis, Vascular disease Subject Terms: Magnetic Resonance Imaging (MRI), Ischemic stroke, Atherosclerosis, Stenosis, Vascular disease © The Author(s). Abstract 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: zhangbing_nanjing@vip.163.com; xihaizhao@tsinghua.edu.cn †Yang Liu, Maoxue Wang, Bing Zhang and Xihai Zhao are contributed equally to this work. †Yang Liu and Maoxue Wang are co-first authors. 3Department of Radiology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China 2Department of Biomedical Engineering, Center for Biomedical Imaging Research, Tsinghua University School of Medicine, Beijing, China Full list of author information is available at the end of the article CMR imaging protocol f d CMR imaging protocol CMR was performed on 3 T CMR scanners (Achieva TX, Philips Healthcare, Best, The Netherlands) with 8- channel phase array carotid coils and 8-channel head coils. All the imaging sites utilized CMR scanners with the same vendor and model. The carotid artery vessel wall was imaged by multicontrast imaging protocol including three dimensional (3D) TOF, two-dimensional T1W and T2-weighted (T2W), and 3D MPRAGE se- quences with the following parameters: TOF: fast field echo (FFE), repeat time (TR) / echo time (TE) 20 ms/4.9 ms, flip angle 20°, field of view (FOV) 14 × 14 cm2, matrix size 256 × 256, and slice thickness 1 mm; T1W: turbo spin echo (TSE), TE/TR 800 ms/10 ms, FOV 14 × 14 cm2, matrix size 256 × 256, and slice thickness 2 mm; T2W: TSE, TE/TR 4800 ms/50 ms, FOV 14 × 14 cm2, matrix size 256 × 256, and slice thickness 2 mm; and MPRAGE: FFE, TE/TR 8.8 ms/5.3 ms, flip angle 15°, FOV 14 × 14 cm2, matrix size 256 × 256, and slice thick- ness 1 mm. A clinically routine imaging protocol was used to acquire brain images with the following parame- ters: T1W: FFE, TE/TR 308 ms/4.6 ms, FOV 23 × 23 cm2, matrix size 400 × 256, and slice thickness 5.5 mm; T2W: TSE, TE/TR 3000 ms/80 ms, FOV 23 × 23 cm2, matrix size 400 × 255, and slice thickness 5.5 mm; and diffusion weighted imaging (DWI): echo planar imaging, TR/TE 2724 ms/86 ms, FOV 23 × 23 cm2, matrix size 128 × 126, and slice thickness 5.5 mm. The gadolinium-based contrast agent was not administrated for carotid CMR imaging in this study. Mutlicontrast cardiovascular magnetic resonance (CMR) vessel wall imaging has been demonstrated to be capable of accurately assessing the morphological and compositional characteristics of carotid atherosclerotic plaques [5]. Among three T1 weighted imaging se- quences of time-of-flight (TOF), T1-weighted (T1W), and magnetization-prepared rapid acquisition gradient echo (MPRAGE), MPRAGE showed the best agreement with histology in evaluating carotid IPH [6]. Therefore, adding an MPRAGE sequence into the multicontrast vessel wall imaging protocol will improve the accuracy of the assessment of carotid IPH. The aim of this study was to determine the usefulness of the size of carotid IPH for determination of risk of ACI in symptomatic patients with carotid plaques using multi-contrast CMR vessel wall imaging. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Page 2 of 9 Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Page 2 of 9 Study population The patients were recruited from a cross-sectional, mul- ticenter, observational study of Chinese Atherosclerosis Risk Evaluation (CARE-II, NCT02017756) which aimed to determine the prevalence of carotid artery high-risk atherosclerotic plaque in symptomatic patients in China. The study design had been published [7]. The inclusion criteria of CARE-II study were as follows: 1) 18 to 80 years; 2) had recent (< 2 weeks) cerebrovascular ischemic events (ischemic stroke or transient ischemic attack); and 3) had atherosclerotic plaques in at least one carotid artery identified by ultrasound imaging. Subjects with CMR imaging protocol f d Since all patients with IPH will be classified into the same risk of developing stroke according to previous evidences, this study would provide additional information for assessing the stroke risk using the size of IPH. Background cardioembolic stroke, hemorrhage stroke, history of radi- ation therapy in the neck, or contraindication to CMR examination were excluded. A flow chart of patient re- cruitment is presented in Fig. 1. The following clinical data were collected from the medical records: age, gender, body mass index (BMI), blood pressure, lipid panel results including total cholesterol, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol, and triglycerides, history of hypertension, smoking, hyperlipidemia, diabetes, and coronary artery disease (CAD). The study protocol was approved by local Ethics Committee at each participating institution and all patients provided written informed consent. g Previous studies have shown that presence of intrapla- que hemorrhage (IPH) in carotid arteries was a strong predictor for future ischemic events [1, 2]. The under- lying mechanism might be based on the hypothesis that IPH accelerates the progression of atherosclerotic plaque and increases the risk of plaque rupture. Physiopatholo- gically, the extracorpuscular hemoglobin released from the phagocytosis of red blood cells promotes local in- flammation and proteolytic enzymes that accelerate the degradation and subsequent disruption of fibrous cap [3]. With the increases in the amount of IPH, the effect of inflammatory factors and proteolytic enzymes might be amplified hypothetically. A most recent study by Wang et al. reported that symptomatic carotid plaques tended to having marginally larger volume of IPH com- pared to those with asymptomatic carotid plaques [4]. However, the usefulness of the size of IPH for determin- ing the risk of acute cerebral infarct (ACI) remains unclear. CMR image analysis CMR images were reviewed by experienced radiologists (> 3 years’ experience in neuroradiology) with consensus who were blinded to clinical information. Brain MR images were reviewed by two radiologists (Y.L. and M.W.) who were blinded to carotid images. Presence or absence of ACI in bilateral hemispheres was determined respectively. The ACI was defined as lesion which shows Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Page 3 of 9 Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Liu et al. Journal of Cardiovascular Magnetic Resonance Page 3 of 9 (2019) 21:36 Fig. 1 Flow chart of patient recruitment for final analysis Fig. 1 Flow chart of patient recruitment for final analysis MPRAGE images. The LRNC showed isointense on TOF and T1W images and hypointense on T2W images within the plaque. The IPH will be determined when there was hyperintense on TOF, T1W, and MPRAGE (1.5 times of signal intensity compared to muscle) images. FCR was identified when there was deficit in fibrous cap or discontinuous surface of the plaque. For arteries with IPH, the age of IPH was also evaluated using the following criteria: fresh IPH: intact red blood cell with intracellular methemoglobin, which showed hy- perintense on T1W or 3D TOF images and hypointense or isointense on T2W images; and recent IPH: lytic red blood cell with extracellular methemoglobin, which showed hyperintense on T1W or 3D TOF images and hyperintense on T2W images [9]. The luminal stenosis of carotid artery was measured on the reconstructed im- ages of TOF CMR angiography with maximum intensity project algorism using North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria [10]. hyperintensity on DWI images but iso- or hypointense on T1W images. g The carotid vessel wall images were interpreted by another two radiologists (Y.H. and X.Z.) who were blinded to brain images using a custom-designed soft- ware (CASCADE, University of Washington, Seattle, Washington, USA). The boundaries of lumen, outer wall and plaque components were outlined manually at each axial location of carotid arteries for measuring the morphology including lumen area, wall area, mean wall thickness (mean WT), IPH area, lipid-rich necrotic core (LRNC) area, and calcification area. Then total vessel area (total vessel area = lumen area + wall area), and normalized wall index (NWI = wall area / total vessel area × 100%) were calculated. CMR image analysis For each subject, the mor- phological measurements were taken from the mean values of each corresponding measurement. The volume of plaque and each plaque component were calculated as the sum of plaque area and each plaque component area with corresponding slices multiplied by slice thick- ness respectively. The percentage of the volume of each plaque component divided by plaque volume was calcu- lated. Carotid atherosclerotic plaque was defined as ec- centric wall thickening on CMR vessel wall images. Presence or absence of atherosclerotic plaque compo- nents including calcification, LRNC, IPH, and fibrous cap rupture (FCR) in carotid arteries were identified using published criteria [8]. Calcification was character- ized by the hypointense on TOF, T1W, T2W, and Characteristics of the study population Of 118 sub- jects who had carotid plaques with IPH, 60 (50.8%) had fresh IPH, 31 (26.3%) had recent IPH, and 27 (22.9%) had both fresh and recent IPHs. There were no signifi- cant differences in the prevalence of fresh IPH, recent IPH and both two types of IPH between patients with and without ACI (all p > 0.05). Characteristics of the study population y p p Figure 1 represents the flow chart of the patient recruit- ment. Of 1047 patients of CARE-II study, 900 were found to have plaque in at least one carotid artery. Of the remaining 900 patients, 213 were excluded due to the following reasons: 1) no MPRAGE images or with poor MPRAGE images (n = 27); 2) no DWI images (n = 186). Of 687 eligible subjects (62.7 ± 10.1 years, 477 (69.4%) males, 516 (75.1%) had hypertension, 380 (55.3%) had hyperlipidemia, 216 (31.4%) had diabetes, 347 (50.5%) had smoke history, and 109 (15.9%) had CAD. Compared with patients without ACI, those with ACI had significantly higher BMI (24.9 ± 3.1 vs. 24.2 ± 3.1 kg/m2, p = 0.010), younger age (60.9 ± 9.8 vs. 63.3 ± 10.1 years, p = 0.005), higher prevalence of males (78.6 vs. 65.8%, p = 0.001), smoke (58.2 vs. 47.5%, p = 0.011), and diabetes (39.3 vs. 28.3%, p = 0.005), and lower LDL (2.8 ± 0.9 vs. 3.1 ± 1.0 years, p = 0.004). The demo- graphic and clinical characteristics are detailed in Table 1. ACI Acute cerebral infarction, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, LDL low density lipoprotein, HDL high density lipoprotein, TC total cholesterol, TG triglycerides, CAD coronary artery disease ACI Acute cerebral infarction, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, LDL low density lipoprotein, HDL high density lipoprotein, TC total cholesterol, TG triglycerides, CAD coronary artery disease ACI had marginally higher prevalence of IPH (21.4% vs. 15.5%, p = 0.062) compared with those without ACI. No significant differences in presence of calcification, LRNC, and FCR between subjects with and without ACI (all p > 0.05). Patients with ACI had significantly larger volume of IPH (164.9 ± 200.5 vs. 71.4 ± 67.7 mm3, p = 0.006) (Fig. 2), larger percent of IPH volume (12.1 ± 10.5% vs. 7.0 ± 5.9%, p = 0.014), and lower percent of calcification volume (3.3 ± 4.2% vs. 4.0 ± 4.0%, p = 0.046) compared with those without ACI. There were no significant differ- ences in the volume of calcification (Fig. 2), volume of LRNC (Fig. 2), and percent of LRNC volume between patients with and without ACI (all p > 0.05). Statistical analysis The confound- ing factors include age, gender, and those clinical fac- tors which were associated with presence of ACI. Two-sided tests were used and p value < 0.05 was considered as statistically significant. All statistical analyses were performed using SPSS 16.0 (Statistical Package for the Social Sciences (SSPS) International Business Machines, Inc., Armonk, New York, USA). Statistical analysis Continuous variables were summarized as mean ± stand- ard deviation (SD) and the categorical variables were described as percentage. For patients with bilateral carotid plaques, the CMR imaging features in plaques with larger plaque burden (plaque volume) were selected for analysis. The clinical characteristics and plaque features in carotid artery were compared be- tween patients with and without ipsilateral ACI using Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Liu et al. Journal of Cardiovascular Magnetic Resonance Page 4 of 9 Table 1 Comparison of clinical characteristics of study population Mean ± SD or n (%) p Patients with ACI Patients without ACI (n = 196) (n = 491) Age, years 60.9 ± 9.8 63.3 ± 10.1 0.005 Gender, male 154 (78.6) 323 (65.8) 0.001 BMI, kg/m2 24.9 ± 3.1 24.2 ± 3.1 0.010 Smoke 114 (58.2) 233 (47.5) 0.011 Diabetes 77 (39.3) 139 (28.3) 0.005 Hypertension 149 (76.0) 367 (74.7) 0.727 SBP, mmHg 145.8 ± 24.7 144.7 ± 20.7 0.999 DBP, mmHg 88.4 ± 13.7 86.9 ± 13.4 0.138 Hyperlipidemia 108 (55.1) 272 (55.4) 0.944 TC, mmol/L 4.5 ± 1.2 4.7 ± 1.1 0.093 TG, mmol/L 1.9 ± 1.2 1.8 ± 1.0 0.833 HDL, mmol/L 1.1 ± 0.4 1.1 ± 0.3 0.089 LDL, mmol/L 2.8 ± 0.9 3.1 ± 1.0 0.004 CAD 34 (17.3) 75 (15.3) 0.502 ACI Acute cerebral infarction, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, LDL low density lipoprotein, HDL high density lipoprotein, TC total cholesterol, TG triglycerides, CAD coronary artery disease Table 1 Comparison of clinical characteristics of study population independent-sample t test, Mann-Whitney U test, Chi-square test, or Fisher’s test as appropriate. The odds ratio (OR) and corresponding 95% confidence interval (CI) of plaque features in discriminating ACI were calculated using logistic regression before and after adjusted for confounding factors. The confound- ing factors include age, gender, and those clinical fac- tors which were associated with presence of ACI. Two-sided tests were used and p value < 0.05 was considered as statistically significant. All statistical analyses were performed using SPSS 16.0 (Statistical Package for the Social Sciences (SSPS) International Business Machines, Inc., Armonk, New York, USA). independent-sample t test, Mann-Whitney U test, Chi-square test, or Fisher’s test as appropriate. The odds ratio (OR) and corresponding 95% confidence interval (CI) of plaque features in discriminating ACI were calculated using logistic regression before and after adjusted for confounding factors. Comparison of plaque features between patients with and without ACI The morphological and compositional characteristics of carotid plaque are detailed in Table 2. Of 687 patients with plaque in carotid arteries, 196 (28.5%) had ACI in the ipsilateral hemispheres. Compared with patients without ACI, those with ACI had significantly greater carotid wall area (35.4 ± 11.6 vs. 32.5 ± 11.0 mm2, p = 0.002), total vessel area (80.7 ± 19.5 vs. 77.0 ± 20.7 mm2, p = 0.016), mean WT (1.3 ± 0.5 vs. 1.2 ± 0.4 mm, p = 0.007), and marginally larger luminal stenosis (26.4 ± 40.0% vs. 16.1 ± 27.5%, p = 0.058). No significant differ- ences were found in the lumen area, normalized wall index, and plaque volume between patients with and without ACI (all p > 0.05). Association of carotid plaque features with ipsilateral ACI The results of logistic regression analysis are detailed in Table 3. Univariate logistic regression showed that wall area (OR, 1.269; 95%CI, 1.081–1.489; p = 0.014), total vessel area (OR, 1.194; 95%CI, 1.014–1.406; p = 0.034), mean WT (OR, 1.293; 95%CI, 1.103–1.515; p = 0.002), volume of LRNC (OR, 1.297; 95%CI, 1.084–1.553; p = 0.05), and volume of IPH (OR, 2.514; 95% CI, 1.363– In this study population, of all 687 subjects, 348 (50.7%) had calcification, 528 (76.9%) had LRNC, 118 (17.2%) had IPH, and 58 (8.4%) had FCR. Patients with Liu et al. Comparison of plaque features between patients with and without ACI Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Page 5 of 9 Table 2 Comparison of carotid plaque features between patients with and without ACI Mean ± SD, or n (%) p Patients with ACI (n = 196) Patients without ACI (n = 491) Plaque morphology Lumen area, mm2 45.3 ± 15.2 44.4 ± 14.9 0.443 Wall area, mm2 35.4 ± 11.6 32.5 ± 11.0 0.002 Total vessel area, mm2 80.7 ± 19.5 77.0 ± 20.7 0.016 Mean wall thickness, mm 1.3 ± 0.5 1.2 ± 0.4 0.007 Normalized wall index, % 44.2 ± 10.7 42.6 ± 9.3 0.122 Luminal stenosis, % 26.4 ± 40.0 16.1 ± 27.5 0.058 a Plaque volume, mm3 722.5 ± 475.3 667.3 ± 426.9 0.320 Presence of plaque compositions Calcification 101 (51.5) 247 (50.3) 0.772 Fibrous cap rupture 18 (9.2) 40 (8.1) 0.659 LRNC 148 (75.5) 380 (77.4) 0.597 All IPH 42 (21.4) 76 (15.5) 0.062 Fresh IPH 22 (11.2) 38 (0.8) 0.804 Recent IPH 8 (0.4) 23 (0.5) 0.185 Both two types of IPH 12 (0.6) 15 (0.3) 0.274 b Size of plaque compositions Volume of calcification, mm3 37.1 ± 76.2 38.7 ± 51.8 0.194 % calcification volume, % 3.3 ± 4.2 4.0 ± 4.0 0.046 Volume of LRNC, mm3 166.5 ± 243.9 113.4 ± 153.7 0.083 % LRNC volume, % 14.9 ± 14.2 12.1 ± 11.1 0.105 Volume of IPH, mm3 164.9 ± 200.5 71.4 ± 67.7 0.006 % IPH volume, % 12.1 ± 10.5 7.0 ± 5.9 0.014 LRNC lipid-rich necrotic core, IPH intraplaque hemorrhage. a The value was calculated from the arterial slices with atherosclerotic plaque. b The value was included from the arterial slices with the corresponding plaque composition, respectively Table 2 Comparison of carotid plaque features between patients with and without ACI Mean ± SD, or n (%) Patients with ACI (n 196) Patie Table 2 Comparison of carotid plaque features between patients with and without ACI LRNC lipid-rich necrotic core, IPH intraplaque hemorrhage. a The value was calculated from the arterial slices with atherosclerotic plaque. b The value was included from the arterial slices with the corresponding plaque composition, respectively LRNC lipid-rich necrotic core, IPH intraplaque hemorrhage. Comparison of plaque features between patients with and without ACI a The value was calcula from the arterial slices with the corresponding plaque composition, respectively 4.637; p = 0.003) with increment of 1 SD, and luminal stenosis (OR, 1.098; 95%CI, 1.044–1.155; p < 0.001), % LRNC volume (OR, 1.199; 95%CI, 1.032–1.393; p = 0.018), and %IPH volume (OR, 2.202; 95%CI, 1.317– 3.680; p = 0.003) with increment of 10% were signifi- cantly associated with presence of ACI. Multivariate logistic regression revealed that the associations of ACI with wall area, mean WT, luminal stenosis, volume of LRNC, % LRNC volume, volume of IPH, and % IPH volume remained statistically significant after adjusted for clinical confounding factors of age, gender, BMI, hypertension, hyperlipidemia, diabetes, and smoke, and CHD (model 1). After further adjusted for plaque vol- ume (model 2), significant associations of presence of ACI with volume of LRNC (OR, 1.398; 95% CI, 1.045– 1.870; p = 0.024) and volume of IPH (OR, 2.813; 95% CI, 1.212–6.528; p = 0.016) can be found (Table 3). More- over, the association of ACI with volume of IPH (OR, 4.044; 95% CI, 1.200–13.626; p = 0.024) remained statis- tically significant after adjusted for the clinical risk fac- tors, plaque volume, and volume of LRNC. In contrast, the association of ACI with LRNC volume was not statistically significant after adjusted for clinical risk fac- tors, plaque volume, and volume of IPH. There were no significant associations between the presence of calcifica- tion, FCR, LRNC, and IPH, volume of calcification, and percent volume of calcification and the presence of ACI before and after adjusting for confounding factors (all p > 0.05). Figure 3 represents an example of a patient who had large volume of IPH in carotid artery and ipsilateral ACI. In addition, logistic regression showed that the presence of carotid IPH (OR, 15.698; 95% CI, 8.575–28.739; p < 0.001) and volume of IPH with in- crement of 1 SD (OR, 3.322; 95% CI, 1.685–6.548; p = 0.001) were significantly associated with presence of FCR. Discussion The present study investigated the relationship between atherosclerotic plaque characteristics and risk of ACI in symptomatic patients with carotid plaque using multi- contrast CMR vessel wall imaging. We found that both Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Page 6 of 9 Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Liu et al. Journal of Cardiovascular Magnetic Resonance Page 6 of 9 (2019) 21:36 Fig. 2 A bar graph of volume of carotid plaque components including calcification (CA), lipid-rich necrotic core (LRNC), and intraplaque hemorrhage (IPH). Volume of and IPH was significantly associated with the presence of acute cerebral infarct h of volume of carotid plaque components including calcification (CA), lipid-rich necrotic core (LRNC), and intraplaque Volume of and IPH was significantly associated with the presence of acute cerebral infarct Fig. 2 A bar graph of volume of carotid plaque components including calcification (CA), lipid-rich necrotic core (LRNC), and intraplaque hemorrhage (IPH). Volume of and IPH was significantly associated with the presence of acute cerebral infarct Fig. 2 A bar graph of volume of carotid plaque components including calcification (CA), lipid-rich necrotic core hemorrhage (IPH). NWI normalized wall index, FCR fibrous cap rupture, LRNC lipid-rich necrotic core, IPH intraplaque hemorrhage. a The increment for plaque burden was 1 SD. b The increment for NWI and luminal stenosis was 10% NWI and stenosis. c Only for patients with the corresponding plaque compositional feature with increment of 1 SD for composition size and 10% for percent of composition volume. d Model 1: adjustment for age, gender, BMI, hypertension, hyperlipidemia, CHD, diabetes, and smoke. e Model 2: adjustment for the former clinical factors and plaque volume Discussion Multicontrast MR images show an atherosclerotic plaque with large IPH (hyperintensity within the vessel wall, white arrows) in the right internal carotid artery. On Brain images of the same patient, acute cerebral infarct which shows hypointensity on T1W image and hyperintensity on DWI image can be seen in the right hemisphere Fig. 3 An example of a patient with carotid intraplaque hemorrhage (IPH) and ipsilateral acute cerebral infarct (ACI). Multicontrast MR images show an atherosclerotic plaque with large IPH (hyperintensity within the vessel wall, white arrows) in the right internal carotid artery. On Brain images of the same patient, acute cerebral infarct which shows hypointensity on T1W image and hyperintensity on DWI image can be seen in the right hemisphere carotid plaque burden and the size of IPH and LRNC were significantly associated with the presence of ipsilat- eral ACI before and after adjusted for age, gender, BMI, smoke, diabetes, hyperintense, hyperlipidemia, and CAD. In addition, after further adjusted for plaque volume and volume of LRNC, the association between volume of IPH and presence of ACI remained statisti- cally significant. Our findings suggest that the associ- ation between volume of IPH and risk of ACI was independent of plaque burden and LRNC volume. greater in patients with recurrent stroke than that in those with first-time stroke [12]. Zhao et al. reported that carotid morphological measurements, such as max- imum WT (β = 11.1;95% CI, 3.4–19.2; p = 0.005) and % wall volume (β = 33.5;95% CI, 14.1–56.2; p = 0.001), were significantly associated with ipsilateral acute cerebral in- farction volume [13]. Our findings further compel the evidence that carotid plaque burden is an independent indicator for risk of ACI. In the present study, volume of LRNC and % LRNC volume can be observed to be correlated with the pres- ence of ACI before and after adjusted for clinical factors. Moreover, the association between presence of ACI and volume of LRNC was remained statistically significant after further adjusted for plaque volume. However, this association was not statistically significant after adjusted for clinical risk factors, plaque volume, and volume of IPH. This may be due to that enlargement of IPH volume would augment the size of LRNC. IPH was as- sumed to occur within LRNC. Hence, the size of LRNC was heavily driven by the size of IPH in our data. Discussion Volume of and IPH was significantly associated with the presence of acute cerebral infarct Table 3 Association of carotid plaque features with ipsilateral ACI Presence of ACI Univariate regression Multivariate regression model 1d Multivariate regression model 2 e OR 95% CI p OR 95% CI p OR 95% CI p a Lumen area, mm2 1.058 0.898–1.247 0.501 0.975 0.819–1.161 0.776 a Wall area, mm2 1.269 1.081–1.489 0.004 1.257 1.047–1.510 0.014 a Total vessel area, mm2 1.194 1.014–1.406 0.034 1.109 0.924–1.331 0.268 a Mean wall thickness, mm2 1.293 1.103–1.515 0.002 1.315 1.103–1.568 0.002 b NWI, % 1.177 0.997–1.390 0.054 1.202 1.008–1.434 0.040 a Plaque volume, mm3 1.130 0.961–1.329 0.140 1.087 0.903–1.308 0.378 b Luminal stenosis, % 1.098 1.044–1.155 < 0.001 1.096 1.039–1.155 0.001 Presence of calcification 1.050 0.754–1.463 0.772 1.094 0.757–1.582 0.632 1.024 0.680–1.541 0.910 Presence of FCR 1.140 0.637–2.042 0.659 1.183 0.646–2.167 0.586 1.095 0.578–2.074 0.780 Presence of LRNC 0.901 0.611–6.328 0.597 0.780 0.516–1.180 0.240 0.663 0.417–1.054 0.083 Presence of IPH 1.489 0.979–2.266 0.063 1.515 0.963–2.384 0.072 1.485 0.907–2.432 0.116 c Volume of calcification, mm3 0.971 0.763–1.234 0.808 1.023 0.794–1.318 0.859 0.953 0.702–1.294 0.759 c % calcification volume, % 0.605 0.314–1.166 0.133 0.731 0.372–1.436 0.363 c Volume of LRNC, mm3 1.297 1.084–1.553 0.005 1.312 1.082–1.591 0.006 1.398 1.045–1.870 0.024 c % LRNC volume, % 1.199 1.032–1.393 0.018 1.190 1.014–1.398 0.034 c Volume of IPH, mm3 2.514 1.363–4.637 0.003 2.907 1.341–6.301 0.007 2.813 1.212–6.528 0.016 c % IPH volume, % 2.202 1.317–3.680 0.003 2.374 1.324–4.257 0.004 NWI normalized wall index, FCR fibrous cap rupture, LRNC lipid-rich necrotic core, IPH intraplaque hemorrhage. a The increment for plaque burden was 1 SD. b The increment for NWI and luminal stenosis was 10% NWI and stenosis. c Only for patients with the corresponding plaque compositional feature with increment of 1 SD for composition size and 10% for percent of composition volume. d Model 1: adjustment for age, gender, BMI, hypertension, hyperlipidemia, CHD, diabetes, and smoke. e Model 2: adjustment for the former clinical factors and plaque volume Table 3 Association of carotid plaque features with ipsilateral ACI Page 7 of 9 Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Fig. 3 An example of a patient with carotid intraplaque hemorrhage (IPH) and ipsilateral acute cerebral infarct (ACI). Discussion Inves- tigators found that IPH can accelerate the progression of carotid plaques and LRNC size may govern the risk of future surface disruption, which was associated with a higher overall volume of cerebral injury [14–16]. Zhao et al. reported that LRNC volume was significantly asso- ciated with severity of cerebral infarction as measured In this study, plaque burden in carotid artery with atherosclerotic plaque was found to be associated with the risk of ACI. The relationship between carotid plaque burden and stroke risk has been well demonstrated in previous studies. The degree of carotid stenosis is recog- nized as an important risk factor for cerebral infarction. Gunduz et al. studied 52 patients with 50–69 and > 70% carotid stenosis and found that the occurrence of new ischemic lesions was significantly related to carotid sten- osis determined by carotid ultrasonography [11]. Beyond luminal stenosis, investigators also found that the vessel wall burden measured by ultrasound and CMR was as- sociated with stroke risk. In a study by Liu et al., carotid plaque burden including mean WT (1.0 ± 0.2vs. 1.1 ± 0.4 mm, p = 0.043), mean wall area (26.2 ± 5.6 vs. 29.8 ± 9.8 mm2, p = 0.043), and percent volume of wall volume (35.5 ± 7.4 vs. 40.7 ± 11.9%, p = 0.021) was found to be Page 8 of 9 Liu et al. Journal of Cardiovascular Magnetic Resonance Liu et al. Journal of Cardiovascular Magnetic Resonance (2019) 21:36 Page 8 of 9 by DWI lesions [13]. In addition, in the present study, we found that the presence of LRNC was insignificantly related to ACI, which is consistent with previous studies [17]. Our findings suggest that the size of LRNC is an effective indicator for the risk of ACI independent of plaque size but not IPH volume. atherosclerosis and LDL has been demonstrated to be an independent risk factor for ACI [24, 25]. However, our study found that patients with ACI had signifi- cantly lower LDL compared with patients without ACI. It may be due to the use of statins. A systematic and meta-analysis documented that statins can lower LDL cholesterol concentration [26]. We found that the association of IPH volume in carotid arteries with ACI was independent of plaque size and LRNC volume. Discussion Moreover, logistic regression re- vealed that size of IPH had higher OR value than the size of LRNC, suggesting that IPH was more dangerous than fat loading of the plaque in determining the risk of ACI. This suggests that in carotid plaques with similar volume, the size of IPH may be used to further deter- mine the risk of ACI. We also found that the presence of IPH was marginally significantly associated with ACI. A study using integrated backscatter ultrasound and CMR demonstrated that carotid plaques in the early stage after the onset of symptoms had a higher percent volume of lipid pool and IPH (57.8 ± 25.1% vs. 46.8 ± 25.1%, p = 0.036) compared with plaques that were stabi- lized by treatment for 30–180 days after the onset of symptoms [18]. In a most recent study, investigators found the symptomatic carotid plaques tended to having larger volume of IPH compared with asymptomatic pla- ques (150 ± 199 vs. 88 ± 106 mm3, p = 0.071) but the difference was not statistically significant which may be due to the smaller sample size (n = 31) [4]. As men- tioned above, the presence of IPH might be a stimulator for plaque progression and future fibrous cap rupture [14, 19, 20]. A study by Cui et al. documented that vol- ume of IPH was associated with minor fibrous cap dis- ruption in carotid arteries (OR, 2.867; 95% CI, 1.505– 5.461; p = 0.001) after adjusted for clinical confounding factors and plaque burden [21]. The inflammatory activ- ity, coagulation proteases, leukocyte serine proteases, and gelatinase within atherosclerotic plaque with IPH may play important roles in degradation of fibrous cap and subsequent plaque rupture [3]. In addition, the bio- mechanical propriety of IPH might be contributable to the vulnerability of plaque surface. And a study by Sadat et al. found that hemorrhagic plaques had higher bio- mechanical stresses than non-hemorrhagic plaques [22]. To the best of our knowledge, disruption of carotid plaques triggers the formation of thromboembolus and subsequent ischemic stroke. In addition, some previous studies documented that no association can be found between the presence of IPH and ischemic stroke [23]. Our findings suggest that quantification of the size of IPH is warranted in patients with carotid plaques in stratification of stroke risk beyond the mere presence of IPH. Acknowledgments We thank all the CARE-II investigators for their contributions to the patient recruitment. Funding h d g This study is funded by the grants of National Natural Science Foundation of China (81771825, 81720108022, 91649116, and 81571040) and Ministry of Science and Technology of China (2017YFC1307904). Discussion Previous studies showed that LDL participated in th ti d l t d i f There are several limitations in our study. First, this is a cross-sectional study that cannot determine the relationship between the progression of IPH volume in carotid arteries and ACI. Second, the current in-plane spatial resolution utilized in the present study was 0.55 mm which is limited to accurate quantify IPH with small size. To improve the spatial resolution is needed in future studies. Third, carotid vessel wall imaging of our study was performed with 2D multicontrast CMR im- aging protocol with limited longitudinal coverage which may not capture the atherosclerotic plaque in more proximal or distal segments of extracranial carotid arteries. Wang et al. [27] proposed a simultaneous non- contrast angiography and intraplaque hemorrhage se- quence with large longitudinal coverage [23] which could be used to evaluate carotid IPH in future studies. Authors’ contributions XZ contributed substantially to the conception and design of the study; CY, and BZ contributed substantially to the interpretation and technical assistance; YL and MW analyzed and interpreted the patient data, and drafted the manuscript; WW, YX, and XZ contributed to data interpretation and critically revised the manuscript. All authors read and approved the final manuscript. Abbreviations 3D Th di 3D: Three dimensional; ACI: Acute cerebral infarct; BMI: Body mass index; CAD: Coronary artery disease; CI: Confidence interval; CMR: Cardiovascular magnetic resonance; DWI: Diffusion weighted imaging; FCR: Fibrous cap rupture; FFE: Fast field echo; FOV: Field of view; IPH: Intraplaque hemorrhage; LDL: Low density lipoprotein; LRNC: Lipid-rich necrotic core; MPRAGE: Magnetization-prepared rapid acquisition gradient echo; NASCET: North American Symptomatic Carotid Endarterectomy Trial; NWI: Normalized wall index; OR: Odds ratio; SD: Standard deviation; T1W: T1 weighted; T2W: T2 weighted; TE: Echo time; TOF: Time-of-flight; TR: Repetition time; TSE: Turbo spin echo; WT: Wall thickness Conclusions In patients with carotid atherosclerotic plaques, the size of IPH is independently associated with ipsilateral ACI, suggesting the size of IPH might be a useful indicator for the risk of ACI. References Al f N 1. 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Ethics approval and consent to participate patients with first-time and recurrent acute ischemic stroke. AJNR Am J Neuroradiol. 2012;33:1257–61. patients with first-time and recurrent acute ischemic stroke. AJNR Am J Neuroradiol. 2012;33:1257–61. patients with first-time and recurrent acute ischemic stroke. AJNR Am J Neuroradiol. 2012;33:1257–61. The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Tsinghua University and other local Ethics Committee at each participating hospital and all patients provided written informed consent. 13. Zhao H, Zhao X, Liu X, Cao Y, Hippe DS, Sun J, et al. Association of carotid atherosclerotic plaque features with acute ischemic stroke: a magnetic resonance imaging study. Eur J Radiol. 2013;82:e465–70. 14. Takaya N, Yuan C, Chu B, Saam T, Polissar NL, Jarvik GP, et al. Presence of intraplaque hemorrhage stimulates progression of carotid atherosclerotic plaques: a high-resolution magnetic resonance imaging study. 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North American symptomatic carotid endarterectomy trial collaborators. N Engl J Med. 1998;339:1415–25. 11. Gunduz Y, Akdemir R, Ayhan LT, Keser N. Can Doppler flow parameters of carotid stenosis predict the occurrence of new ischemic brain lesions detected by diffusion-weighted MR imaging after filter-protected internal carotid artery stenting? AJNR Am J Neuroradiol. 2014;35:760–5. 12. Liu XS, Zhao HL, Cao Y, Lu Q, Xu JR. Comparison of carotid atherosclerotic plaque characteristics by high-resolution black-blood MR imaging between
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Lack of association between vascular dementia and Chlamydia pneumoniae infection: a case-control study
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BioMed Central BioMed Central BioMed Central Ope Research article Lack of association between vascular dementia and Chlamydia pneumoniae infection: a case-control study Soo Chan Carusone1, Marek Smieja1,2,3, William Molloy4,5, Charlie H Goldsmith1, Jim Mahony2,3, Max Chernesky2,3, Judy Gnarpe6, Tim Standish4, Stephanie Smith2 and Mark Loeb*1,2,3 Address: 1Department of Clinical Epidemiology and Biostatistics, McMaster University, 1200 Main St. W., L8N 3Z5 Hamilton, Canada, 2Department of Pathology and Molecular Medicine, McMaster University, 1200 Main St. W., L8N 3Z5 Hamilton, Canada, 3Hamilton Regional Laboratory Medicine Program, 50 Charlton Ave. E., L8N 4A6 Hamilton, Canada, 4St. Peter's Centre for Studies in Aging, St. Peter's Hospital, 88 Maplewood Ave., L8M 1W9 Hamilton, Canada, 5Department of Medicine, McMaster University, 1200 Main St. W., L8N 3Z5 Hamilton, Canada and 6Department of Medical Microbiology and Immunology, University of Alberta, T6G 2H7 Edmonton, Canada Email: Soo Chan Carusone - chansy@mcmaster.ca; Marek Smieja - smiejam@mcmaster.ca; William Molloy - wmolloy@stpetes.ca; Charlie H Goldsmith - goldsmit@mcmaster.ca; Jim Mahony - mahonyj@mcmaster.ca; Max Chernesky - chernesk@mcmaster.ca; Judy Gnarpe - judy.gnarpe@ualberta.ca; Tim Standish - tstandish@stpetes.ca; Stephanie Smith - smithste@mcmaster.ca; Mark Loeb* - loebm@mcmaster.ca * Corresponding author Received: 03 June 2004 Accepted: 12 October 2004 Received: 03 June 2004 Accepted: 12 October 2004 BMC Neurology 2004, 4:15 doi:10.1186/1471-2377-4-15 This article is available from: http://www.biomedcentral.com/1471-2377/4/15 This article is available from: http://www.biomedcentral.co This article is available from: http://www.biomedcentral.com/1471-2377/4/15 © 2004 Carusone et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Open Research article Lack of association between vascular dementia and Chlamydia pneumoniae infection: a case-control study Soo Chan Carusone1, Marek Smieja1,2,3, William Molloy4,5, Charlie H Goldsmith1, Jim Mahony2,3, Max Chernesky2,3, Judy Gnarpe6, Tim Standish4, Stephanie Smith2 and Mark Loeb*1,2,3 BMC Neurology Open Access Abstract Background: Chronic inflammation appears to play a role in the pathogenesis of vascular dementia. Given the association between Chlamydia pneumoniae and stroke, the possibility exists that previous exposure to C. pneumoniae may play a role in vascular dementia. The objective of this study was to determine if there was an association between serological evidence of C. pneumoniae infection or inflammatory markers with vascular dementia. Methods: 28 case-patients with vascular dementia at a geriatric clinic and 24 caregiver-controls were tested for C. pneumoniae IgG and IgA antibodies. The association between vascular dementia and C. pneumoniae titres as well as inflammatory markers was estimated by using both conditional logistic regression and stratified logistic regression. Results: When matched cases were compared to controls, there was no significant difference in elevated C. pneumoniae specific IgG antibodies (titre ≥ 1:32), odds ratio [OR] 1.3 (95% confidence intervals [CI] 0.3 to 6.0), p = 0.71, or in elevated C. pneumoniae specific IgA antibodies (titre ≥ 1:16), OR 2.0 (95%CI 0.5 to 8.0), p = 0.33 indicative of past or persistent C. pneumoniae infection. Similarly, no difference in high IgG or IgA antibody levels (IgG titre ≥ 1:512 or IgA titre ≥ 1:64) between the two groups, indicative of recent C. pneumoniae infection, was found, OR 0.4 (95%CI 0.1 to 2.1), p = 0.27. For C-reactive protein (CRP), the mean difference between 18 matched pairs (case – control) was – 3.33 mg/ L. There was no significant difference between cases and controls when comparing log transformed values, OR 0.03 (95%CI 0.00 to 2.89), p = 0.13 or comparing CRP values above or below the median, OR 0.8 (95%CI 0.2 to 3.4), p = 0.71. For fibrinogen, the mean difference between pairs (case – control) was -0.07 g/L. There was no statistical difference between cases and controls when comparing log transformed values, OR 0.6 (95%CI 0.0 to 31.2), p = 0.79 or between fibrinogen values above and below the median, OR = 0.5 (95%CI 0.1 to 2.0), p = 0.50. Conclusion: We found no evidence for a significant association between C. pneumoniae infection, inflammatory markers such as CRP and fibrinogen, and vascular dementia. Conclusion: We found no evidence for a significant association between C. pneumoniae infection, inflammatory markers such as CRP and fibrinogen, and vascular dementia. Study design y g Patients with vascular dementia were enrolled from the Geriatric Clinic at Henderson Hospital, an outpatient clinic affiliated with a tertiary hospital in Hamilton, Ontario. The diagnosis of vascular dementia for partici- pants enrolled was determined in accordance with criteria established by the Neuroepidemiology Branch of the National Institute of Neurological Disorders and Stroke and Association Internationale pour la Recherche et l'Enseignement en Neurosciences (NINDS-AIREN) Inter- national workshop [13]. This includes both physical and imaging evidence of strokes, and a temporal relationship between stroke and dementia [3]. Case-patients meeting any of the following criteria were excluded: 1) cognitive impairment due to acute cerebral trauma, hypoxic cere- bral damage post cardiac arrest, vitamin deficiency states, central nervous system infection, cerebral neoplasia, sig- nificant endocrine or metabolic disease, mental retarda- tion; 2) stroke within the last 6 weeks; 3) patients known, in the past 3 months, to have taken a 7 day or more course of antibiotics with activity against C. pneumoniae (erythro- mycin, clarithromycin, azithromycin, levofloxacin, trova- floxacin, doxycycline, or tetracycline). Laboratory methods For C. pneumoniae IgG and IgA antibody detection, all sera were titrated at two-fold dilutions from 1:16 to endpoint. Samples were analyzed by microimmunofluorescence (MIF), using a 16 hour incubation of serum and substrate at 4–8°C with the same batch of C. pneumoniae IgG/IgM MIF slides (LabSystems OY, Helsinki) (23). To prevent IgG interference, sera used for IgA detection were first treated with goat anti-human IgG antibodies (GullSorb; Gull Laboratories, Salt Lake City, UT, USA). CRP was measured using a high sensitivity automated rate neph- elometric immunoassay (Dade Behring high-sensitivity CRP, BNII Nephelometer System, Marburc, DE). Fibrino- gen was assayed using an automated STA fibrinogen assay (von Clauss method) on a Roche/Stago (Diagnostica Stago SA). Background the study, regardless of the diagnosis of their spouse or family member. For each case, one caregiver matched for age (within five years) and sex was selected. Caregivers were excluded if they had a diagnosis that included any of the following: dementia, stroke, or cognitive impairment as determined by a Standardized Mini-Mental Status Examination score of < 27 [14]. Enrolment was from July 1999 to October 2001. All eligible cases and controls who attended the clinic during the study period were approached for consent to participate in the study. Demo- graphic data (age, sex), medical history, and smoking his- tory were collected as well as blood samples for C. pneumoniae IgG and IgA antibodies, CRP, and fibrinogen. g Vascular dementia is characterized by a loss of cognitive function and social adaptive functions in individuals with cerebrovascular disease [1,2]. Vascular dementia is the second most common cause of dementia and accounts for 10% to 15% of all cases [3]. The clinical presentation of this illness is variable, depending on the site and extent of the lesion or infarct [2]. The pathogenesis of vascular dementia has not been well defined [1,3]. Chronic inflammation and cytokine dysregulation may play a role [4] similar to that seen in Alzheimer's disease [5]. Recent data from serological and PCR studies support an association between Chlamydia pneumoniae and cerebrov- ascular disease. C. pneumoniae has been associated with stroke, transient cerebral ischemia, and atherosclerosis in the middle cerebral artery in both prospective and case- control studies [6-12]. Since stroke is an important precur- sor to vascular dementia, these data raise the possibility that C. pneumoniae infection may also be a risk factor for vascular dementia. To our knowledge, this potential rela- tionship has not previously been assessed. We conducted a pilot case-control study to determine an association between serological evidence of C. pneumoniae infection and vascular dementia. We also sought to determine if the inflammatory markers, C-reactive protein (CRP) and fibrinogen were associated with this illness. This study was approved by the research ethics board at McMaster University. Signed consent was obtained for all participants (proxy consent was utilized for participants considered decisionally impaired). Abstract Page 1 of 7 (page number not for citation purposes) Page 1 of 7 Page 1 of 7 itation purposes) (page number not for citation purposes) (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/4/15 BMC Neurology 2004, 4:15 Analysis The presence of elevated antibody levels, indicative of past or persistent C. pneumoniae infection, was defined as an IgG titre of 1:32 or greater and IgA of 1:16 or greater [15,16]. High antibody titres to C. pneumoniae, suggesting a more recent infection, was defined by IgG titres of 1:512 or greater or IgA titres of 1:64 or greater [16]. Because a skewed distribution was anticipated and a linear relation- ship with risk was not expected, CRP and fibrinogen were analyzed in two ways: using a log transformation of the values and dichotomizing at the median. The association between vascular dementia and C. pneu- moniae titres and inflammatory markers was estimated using a matched analysis. Conditional logistic regression analyses were performed for antibody levels, dichot- omized and log transformed CRP, as well as dichot- omized and log transformed fibrinogen. The controls for this study were chosen from a list of all caregivers who attended the geriatric clinic at the time of Page 2 of 7 (page number not for citation purposes) Page 2 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/4/15 http://www.biomedcentral.com/1471-2377/4/15 BMC Neurology 2004, 4:15 Table 1: Comparison of analyses used to assess for associations of C. pneumoniae specific serology and vascular dementia Variable Conditional Stratified* Adjusted** OR p n1 OR p n2 OR p n2 IgG response 1.5 0.66 15 1.8 0.46 48 1.7 0.50 48 IgA response 1.7 0.48 15 2.8 0.13 48 3.0 0.11 48 High titre response 0.4 0.27 15 0.6 0.40 48 0.5 0.34 48 1 Number of pairs included in the analysis 2 Number of individuals included in the analysis * Stratified on age (≤ 70 years, 71–80 years, 81–90 years) and gender **Stratified analysis with adjustment for current smoking status Table 1: Comparison of analyses used to assess for associations of C. pneumoniae specific serology and vascular dementia A stratified logistic regression analysis was also conducted for C. pneumoniae titres and inflammatory markers (both log transformed and dichotomized at the median), strati- fying by age and sex (the following age strata were used: ≤ 70 years, 71–80 years, 81–90 years). All analyses for C. pneumoniae titres and inflammatory markers were also performed with adjustment for current smoking status. Data analyses were performed with SPSS version 10 or Egret for Windows version 2.0.3. Participants Similarly, no statistically significant difference between elevated C. pneumoniae specific IgA antibodies was found between matched pairs, OR = 2.0 (95%CI 0.5 to 8.0), p = 0.33. The stratified analysis produced slightly higher odds ratio estimates, although not statistically significant. For the unadjusted analysis, OR = 2.8 (95%CI 0.7 to 10.4), p = 0.13 and for the adjusted analysis, OR = 2.7 (95%CI 0.5 to 14.2), p = 0.24 (see Table 1). A total of 28 case-patients were enrolled: mean age 76.2 years (minimum to maximum: 56 to 90 years); 18 (64%) were male. Nine of the 28 cases had at least one comor- bidity (including angina, coronary heart disease, vascular disease, liver disease and renal disease); 1 case had 3 or more comorbidities. Thirteen of the cases were current smokers. Twenty of the 28 cases could be matched to caregiver-con- trols, for a total of 20 case-control pairs. Of these 20 car- egiver-controls, 16 were unrelated to a case, and four were spouses of a case. However, none of these four were matched to their spouse. Four additional caregiver-con- trols were selected for the unmatched analyses, so that data on a total of 24 caregiver-controls was obtained. There was also no statistical difference in high antibody levels between matched pairs, OR = 0.4 (95%CI 0.1 to 2.1), p = 0.27 or in the stratified analysis, OR = 0.6 (95%CI 0.2 to 2.0), p = 0.40 for the unadjusted analysis and OR = 0.5 (95%CI 0.1 to 2.5), p = 0.41 for the adjusted analysis (see Table 1). Inflammatory markers Where there was incomplete data on antibody or inflam- matory marker levels, those pairs were excluded from matched analyses. Incomplete information on cases and controls occurred when individuals consented to partici- pate in the study and provided medical information but did not attend the out-patient clinic for the required blood collection. All individuals with complete data were included in the unmatched analyses. A matched analysis of CRP performed on 18 of the 20 matched pairs (all pairs with complete data) revealed no significant difference between log-transformed values, OR of 0.03 (95%CI 0.00 to 2.89), p = 0.13. Similarly, there was no difference comparing matched cases and controls with CRP values above or below the median, OR = 0.8 (95%CI 0.2 to 3.4), p = 0.71. In the stratified analysis (Table 2), the log transformed CRP variable and the CRP variable dichotomized at the median were not statistically significant in both the unadjusted (OR = 0.5 (95%CI 0.1 to 3.6), p = 0.50 and OR = 2.2 (95%CI 0.7 to 7.2), p = Analysis The original protocol involved two concurrent case-con- trol studies: one including 30 vascular dementia patients and 30 controls and the other with 30 Alzheimer disease patients and 30 controls. The analysis was to include the additional 30 Alzheimer disease controls in the vascular dementia analysis (giving a 1:2 case:control ratio). Assum- ing that one third of controls would have elevated C. pneu- moniae titres, for an alpha of 0.05 and 80% power, matching 30 cases to 60 controls would allow for detec- tion of an odds ratio of 3.8 or higher. As the study pro- ceeded, it became apparent that enrolling the Alzheimer's patients was not feasible. We decided then to limit the analysis to a 1:1 case:control ratio focusing on 30 patients with vascular dementia. vated antibody levels between matched case-patients and controls, odds ratio [OR] = 1.3 (95% confidence intervals [CI] 0.3 to 6.0), p = 0.71. When pairs were broken and stratified analyses were performed the difference was not statistically significant, OR = 1.8 (95%CI 0.4 to 8.3), p = 0.46. The analysis was also performed with adjustment for participants' current smoking status, OR = 1.8 (95%CI 0.2 to 12.2), p = 0.57 (see Table 1). http://www.biomedcentral.com/1471-2377/4/15 BMC Neurology 2004, 4:15 Table 2: Comparison of analyses used to assess for associations of inflammatory markers and vascular dementia Variable Conditional Stratified* Adjusted** OR p n1 OR p n2 OR p n2 LogCRP 0.0 0.13 18 0.5 0.50 49 0.5 0.52 49 CRP 0.8 0.71 18 2.2 0.20 49 2.2 0.19 49 LogFibrinogen 0.6 0.79 18 0.8 0.92 49 0.6 0.83 49 Fibrinogen 0.5 0.33 18 0.6 0.38 49 0.5 0.32 49 1 Number of pairs included in the analysis 2 Number of individuals included in the analysis * Stratified on age (≤ 70 years, 71–80 years, 81–90 years) and gender **Stratified analysis with adjustment for current smoking status Table 2: Comparison of analyses used to assess for associations of inflammatory markers and vascular dementia elevated IgA and IgG antibodies do not definitively rule out an association. In fact, we powered this study to detect a minimally important association between antibodies and vascular dementia of 3.8. Given that the odds ratio 95% confidence interval of IgG is from 0.3 to 6.0, and 0.5 to 8.0 for IgA, our data do not rule out clinically important associations. The point estimates for elevated IgA and IgG antibodies (2.0 and 1.3, respectively) are similar to the recent meta-analysis odds ratio estimates for coronary heart disease of 1.25 (95% CI 1.03 to 1.53) and 1.15 (95% CI 0.97 to 1.36), respectively [21,22]. In both cases the odds ratio estimate for IgA titres is slightly higher than IgG titres, but not statistically different. The meaning of this difference is uncertain. Danesh et al [21] suggest that these differences are likely due to chance, selection biases, or selective emphasis on particular reports. In contrast, other studies have suggested that IgA titres are more strongly associated with disease outcomes because they are a better indicator of chronic C. pneumoniae infection [23,15,10]. 0.20, respectively) and the adjusted analysis (OR = 0.4 (95%CI 0.04 to 4.7), p = 0.49 and OR = 1.4 (95%CI 0.3 to 6.3), p = 0.64, respectively). A matched analysis of fibrinogen performed on the same 18 pairs revealed no significant difference between the log-transformed values of the two groups, OR = 0.6 (95%CI 0.0 to 31.2), p = 0.79. Similarly, there was no dif- ference when comparing the pairs on fibrinogen values above and below the median, OR = 0.5 (95%CI 0.1 to 2.0), p = 0.33. http://www.biomedcentral.com/1471-2377/4/15 In the stratified analysis (Table 2), the log transformed fibrinogen variable and the fibrinogen varia- ble dichotomized at the median were not statistically sig- nificant in both the unadjusted (OR = 0.8 (95%CI 0.0 to 71.6), p = 0.92 and OR = 0.6 (95%CI 0.2 to 2.0), p = 0.38, respectively) and the adjusted analysis (OR = 0.1 (95%CI 0.0 to 73.9), p = 0.43 and OR = 0.3 (95%CI 0.1 to 1.6), p = 0.17, respectively). Vascular dementia is the second most common cause of dementia, second only to Alzheimer's disease. It was pre- viously believed that most cases of dementia were the out- come of one of these two distinct diseases. However, the clear division between them has recently been challenged. It is now widely believed that vascular risk factors are also associated with Alzheimer's disease and Alzheimer's and vascular dementia may share many common clinical and pathological characteristics [3,24-26]. A number of stud- ies have examined the association between Alzheimer's disease and C. pneumoniae infection. In 1998 Balin et al [27] found an extremely high association between the presence of C. pneumoniae in post-mortem brain samples and late-onset Alzheimer's disease. However, more recent studies have not repeated these findings [28-31]. A recent randomized controlled clinical trial [16], based on the hypothesis that chronic C. pneumoniae infection contrib- utes to Alzheimer's disease, found an improved long-term cognitive state in patients with mild to moderate Alzhe- imer's disease who had been treated with doxycycline and rifampin. However, the serological data did not suggest that this clinical effect was due to treatment of chronic C. pneumoniae infection. One study has looked for C. pneu- moniae in brain samples of vascular dementia patients. This study, like the later AD studies, did not identify C. pneumoniae in any of the brain samples [32]. These results suggest that the presence of C. pneumoniae in the brains is not strongly associated with late-onset Alzheimer's dis- ease or vascular dementia. C. pneumoniae serology Univariate analysis of C. pneumoniae specific IgG antibod- ies showed no statistically significant difference in ele- Page 3 of 7 (page number not for citation purposes) Page 3 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/4/15 http://www.biomedcentral.com/1471-2377/4/15 BMC Neurology 2004, 4:15 infections. As a result, different groups have used different criteria making comparisons across studies more difficult. However, the importance of this inconsistency is unclear. In the meta-analysis reported by Danesh et al [21] no sig- nificant heterogeneity was found among the studies even though four different cut-off titres were used to determine seropositivity in the microimmunofluorescence assays. An alternative test, that may prove to be more reliable, involves the detection of C. pneumoniae DNA in peripheral blood mononuclear cells [45]. Another potential limita- tion is the choice of controls; because C. pneumoniae is infectious an increased exposure in the caregivers could potentially mask a statistically significant association between the patients and controls. There is also evidence that caregivers, because of stress, may have altered immune systems [46] which could interfere with their generation of antibodies and inflammatory markers [47- 49]. predict cardiovascular disease [34] and dementia [33], and have been associated with stroke patients [35]. Recently, an association between inflammatory markers alpha 1-antichymotrypsin, interleukin 6, and, to a lesser extent, C-reactive protein were associated with an increased risk of dementia [36]. In this study we did not find a significant difference in CRP levels between the cases and controls. This most likely was due to the limited power in the study and the limitations of measuring serum CRP. Although CRP was originally thought to be produced almost exclusively by hepatocytes, CRP is now known to be synthesized in brain cells and upregulated in Alzheimer tissue [37,38]. Consequently, localized increases in CRP may be associated with vascular demen- tia but not detected with serum measurements. We found no significant association between increased fibrinogen levels and vascular dementia. Abnormalities of haemostasis are thought to be important in the pathogen- esis of cardiovascular disease, ischaemic stroke, and vascu- lar dementia. Within the pathways of coagulation and fibrinolysis, fibrinogen represents an important marker. Elevated levels of fibrinogen are associated with increased risks of cardiovascular disease and ischaemic stroke [39,40] but the results are less conclusive for vascular dementia [41,42]. Lowe and Haverkate [43] believe that because vascular dementia is only one phenotype of the systemic atherothrombosis disease, associations between haemostatic variables and any given phenotype should be interpreted with caution. To show a specific association with a single phenotype, a study would need an extremely large sample size to overcome the overlap in phenotypes and risk factors seen in atherothrombosis. Conclusions In summary, a case-control study of vascular dementia patients suggests that there is no significant association between C. pneumoniae antibodies and vascular dementia. We found no evidence for a significant association between systemic inflammatory markers and vascular dementia. While this study can rule out a strong associa- tion, larger studies are necessary to determine if a weak association exists. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors' contributions ML, MS, WM, CG, JM, and MC conceived and designed the original study. SCC conducted the analysis of data and drafted the manuscript. SS and TS coordinated the study and collected data. JG conducted the serological testing. All authors offered critical input into the manuscript and all have read and approved the final version. We acknowledge several limitations of this study. Because of the relatively small sample size, the analyses were adjusted for only a small number of potentially important covariates and the analysis of CRP and fibrinogen was restricted to above and below the median (while quartiles would have been more sensitive). To adjust for variables that were not used as matching criteria and to maximize the data collected a stratified analysis was also done. The additional stratified analysis adjusted for current smoking status. We adjusted for smoking status because there is a known strong association between smoking and C. pneu- moniae titres; and between smoking and vascular demen- tia [44]. However, it may also be important to adjust for additional factors that may affect inflammatory markers. We also acknowledge that C. pneumoniae serology is an imperfect test of C. pneumoniae exposure and chronic infection. First, the high prevalence of C. pneumoniae exposure makes it difficult to detect true serological differ- ences between cases and controls. Second, it is unclear what the appropriate serological cut-offs should be for identifying exposure versus chronic infection or recent Discussion In this case-control study, we found no significant associ- ation between elevated or high C. pneumoniae specific IgG or IgA antibodies and vascular dementia. To our knowl- edge, this is the first epidemiologic study to test for an association between vascular dementia and infection with C. pneumoniae. We conducted this study on the basis of evidence linking C. pneumoniae to cardiovascular disease and stroke. There is an extensive literature supporting an association between C. pneumoniae and atherosclerosis [17-19]. Although the majority of these studies initially focused on coronary heart disease more recent evidence also supports an association with stroke [6-12,20]. However, the clinical importance of this association is uncertain. Inflammatory responses are also known to be associated with cardiovascular disease and have recently been impli- cated in dementia [33]. Elevated levels of serum C-reactive protein (CRP), a non-specific marker of inflammation, Although no significant associations were noted, the rela- tively small sample size and the odds ratio estimates for Page 4 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/4/15 References McGeer PL, McGeer EG, Yasojima K: Alzheimer disease and neuroinflammation. J Neural Transm Suppl 2000, 59:53-57. 15. Wong BYL, Gnarpe J, Teo KK, Ohman EM, Prosser C, Gibler WB, Langer A, Chang W-C, Armstrong PW: Does chronic Chlamydia pneumoniae infection increase the risk of myocardial injury? Insights from patients with non-ST-elevation acute coronary syndromes. Am Heart J 2002, 144:987-994. 38. Yasojima K, Schwab C, McGeer EG, McGeer PL: Human neurons generate C-reactive protein and amyloid P: upregulation in Alzheimer's disease. 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Cerebrovasc Dis 2003, 15(1–2):11-16. y 43. Lowe GD, Haverkate F: Specificity of haemostasis abnormali- ties for vascular phenotypes. Haemostasis 1998, 28:223-227. p yp 44. References 1. Strub R: Vascular dementia. South Med J 2003, 96(4):363-366. 2. de Leeuw FE, van Gijn J: Vascular dementia. Practical Neurology 2003, 3:86-91. 3. Quinn J: Vascular dementia. J Am Med Dir Assoc 2003, 4(6 suppl):S155-S161. 3. Quinn J: Vascular dementia. J Am Med Dir Assoc 2003, 4(6 suppl):S155-S161. pp ) 4. Wilson CJ, Finch CE, Cohen HJ: Cytokines and cognition – the case for a head-to-toe inflammatory paradigm. J Am Geriatr Soc 2002, 50:2041-2056. pp ) 4. Wilson CJ, Finch CE, Cohen HJ: Cytokines and cognition – the case for a head-to-toe inflammatory paradigm. J Am Geriatr Soc 2002, 50:2041-2056. 5. Hull M, Strauss S, Berger M, Volk B, Bauer J: Inflammatory mech- anisms in Alzheimer's disease. Eur Arch Psychiatry Clin Neurosci 1996, 246(3):124-128. 6. Madre JG, Garcia JL, Gonzalez RC, Montero JM, Paniagua EB, Escrib- ano JR, Martinez JD, Cenjor RF: Association between seroposi- tivity to Chlamydia pneumoniae and acute ischaemic stroke. Eur J Neurol 2002, 9(3):303-306. J ( ) 7. Kawamoto R, Kajiwara T, Oka Y, Takagi Y: An association between an antibody against Chlamydia pneumoniae and ischemic stroke in elderly Japanese. Intern Med 2003, 42(7):571-575. Page 5 of 7 (page number not for citation purposes) Page 5 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2377/4/15 BMC Neurology 2004, 4:15 8. Wolf SC, Mayer O, Jürgens S, Vonthein R, Schultze G, Risler T, Brehm BR: Chlamydia pneumoniae IgA seropositivity is associated with increased risk for atherosclerotic vascular disease, myo- cardial infarction and stroke in dialysis patients. Clin Nephrol 2003, 59(4):273-279. of Chlamydia pneumoniae in the Alzheimer's brain. Med Micro- biol Immunol (Berl) 1998, 187(1):23-42. 28. Gieffers J, Reusche E, Solbach W, Maass M: Failure to detect Chlamydia pneumoniae in brain sections of Alzheimer's dis- ease patients. J Clin Microbiol 2000, 38(2):881-882. 29. ( ) 9. Virok D, Kis Z, Karai L, Intzedy L, Burian K, Szabo A, Ivanyi B, Gonc- zol E: Chlamydia pneumoniae in artherosclerotic middle cere- bral artery. Stroke 2001, 32(9):1973-1976. p J ( ) 29. Nochlin D, Shaw CM, Campbell LA, Kuo CC: Failure to detect Chlamydia pneumoniae in brain tissue of Alzheimer's disease. Neurology 1999, 53:1888. y ( ) 10. Elkind MSV, Lin I-F, Grayston JT, Sacco RL: Chlamydia pneumoniae and the risk of first ischemic stroke: the Northern Manhat- tan Stroke Study. Stroke 2000, 31(7):1521-1525. 30. Ring RH, Lyons JM: Failure to detect Chlamydia pneumoniae in the late-onset Alzheimer's brain. References J Clin Microbiol 2000, 38(7):2591-2594. ( ) 31. Taylor GS, Vipond IB, Paul ID, Matthews S, Wilcock GK, Caul EO: Failure to correlate C. pneumoniae with late onset Alzhe- imer's disease. Neurology 2002, 59:142-143. 11. Fagerberg B, Gnarpe J, Gnarpe H, Agewall S, Wikstrand J: Chlamydia pneumoniae but not cytomegalovirus antibodies are associ- ated with future risk of stroke and cardiovascular disease: a prospective study in middle-aged to elderly men with treated hypertension. Stroke 1999, 30(2):299-305. gy 32. Wozniak MA, Cookson A, Wilcock GK, Itzhaki RF: Absence of Chlamydia pneumoniae in brain of vascular dementia patients. Neurobiol Aging 2003, 24(6):761-765. yp ( ) 12. Cook PJ, Honeybourne D, Lip GY, Beevers DG, Wise R, Davies P: Chlamydia pneumoniae antibody titers are significantly asso- ciated with acute stroke and transient cerebral ischemia: the West Birmingham Stroke Project. Stroke 1998, 29(2):404-410. p g g ( ) 33. Schmidt R, Schmidt H, Curb JD, Masaki K, White LR, Launer LJ: Early inflammation and dementia: a 25-year follow-up of the Honolulu-Asia Aging Study. Ann Neurol 2002, 52(2):168-174. g j , ( ) 13. Roman GC, Tatemichi TK, Erkinjuntti T, Cummings JL, Masdeu JC, Garcia JH, Amaducci L, Orgogozo JM, Brun A, Hofman A, Moody DM, O'Brien MD, Yamaguchi T, Grafman J, Drayer BP, Bennett DA, Fisher M, Ogata J, Kokmen E, Bermejo F, Wolf PA, Gorelick PB, Bick KL, Pajeau AK, Bell MA, DeCarli C, Culebras A, Korczyn AD, Bogous- slavsky J, Hartmann A, Scheinberg P: Vascular dementia: diagnos- tic criteria for research studies. Report of the NINDS- AIREN International Workshop. Neurology 1993, 43(2):250-260. g g y ( ) 34. Strandberg TE, Tilvis RS: C-reactive protein, cardiovascular risk factors, and mortality in a prospective study in the elderly. Arterioscler Thromb Vasc Biol 2000, 20(4):1057-1060. 35. Sanchez-Moreno C, Dashe JF, Scott T, Thaler D, Folstein MF, Martin A: Decreased levels of plasma vitamin C and increased con- centrations of inflammatory and oxidative stress markers after stroke. Stroke 2004, 35(1):163-168. ( ) 36. Engelhart MJ, Geerlings MI, Meijer J, Kiliaan A, Ruitenberg A, van Swi- eten JC, Stijnen T, Hofman A, Witteman JCM, Breteler MMB: Inflam- matory proteins in plasma and the risk of dementia: the Rotterdam Study. Arch Neurol 2004, 61:668-672. p gy ( ) 14. Molloy DW, Alemayehu E, Roberts R: Reliability of a Standard- ized Mini-Mental State Examination compared with the tra- ditional Mini-Mental State Examination. Am J Psychiatry 1991, 148:102-105. y 37. References Smieja M, Gnarpe J, Lonn E, Gnarpe H, Olsson G, Yi Q, Dzavik V, McQueen M, Yusuf S: Multiple infections and subsequent cardiovascular events in the Heart Outcomes Prevention Evaluation (HOPE) study. Circulation 2003, 107:251-257. 21. Danesh J, Whincup P, Lewington S, Walker M, Lennon L, Thomson A, Wong Y-K, Zhou X, Ward M: Chlamydia pneumoniae IgA titres and coronary heart disease: prospective study and meta- analysis. Eur Heart J 2002, 23:371-375. ( ) y 45. Smieja M, Mahony J, Petrich A, Boman J, Chernesky M: Association of circulating Chlamydia pneumoniae DNA with cardiovascu- lar disease: a systematic review. BMC Infect Dis 2002, 2:21. 22. Danesh J, Whincup P, Walker M, Lennon L, Thomson A, Appleby P, Wong Y, Bernardes-Silva M, Ward M: Chlamydia pneumoniae IgG titres and coronary heart disease: prospective study and meta-analysis. BMJ 2000, 321(7255):208-213. 46. Kiecolt-Glaser JK, Preacher KJ, MacCallum RC, Atkinson C, Malarkey WB, Glaser R: Chronic stress and age-related increases in the proinflammatory cytokine IL-6. Proc Natl Acad Sci USA 2003, 100(15):9090-9095. J ( ) 23. Strachan DP, Carrington D, Mendall MA, Ballam L, Morris J, Butland BK, Sweetnam PM, Elwood PC: Relation of Chlamydia pneumo- niae serology to mortality and incidence of ischaemic heart disease over 13 years in the Caerphilly prospective heart dis- ease study. BMJ 1999, 318(7190):1035-1040. 47. von Känel R, Mills PJ, Fainman C, Dimsdale JE: Effects of psycholog- ical stress and psychiatric disorders on blood coagulation and fibrinolysis: a biobehavioral pathway to coronary artery disease? Psychosom Med 2001, 63:531-544. y J ( ) 24. Breteler MM: Vascular risk factors for Alzheimer's disease: an epidemiologic perspective. Neurobiol Aging 2000, 21:153-160. y 48. Kiecolt-Glaser JK, Glaser R, Gravenstein S, Malarkey WB, Sheridan J: Chronic stress alters the immune response to influenza virus vaccine in older adults. Proc Natl Acad Sci USA 1996, 93(7):3043-3047. p g p p g g 25. Pansari K, Gupta A, Thomas P: Alzheimer's disease and vascular factors: facts and theories. Int J Clin Pract 2002, 56(3):197-203. 26 Shi J P G S i h MA F i dl d RP V l b li i J , ( ) 26. Shi J, Perry G, Smith MA, Friedland RP: Vascular abnormalities: the insidious pathogenesis of Alzheimer's disease. Neurobiol Aging 2000, 21:357-361. ( ) 49. 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Balin BJ, Gerard HC, Arking EJ, Appelt DM, Branigan PJ, Abrams JT, Whittum-Hudson JA, Hudson AP: Identification and localization Page 6 of 7 (page number not for citation purposes) Page 6 of 7 http://www.biomedcentral.com/1471-2377/4/15 http://www.biomedcentral.com/1471-2377/4/15 http://www.biomedcentral.com/1471-2377/4/15 BMC Neurology 2004, 4:15 Pre-publication history Pre-publication history p y The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2377/4/15/prepub http://www.biomedcentral.com/1471-2377/4/15/prepub Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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Divine Smells: Odorama, Melodrama, and the Body in John Waters' Polyester
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Rosen, I 2022 Divine Smells: Odorama, Melodrama and the Body in John Waters’ Polyester. Open Screens, 5(1): 10, pp. 1–17. DOI: https://doi.org/10.16995/os.8503 Divine Smells: Odorama, Melodrama and the Body in John Waters’ Polyester Ido Rosen, Asian and Middle Eastern Studies, University of Cambridge, UK, ir311@cam.ac.uk The comedy Polyester (John Waters, 1981) introduced a new cinematic experience. The screenings were accompanied by the Odorama technique in the form of a ‘scratch and sniff’ card that was handed to viewers in the movie theater. There has yet to be a serious examination of Odorama, which is usually dismissed as nothing more than a gag. This essay shows that Odorama has sophisticated subversive qualities. It confirms scholars’ and critics’ view that Polyester was a turning point in the career of Waters, one of the most important queer filmmakers of all times. The film is frequently seen as his transition from the realm of anarchistic midnight movies to mainstream cinema. This shift was disappointing to many fans, some of whom even considered it betrayal. By contrast, it is argued here that although the film was made by a distinguished auteur, it is also a parody of classic Hollywood melodramas, and playfully adopts the genre’s conventions. Unlike Waters’ previous films, in Polyester the critical ideas are all beneath the surface. It criticizes social norms, middle class values, hypocritical and fraudulent images, ‘conventional’ families, and gender dichotomies in society and their representations in the cinema. However, this is disguised in a borrowed aesthetic, and expressed through a cunning tactic which some audiences and critics missed entirely. Open Screens is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Open Screens is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Open Screens is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The Author(s). Divine Smells: Odorama, Melodrama and the Body in John Waters’ Polyester This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS 2 2 Polyester, which was released in 1981, is considered by many to be a turning point in the career of its director and writer, the idiosyncratic auteur John Waters. In Waters’ previous films such as Pink Flamingos (1972), his deliberate violation of good taste is smeared all over the screen, sometimes almost literally, in an explicit attempt to shock his viewers (Hoberman and Rosenbaum 1983: 138). Early films such as Mondo Trasho (1969) and Multiple Maniacs (1970) consist of a jumble of scenes collated together, with loose plots that unfold randomly. They feature a series of crimes, perversions and anti-social behavior presented in a lighthearted and rebellious manner. Waters has been holding a notorious reputation as ‘the pope of trash’ (Egan 2011: Xiii, 192, 227): however, during the 1980s it was undermined. The earlier films, from the 1960s–70s, were independent, low budget, and outrageous. Mondo Trasho was made for only $2000. Multiple Maniacs for $5000. The budget for Female Trouble (1974) was $26,000 and for Desperate Living (1977) it was $65,000 (Egan 2011: 45, 50). The films from the 1980s onwards were funded by studios and targeted wider audiences. Polyester was produced by New Line Cinema and enjoyed a budget of $320,000 plus a couple hundred thousand dollars that financed the manufacturing of the accompanying Odorama cards that were handed to viewers (Egan 2011: 107). Cry-Baby (1990) was produced by Imagine Entertainment and distributed by Universal Studios, and its budget was $8 Million. (Egan 2011: 117) The bigger budgets enabled higher production values. The narratives and characters were more developed. Instead of his usual gang of non-professionals who played the leading roles in the early films (marginals and friends from Baltimore, known as ‘Dreamlanders’), the later productions featured Hollywood stars. These included Kathleen Turner in Serial Mom (1994), Edward Furlong and Christina Ricci in Pecker (1998), Melanie Griffith in Cecil B. Demented (2000), and Tracey Ullman and Selma Blair in A Dirty Shame (2004). The offensiveness of the ‘prince of puke’ had apparently been tamed. His productions were no longer rated X by the MPAA, and Hairspray (1988) was even rated PG. Divine Smells: Odorama, Melodrama and the Body in John Waters’ Polyester Although the films maintained a level of eccentricity, they are considered mainstream (Kane-Meddock 2012: 205). James Hoberman and Jonathan Rosenbaum, for example, have expressed some disappointment in Polyester, claiming it was only slightly edgier than sitcoms on major television networks at the time (1983: 171). Elisa Padilla argues that ‘Waters’ career has been traditionally understood as a process of domestication or assimilation into the mainstream’, and his drive to reach a larger audience was perceived as betraying the exclusivity of cult reception (2000: 1). Walter Metz considers the film to be ‘a pivotal moment in the evolution of the underground cinema in the United States’ and that it proved that successful transition from midnight movies to a conventional Hollywood product could take place (2003: 157). 3 3 Some critics have argued that the historical context of Polyester’s release was another possible reason for the shift in Waters’ career. According to Eve Kosofsky Sedgwick and Michael Moon, 1981 was ‘the year that many people in and around urban communities in Western Europe and the United States began to register the scope of the threat and reality of AIDS” (1993: 246). Matthew Tinkcom considers Polyester to presage the staging of the crisis of family values (2002: 170). Elana Gorfinkel has analyzed the representation of non-mainstream cinema in Polyester that includes porn theaters, independent productions, arthouse films, and drive-ins, all of which can be connected in one way or another to Waters’ early films. However, ‘with the onset of the Reagan era, a different kind of cinema may have been required’ (2019). Polyester indeed taps a different style but never abandons its defiant approach. To a certain extent, Waters willingly gives up his artistic signature to adopt and lampoon the style of another auteur, Douglas Sirk. In a documentary entitled In Bad Taste (Yeager 1999), the cinematographer David Insley recalled that when he asked Waters what look he was interested in for Polyester, the director told him to watch Sirk’s Written on the Wind (1956). Waters himself stated in the documentary that Sirk was one of his favorite directors, and that he thought that Sirk’s best films dealt with suburban lives, the people locked up in them, and the irony encapsulated in this lifestyle. Divine Smells: Odorama, Melodrama and the Body in John Waters’ Polyester This observation is shared by Nieves Alberola Crespo and José Javier Juan Checa, who claim that Sirk was at his best when he ‘expertly dissects American suburban life from the 1950s with its upper-class conventions, faux formalities and conformity’ (2021: 118). The genre that Sirk is most closely identified with is melodrama. The generic conventions of melodrama force Waters to morph from a blunt attack on the ‘normative’ lifestyle to one that seethes beneath the surface. Geoffrey Nowell-Smith discusses excess in melodrama and claims that ‘the undischarged emotion which cannot be accommodated within the action… is traditionally expressed in the music and… the mise en scène’ (1987: 73). This time, it is also expressed through smells. In Polyester Waters does bring something brand new to the table in a sensory technique known as Odorama. Each admission ticket was attached to a scratch and sniff card whose scents were related to key scenes. At the proper moment, a number flashed in the corner of the screen, signaling to viewers what part of the card to scratch. Odorama was inspired by previous prototypes by other filmmakers, and other methods of adding odors to films such as AromaRama, that was used in the American release of Behind the Great Wall (Lizzani 1959) or Smell-O-Vision, that was used in the film Scent of Mystery (Cardiff 1960). However, with Odorama, smells, and 4 the sense of smell, both within the diegesis and in the cinematic viewing experience, are used to expose and convey the films’ subversive ideas. The literature on Odorama shows that it has never been subject to an in-depth analysis. Whether in cinematic studies or more technological works, Odorama is only briefly mentioned as an anecdote. Most scholars seem to dismiss it as just another gag in the film (Banes 2001: 71; Biocca and Delaney 1995: 43,64; DeLahunta 2003: 86; Ghinea and Ademoye 2011: 613; Gorfinkel 2019; Hanich 2009: 299; Khamsi 2009: 513; Krueger 2013: 59; Nakamura 2013: 134; Niedenthal 2012: 108–109; Olofsson et al. 2017: 458; Paterson 2006: 359; Spence 2020: 8–9; Spencer 2006: 169; Spencer 2004: 140; Wells 2000: R172-R173). When Waters himself addressed the issue in interviews, he also tended to underplay Odorama’s importance. However, as a popular interviewee, who was frequently invited to talk shows and commentators’ panels, Waters usually delivered snappy comebacks and sardonic remarks aiming to amuse, not to educate. Divine Smells: Odorama, Melodrama and the Body in John Waters’ Polyester His interviewers, and his fan base, would probably not expect him to provide lengthy commentary on cinematic theory. In an anthology of interviews his answers were inconsistent, if not contradictory. He gave different explanations of Odorama to different interviewers on different occasions. At times he described it as a gimmick meant to attract investors. In others he claimed it was a gesture towards B-movies director William Castle, who was famous for his innovative publicity stunts. Sometimes Waters saw Odorama as a practical joke at the viewers’ expense. He made fun of people who watch repulsive situations on the screen but still choose to scratch their cards and sniff the smells, even though some were disgusting, for example, skunk spray and flatulent stench (Egan 2011: 74, 87, 107). One reason why the film has been overlooked is related to the low-brow stigma associated with gimmicks. Sianne Ngai nevertheless argues that this attitude did not prevent condescending critics from being fooled: ‘Protected by its own slickness, as a thing whose sheer stupidity cleverly neutralizes the critical feeling it incites, the gimmick defends itself from intellectual curiosity in a way that puts any person seeking to analyze it at a comical disadvantage’ (2020: 9). Thus, why would Waters himself, who had no problem dealing with the despised, choose to downplay his own gimmick? One possible explanation is that by preserving its inferiority he was simply confirming his trademark rejection of good taste. ‘Gimmicks seem to provoke contempt… mere tools that have a strange way of stealing attention… one that performers “exploit” but in exploiting make their witnesses feel exploited, too’ (Ngai 2020: 52). However, the ‘irritating’ qualities of gimmicks do not appear to be a satisfactory justification for their use in this film. Rather, the olfactory gimmick serves, as Ngai suggests, ‘as 5 5 affective speech capable of being put to critical or even political uses in a way other negative aesthetic judgments are not’ (2020: 17). As shown below, Waters in fact cleverly used the generic conventions of the melodrama, along with an enhanced phenomenological viewing experience and the representation of the female body in film, to rebel against the dominant social values of that era. Meet Your Polyester Queen However, the dowdy housewife who appears on screen has nothing royal about her. Francine sits in front of the mirror. Like TV sets, the mirror is also a motif in Sirk’s films. It reminds viewers that the characters ‘live in a world where privacy is virtually non-existent. The characters are aware of being under scrutiny, so their best protection is to try and take command of the situation by determining their own appearance, if necessary even by deliberately putting on an act’ (Willemen 1991: 277). Francine is played by Waters’ frequent collaborator, Divine (born Harris Glenn Milstead). Francine’s appearance, however, does not match the iconic persona of Divine in earlier films. In Pink Flamingos and Female Trouble, for example, Divine wears colorful dresses, heavy makeup, has a partly shaved head, and bizarre accessories. Francine, on the other hand, looks like the women in any suburban 1950s neighborhood. Francine not only looks different, but also acts differently. In this particular scene, she goes through her beauty routine. Unlike Pink Flamingos, where Divine wants to be the ‘filthiest person in the world’, Francine attempts to conform to social dictates and pampers herself. She carefully applies (nonexaggerated) make up and perfume, plucks her eyebrows and nose hair, shaves her armpits, and sprays her body with deodorant. In other words, she gets rid of her bodily emanations. She hides and represses her natural body odors. By doing so she creates a body that is considered by society (and by the ‘woman’s film’ that reflects such values) as more feminine, pretty, and respectable. At the end of the scene, she gets on the scale with a grimace. This reinforces her interest in striving to conform to the ideals of feminine beauty of the time and garnering the approval of others. Derek Kane-Meddock also points out the contrast between Polyester’s protagonist Francine Fishpaw and Pink Flamingos’ protagonist Babs Johnson (a name which the film’s narrator describes as an alias that the notorious criminal Divine took on when she is wanted for murder). Francine, he says, ‘represses her desires, suffering for the good of her deviant family… [and] seeks to contain the smut that threatens her vision of idyllic home life’. The generic shift requires toning down Divine’s excesses, so that she will be able to inhabit the world of family melodrama. Meet Your Polyester Queen Polyester starts with an aerial shot of green fields in a suburban neighborhood. The shot is a nod to Sirk’s melodramas, for example, All That Heaven Allows (1955) in which the view from the height of the church tower presents an ideal image of a peaceful provincial town. The scene is accompanied by a soundtrack in which a cheesy singer touts a kitschy image of the neighborhood. The singer is Tab Hunter, who also stars in the film as Todd Tomorrow, Francine’s lover. In the 1950’s, Hunter was a successful teen idol and a sex symbol. However, in private life, Hunter was gay, and had to keep his true identity in the closet (Hunter and Muller 2005; see also the documentary Tab Hunter Confidential (Schwarz 2015)), just like one of Sirk’s most notable leading actors, Rock Hudson. Both Hudson and Hunter had their star image shaped by the same talent agent, Henry Willson (Klinger 1994: 102). Hudson’s persona was carefully manufactured. His publicity campaigns included articles and photos which cemented his image as, in Barbara Klinger’s words, ‘the continued presence of normality in a sea of disintegrating personalities… Through this kind of iconography, wholesome Hudson appeared as a kind of antidote to an overdose of unstable oddballs—drugged, divorced, and uncertain of their identities’. In typical photos he is seen engaged in ‘manly’ activities, having ‘a happy, normal, good time’ in settings which represent ‘Americana’ (1994: 109). Polyester’s opening song and images convey the same kind of illusion that negates any hint of the abnormal. The camera moves into the Fishpaw family’s private residence, up the stairs, and into the bedroom. An old TV set in the corner of the room is turned off, and the room is reflected in its screen. This recalls Sirk’s use of TV sets to signal the loneliness and misery of the housewives in his films (McNiven 1983: 44–45; Willemen 1991: 277). Bright light shines into the room from the window behind. In the other corner, Francine Fishpaw, the heroine, is standing in front of another window, also bathed in glowing light. Two lamps are also lit on either side of the room. The exaggerated lighting is an articulation of the overstated ironic expressiveness of the film, a typical feature of melodramas. 6 On the soundtrack, the singer praises Francine, whom he calls ‘the polyester queen’. Meet Your Polyester Queen Harries, on the other hand, suggests that Francine is only disguised as ‘weak’ and thus more ‘feminine’, but clearly as powerful as Divine ever was. He argues that Divine’s persona is characterized as a ‘strong woman’. One of the main ways to confirm this is to play with the juxtaposition between feminine qualities and her enormous body: ‘This, in turn, fosters his subversive parody of what a woman should be in terms of size and stature…’. Harries suggests that ‘the primary means of achieving this is the “animalization” of Divine’s persona, equating Divine with large animals… Divine is reduced to an animalized form, a freak and subversive threat who parodies the social order through a grotesque transformation’ (1990: 17). Even though Polyester might seem to present a new, gentle Divine, this is merely a façade. Throughout the film, in moments of excessive emotion, she can no longer restrain herself, and is ‘jolted back into the “monstrous” Divine’, for example, when she aggressively knocks down doors (Harries 1990: 20). Interestingly, Sedgwick and Moon themselves point to Francine’s animalization. In fact, they define her as a sort of human-animal hybrid. However, her animalistic features are not the result of her size, but rather her highly developed olfactory abilities, ‘…a prehensile and almost paranormal receptivity to offensive odor… [she is] darting heavily about her own house… snuffling noisily at bedclothes and the cracks of doors - wriggling uncontrollably… behaving, in short, like any scratch-and-sniffing animal in the world except Homo sapiens domesticus nuclearus’ (1993: 245). Clearly, in the world of melodrama, things are never what they seem, since threats to an ideal image are being repressed, hidden and denied. Waters aims to expose this hypocrisy and asks his viewers not to believe their eyes. Instead, the viewers should rely on all their bodily senses. Linda Williams suggests that melodramas, horror and pornography are all ‘body genres’. She claims that these genres are considered culturally inferior because in all of them ‘the body of the spectator is caught up in an almost involuntary mimicry of the emotion or sensation of the body on the screen along with the fact that the body displayed is female’ (Williams 1991: 4). Waters, who enjoys wallowing in the culturally inferior, encourages viewers to use their whole bodies when watching the film to become what Vivian Sobchack (2000) calls ‘the cinesthetic subject’. Meet Your Polyester Queen Nevertheless, says Kane-Meddock, Waters does not abandon his agenda, and in his version ‘the traditional family becomes the very site of perversity... In this case, filth surrounds the maternal figure instead of deriving from it as in Pink Flamingos’ (2012: 207–208). The documentary I am Divine (Schwartz, 2013) suggests that this was a planned image change, and since Waters had already done Divine as monster, he now wanted to show her as a sympathetic heroine. Other writers have also discussed how Francine diverges from Divine’s previous portrait gallery. Sedgwick and Moon claim that ‘Polyester is the only [Waters] film whose diegesis assumes that Divine’s obesity makes her unlovable and powerless rather than magnetically irresistible’ (1993: 244). Dan M. Harries, on the other hand, suggests that Francine is only disguised as ‘weak’ and thus more ‘feminine’, but clearly as powerful as Divine ever was. He argues that Divine’s persona is characterized as a ‘strong woman’. One of the main ways to confirm this is to play with the juxtaposition between feminine qualities and her enormous body: ‘This, in turn, fosters his subversive parody of what a woman should be in terms of size and stature…’. Harries suggests that ‘the primary means of achieving this is the “animalization” of Divine’s persona, equating Divine with large animals… Divine is reduced to an animalized form, a freak and subversive threat who parodies the social order through a grotesque transformation’ (1990: 17). Even though Polyester might seem to present a new, gentle Divine, this is merely a façade. Throughout the film, in moments of excessive emotion, she can no longer restrain herself, and is ‘jolted back into the “monstrous” Divine’, for example, when she aggressively knocks down doors (Harries 1990: 20). Interestingly, Sedgwick and Moon themselves point to Francine’s animalization. In fact, they define her as a sort of human-animal hybrid. However, her animalistic features are not the result of her size, but rather her highly developed olfactory abilities, ‘…a prehensile and almost paranormal receptivity to offensive odor… [she is] darting heavily about her own house… snuffling noisily at bedclothes and the cracks of doors - wriggling uncontrollably… behaving, in short, like any scratch-and-sniffing animal in the world except Homo sapiens domesticus nuclearus’ (1993: 245). ‘Polyester is the only [Waters] film whose diegesis assumes that Divine’s obesity makes her unlovable and powerless rather than magnetically irresistible’ (1993: 244). Dan M. Whoever Smelt It, Dealt It Throughout Polyester, the viewers, along with the heroine Francine, use their sense of smell to discover that something is amiss before observing it with their own eyes. These include the smell of the glue that Francine’s daughter inhales to get high, or the gas that her husband passes while they lie in their bed. The flatulent stench perhaps symbolizes their rotten relationship. Melodrama is an excessive cinema that assault the senses. Nowell Smith (1987) and Mulvey (1996) claim that in this genre, aesthetic elements like colors, lighting, composition, or soundtrack inflect meaning and should be read as symbolic. They ‘do not just heighten the emotionality of an element of the action: to some extent they substitute for it’ (1987: 73). In this special case, the same also applies for smells. This innovative and amusing form of excess is chosen for its campy qualities. There are many interpretations of ‘camp’, a term which has been constantly eluding critical definitions (Cleto 1999). One which is particularly useful for the purposes of this discussion, is the definition by Chuck Kleinhans. According to him, camp is ‘an ironic and parodic appreciation of an extravagant form that is out of proportion to its content’ (1994: 160). The film chooses to exaggerate smell, which films in general do not even bother with, by aggrandizing it. Viewers see a stinky object somewhere in the space that is presented at a distance on the screen but smell it as though it were right under their noses. Kleinhans adds that ‘when employed intentionally… [camp] relies for its effect on casual excess, deviant decorum, and libidinal obviousness’ (1994: 163), the sort of humor one would expect to find in Waters’ filthy works. However, this is not simply a gag for the purpose of evoking crude bodily responses (shock/laugh), but rather has a deeper purpose. Kleinhans notes that ‘instead of acquiescing in the ideology of a disposable culture that wants to flush away its social problems, Camp can insist on a determined recycling of political agendas as well as aesthetic diversity’ (1994: 171). By refusing to flush away the unpleasant, and instead indulging in its stench, Waters finds another way to expose what melodramas usually avoid facing directly. Thus, the use of Odorama in a parody like Polyester is even more effective than in a generic melodrama precisely because of its campy embrace. Meet Your Polyester Queen This subverts the normative objective vision which reduces the cinematic sensory experience to a purely visual act of watching. The cinesthetic subject undermines identity and perception theories which prioritize the sense of sight and do not take the whole body into account. 8 Whoever Smelt It, Dealt It As Ngai has suggested, comedy ‘has a unique way of bringing out the gimmick’s aesthetic features in explicit linkage to its status as a practical device’ (2020:53). One key scene captures the height of Francine’s suffering. Prior to this, she goes out for a picnic in the woods. While she is away, her daughter’s punk boyfriend shows up at the Fishpaw’s residence with his friend for Trick-or-treating. Francine’s mother does not offer them the desired candies, so they break into the house, trash it, and the situation escalates into a gun fight. Francine’s day out ends abruptly after she is being 9 sprayed by a skunk. At the start of the scene, she returns home to find that her woes have not ceased. As in the Book of Job, ‘while he was yet speaking, there came also another’. First, she discovers that her living room is wrecked. Her injured mother scolds her that it is all her fault. She then notices the dead body of her daughter’s boyfriend. Before Francine can understand what has happened, a mysterious smell rushes her into the kitchen, where she finds her heartbroken daughter shoving her head into the oven. Viewers had already smelled the cooking gas via Odorama before witnessing it on screen. Francine tries to save her daughter, sobbing and praying God to spare the life of her little girl, when her eyes suddenly light on a suicide letter. Strangely, the signature at the bottom is not her daughter’s, but another family member’s. The camera quickly tilts up from the letter to reveal that the family’s dog has hanged himself. This final blow is an unlikely scenario even in a melodrama and foregrounds the parody in what seems to move into wild absurd humor. Nevertheless, as Nowell-Smith writes, in melodrama ‘…the ‘hysterical’ moment of the text can be identified as the point at which the realist representative convention breaks down’ (1987: 74). Francine bursts into tears, gasps agonizing moans which no longer form coherent sentences, and eventually faints. Nowell-Smith draws a parallel between the body of the hysterical patient and the melodramatic body. ‘In hysteria, the energy attached to an idea that has been repressed returns converted into a bodily symptom... In the melodrama... a conversion can take place into the body of the text’ (1987: 73). Francine faints since she can no longer contain all her emotions. Whoever Smelt It, Dealt It But it is not merely her body that is aching, it is also the cinematic text that reeks. In a related way, Williams has noted the way excess in culturally inferior genres, including melodrama, is often figured bodily. According to her, these genres share a focus on ecstatic excess, especially through the female body which embodies excessive pleasure, fear, and pain. Waters celebrates this hysterical excess and exaggerates it. In body genres, the female body becomes a source of sensation mimicked by the spectators’ body. The representations are deployed in over-intimate ways to generate close modes of identification between what is on screen and the spectators, who often physically experience the same bodily functions (or production of bodily fluids – tears, sweat, etc.) as the characters in the film. Williams claims that during such experiences the spectator’s viewpoint is much more flexible than usual. The viewers’ identification is not fixed and moves along the spectrum from feminine to masculine (1991: 4, 8–9). Odorama enhances this experience since it forces the viewers to physically smell what Francine smells. The fact that the female body in the center of this spoof of women’s films is Divine’s body further undermines gender dichotomies, since Divine is a male actor who portrays a female character. 10 Similarly, Laura Mulvey argues that sexualized binarism collapses in women’s pictures. Referring to classic films such as Stella Dallas (Vidor 1937) and Now, Voyager (Rapper 1942), she claims that ‘these movies were, very often, about performance and therefore necessarily draw attention to the artifice of appearance and the process of its production’ (1996: 30–32). In Polyester, this is reinforced by the fact that the heroine is played by Divine, a female impersonator. Mulvey mentions that ‘female impersonators have, for a long time, made use of the accoutrements of masquerade associated with Hollywood stars’. In an interview which appears as a bonus feature on the British DVD edition of The Girl Can’t Help It (Tashlin 1956), Waters says that Divine’s persona was designed as a twisted takeoff on the persona of the glamor girl and actress Jayne Mansfield. Similarly, Kane-Meddock points out Divine’s use of elaborate costuming and makeup in the transformation process for movie roles: ‘This, in conjunction with his growing celebrity, made artifice and performance key subtexts for the audience, as sometimes happens with Hollywood stars who have grown too famous to completely inhabit a character’ (2012: 207). Whoever Smelt It, Dealt It Waters takes this subversion a step further by completely blurring genders, since the ‘female body’ that experiences these female phenomena is the body of Divine, who identifies as a male actor in drag, and who was famous for passing as a woman. When Waters mixes traditional roles which are identified with the female stars of the melodramas with attributes that are identified as manly, he complicates and disrupts the feminist theories cited by Williams and Mulvey which criticize the display of the suffering female body as a spectacle for the sadistic male gaze (1991: 6). However, Judith Butler has identified Waters’ sleight of hand and used his films to support her ideas. Butler’s pioneering work Gender Trouble was named after Waters’ film Female Trouble (despite the fact that Butler later downplayed the role of drag in the theory of gender performativity). Butler praises Divine’s appearance in films such as Female Trouble and Hairspray that ‘implicitly suggests that gender is a kind of persistent impersonation that passes as the real. Her/his performance destabilizes the very distinctions between the natural and the artificial, depth and surface, inner and outer through which discourse about genders almost always operates’. Butler argues that ‘laughter in the face of serious categories is indispensable for feminism’ and explains that when gender practices are placed in parodic context it brings into relief the performative construction of an original and true sex. Divine’s skillful acting raises the question of whether gender is established through the imitation of gender, or a dramatization of the signifying gestures? (2011: xxx-xxxI). Francine Fishpaw is not just another cabaret drag act. This performance is not evaluated as a man who passes as a woman (or a travesty of womanhood, as some critics would say). It is neither just 11 another film in which a male comedian plays a woman for laughs. Instead, Francine can be defined as a woman played by a drag queen who is played by a man. Unlike in Waters’ previous films where Divine remained ‘Divine’ - the icon, the distinct persona - even when playing characters named Babs Johnson or Dawn Davenport, in this film, for the first time, Divine turns to someone else, hence destabilizing even the discourse about Divine. Furthermore, Kane-Meddock also places such satirical and gender- subversive ideas in a broader context within Waters’ filmography. Whoever Smelt It, Dealt It Based on Williams’ distinction between extroverted ‘posing’ which emphasizes artifice and restrained ‘passing’, he compares Babs Johnson’s extravagance in Pink Flamingos to Polyester’s Francine Fishpaw’s subtlety. This leads to a more comprehensive argument about Waters’ transitions throughout his career. With time, he claims, ‘as gender became a more subtle influence on Waters’ work, the exaggeration of generic conventions took on added importance’ (2012: 209–211). In the case of Polyester, the generic conventions are those of the family melodrama, and one exaggeration does not necessarily come at the expense of the other. Some may wish to classify the Odorama-technology pioneering film Polyester under a different label, such as that coined by Tom Gunning: the ‘cinema of attractions’. Sobchack, although critical of Tom Gunning’s term, mentions that carnal responses to the cinema tend to be regarded as too crude. Films that collapse the ‘proper esthetic distance’ between the spectator and the screen are often considered primitive. Their easy thrills, commercial impact, and cultural associations are conflated with other more kinetic forms of amusement such as theme park rides (2000). The use of smell in theatrical experiences has also often been regarded as simplistic or superfluous, ‘merely iconic and illustrative, a weak link in a chain of redundancy across sensory channels that does nothing more than repeat what is already available visually and aurally’ (Banes 2001: 68–69). However, as Sally Banes notes, sometimes smell is used for the exact opposite, ‘to complement or contrast with what is happening in the rest of the performance… in this category of our taxonomy, the odor introduces new or even conflicting information’ (2001: 70). In melodrama, smell signals the gap between what is presented on the surface and what is indirectly conveyed. Whenever Francine, and Polyester’s viewers, sniff a new scent, it is foreshadowing a turn in her life. More often than not, it is a negative twist, that shatters the ideal façade that the all-American housewife works so hard to present to her surroundings and to herself. Francine is in denial about her problems, and therefore she suffers. Throughout the film she becomes disillusioned, mainly due to her sense of smell. The bad smells contradict the pleasant and decent associations that imagery of 1950’s suburbia usually provoke and break the delusion. 12 Smells function in several ways, which might even appear as contradictory. Whoever Smelt It, Dealt It Smell can also be used as a distancing device, which calls attention to itself as an effect, and to the artificiality of the experience. The viewers are aware of the illusion, and the smell undermines, rather than enhances, the realism (Banes 2001: 71). In the case of Odorama there is another layer of complexity since the smells are activated by the user, who is aware and not passive. It is also prompted by Waters’ choice to use many unpleasant smells, such as skunk, or dirty shoes. As Julian Hanich has noted ‘In order to be experienced as disgusting an object must come overly near and penetrate the intimate area of the senses… the integrity and cleanliness of the body seems threatened by a film’ (2009: 295–296). Bad smells are a disturbance, they create discomfort, they alienate the viewers, make them more aware, and enable Waters to convey his critical messages. Hanich notes that ‘because sight and sound are the only senses actively called upon… it is much easier to withdraw and hence to avoid serious displeasure’. The instinct is to shake off what is disgusting, ‘to get rid of the obtrusively close object that constricts us’. Odorama prevents viewers from following this instinct, immerses them in the disturbing experience, and resists their objection. Hence, Odorama bridges over the opposing effects of alienation and identification. Instead of contradicting they are complementing. The smells are provoking intensified intimacy, and at the same time, especially when the smells are unpleasant, the distancing effect creates a critical viewer who reflects on this intimate experience. What My Nostrils Knew When Sobchack (2000) discusses affect and sensory experience while watching a film, she does not refer to cases in which smell is really, physically experienced, as in Polyester. But this does not mean that her observations do not apply to Odorama. Sobchack explains that (in almost all films which do not use Odorama or similar technologies) since viewers cannot smell, touch, or taste the figures on the screen that elicit sensual desires, the body ‘seeking a sensible object to fulfill this sensual solicitation, will reverse its direction  to locate its sensual grasp on something more literally accessible. That more literally accessible sensual object is my own subjective lived body… I will reflexively and carnally turn toward my own carnal being to touch myself touching, smell myself smelling, taste myself tasting, and, in sum, sense my own sensuality’. It is also worth wondering whether since the body can do this, aren’t the scratch-card-produced smells redundant? In a way the cards are, thus turning them into significant excess. The act of smelling, and not looking, opens Francine’s eyes, and enables her to confront her problems, among them: the ungrateful treatment from her family, loveless relationships, dysfunctional kids, low self-image, and constant failure to live the life 13 she desires. Although she manages to solve many of them, Polyester’s finale, as often happens in melodrama, is a pale imitation of a happy ending. Williams claims that each one of the three body genres, and the excessive fantasy or perversion they represent, has its own temporality: the melodrama comes too late, the porn film is right on time, and the horror film is too early (1991: 11). This temporality helps interpret the finale. Towards the end of the film, Francine’s ex-husband and his lover try to murder her. Her children overcome them and kill them. In addition, Francine discovers that her new boyfriend is having an affair with her evil mother, and they plot to take her belongings and admit her to a mental institution. Francine finally realizes the truth, too late. Luckily, however, at the last moment, her best friend Cuddles and her chauffeur drive to the scene, run over her enemies, and save Francine. This ridiculously improbable ending mocks the typical melodramatic endings which sink into artificial and arbitrary solutions (Nowell-Smith 1987: 70, 73). The final sequence exaggerates Williams’ concept of the melodramatic temporality of ‘too late’. Competing Interests The author has no competing interests to declare. Acknowledgements I wish to thank Professor Raz Yosef for his helpful notes on an earlier version of this essay. I wish to thank Professor Raz Yosef for his helpful notes on an earlier version of this essay. What My Nostrils Knew Everything appears to have been resolved. In line with Waters’ macabre humor, the ‘happy end’ is shown on screen as two dead bodies symmetrically and decoratively placed on both sides of the frame. The choice to end the story there, during a temporary moment of relief that only removes the immediate threats, creates the impression of a happy ending. The cheesy theme song returns to dominate the soundtrack, replacing the real sounds of the accident, thus once again sugarcoating the images. One might believe, as Metz put it, ‘at the end of Polyester, as at the end of Sirk’s All That Heaven Allows… the middle-class housewife is offered a chance for happiness’ (2003: 161). But the characters still face many problems which are simply suspended or repressed. The heroic rescue indeed comes too late. Francine’s children have already committed murder. Their father, as nasty as he was, is tragically dead. Their grandmother and Francine’s new boyfriend have also lost their lives. Cuddles, Francine’s best friend, ran over and killed two people, and will probably face charges. Francine lost everything. Is this truly a happy ending? Thus, even though Francine deeply inhales the comforting scent of an air freshener that she sprays around her which is supposed to clear the atmosphere and restore peace and happiness, this is only an illusion. Waters emphasizes the unsatisfying phony artificiality of the genre’s traditional endings. Francine only deceives herself, while still repressing her troubles and unfulfilled desires. Life, according to Waters, always stinks. It may be no coincidence that Odorama was used in Polyester. Although panned by the critics, Odorama should not be treated as a mere gimmick grafted to the film. If Waters had used Odorama in one of his earlier gross-out films it would have been easier to treat it as no more than an attraction, a cheap shtick. However, Polyester, in 14 its unique and lampooning way, is a melodrama. It adopts the generic formula of films in which many times nothing is as it seems, and excess is repressed by the plot only to burst out through aesthetic choices. Waters uses smells, and the act of smelling, to vitiate social norms, and middle-class values, which he identifies as hypocritical and fraudulent, as mirrored in ‘conventional’ families, and gender dichotomies, in society and in its representation in the cinema. 15 References Banes, S., 2001. Olfactory Performances. The Drama Review 45.1, pp. 68–76. DOI: https://doi. org/10.1162/105420401300079040 Biocca, F. and Delaney, B., 1995. Immersive Virtual Reality Technology. In: Communication in the Age of Virtual Reality. Hillsdale: Lawrence Erlbaum Associates P, pp. 57–124. Butler, J., 2011. Gender Trouble: Feminism and the Subversion of Identity. New York: Routledge. DOI: https://doi.org/10.4324/9780203824979 Cleto, F., 1999. Camp: Queer Aesthetics and the Performing Subject: A Reader. Edinburgh: Edinburgh U P. DOI: https://doi.org/10.3998/mpub.11698 Crespo, N.A. and Checa, J.J.J., 2021. Suburbia as a Narrative Space in the Cinematic Universe of Douglas Sirk. Language Value, 14.1, pp.112–132. DOI: https://doi.org/10.6035/languagev.5953 DeLahunta, S., 2003. Sniffable Media. Performance Research 8.3, pp. 85–86. gan, J., 2011. John Waters: Interviews. Jackson: U of Mississippi P. Egan, J., 2011. John Waters: Interviews. Jackson: U of Mississippi P. Ghinea, G. and Ademoye, O. A., 2011. Olfaction-Enhanced Multimedia: Perspectives and Challenges. Multimedia Tools and Applications 55.3, pp. 601–626. DOI: https://doi.org/10.1007/ s11042-010-0581-4 gi s11042-010-0581-4 Gorfinkel, E., 2019. Polyester: The Perils of Francine. The Criterion Collection. Polyester DVD/Bluray Release Essay. Hanich, J., 2009. Dis/liking Disgust: The Revulsion Experience at the Movies. New Review of Film and Television Studies 7.3, pp. 293–309. DOI: https://doi.org/10.1080/17400300903047052 Harries, D. M., 1990. Camping with Lady Divine: Star Persona and Parody. Quarterly Review of Film & Video 12.1–2, pp. 13–22. DOI: https://doi.org/10.1080/10509209009361335 Hoberman, J. and Rosenbaum, J., 1983. Midnight Movies. New York: Harper & Row. Hunter, T. and Muller, E., 2005. Tab Hunter Confidential: The Making of a Movie Star. Chapel Hill: Algonquin Books. Kane-Meddock, D., 2012. Trash Comes Home: Gender/Genre Subversion in the Films of John Waters. In: Gender Meets Genre in Postwar Cinema. Illinois: U of Illinois P, pp. 205–220. Khamsi, R., 2009. Smells Like Green Spirit. Nature 459.7246, p. 513. DOI: https://doi. org/10.1038/459513a Kleinhans, C., 1994. Taking Out the Trash: Camp and the Politics of Parody. In: The Politics and Poetics of Camp. London: Routledge, pp. 157–173. 16 Klinger, B., 1994. Melodrama and Meaning: History, Culture, and the Films of Douglas Sirk. Bloomington: Indiana U P. Krueger, C., 2013. The Scent Trial of ‘Une charogne’. French Forum 38.1, pp. 51–68. DOI: https:// doi.org/10.1353/frf.2013.0003 McNiven, R. D., 1983. The Middle-Class American Home of the Fifties: The Use of Architecture in Nicholas Ray’s “Bigger than Life” and Douglas Sirk’s “All That Heaven Allows”. Cinema Journal 22.4, pp. 38–57. DOI: https://doi.org/10.2307/1224953 Metz, W., 2003. John Waters Goes to Hollywood. In: Authorship and Film. New York: Routledge, pp. 157–174. DOI: https://doi.org/10.4324/9780203698976-8 Mulvey, L., 1996. Social Hieroglyphics: Reflections on Two Films by Douglas Sirk. In: Fetishism and Curiosity. Bloomington: BFI, Indiana U P, pp. 29–39. Ngai, S. 2020. Theory of the Gimmick: Aesthetic Judgment and Capitalist Form. Cambridge: Harvard U P. DOI: https://doi.org/10.4159/9780674245365 Nakamura, K., 2013. Making Sense of Sensory Ethnography: The Sensual and the Multisensory. American Anthropologist 115.1, pp. 132–135. DOI: https://doi.org/10.1111/j.1548- 1433.2012.01544.x Niedenthal, S., 2012. Skin Games: Fragrant Play, Scented Media and the Stench of Digital Games. Eludamos: Journal for Computer Game Culture 6.1, pp. 101–131. DOI: https://doi. org/10.7557/23.6141 Nowell-Smith, G., 1987. Minnelli and Melodrama. In: Home is Where the Heart is: Studies in Melodrama and Woman’s Film. London: BFI P, pp. 70–74. Olofsson, J.K., Niedenthal, S., Ehrndal, M., Zakrzewska, M., Wartel, A. and Larsson, M., 2017. Beyond Smell-O-Vision: Possibilities for Smell-Based Digital Media. Simulation & Gaming 48.4, pp. 455–479. DOI: https://doi.org/10.1177/1046878117702184 Padilla, E., 2020. gi s11042-010-0581-4 The Baltimore Artist: Taste, Class, and Distinction in John Waters’ Pecker. Quarterly Review of Film and Video, pp.1–19. DOI: https://doi.org/10.1080/10509208.2020.1842093 Paterson, M. W., 2006. Digital Scratch and Virtual Sniff: Simulating Scents. In: The Smell Culture Reader. Oxford: Berg, pp. 358–367. Sedgwick, E.K. and Moon, M., 1993. Divinity: A Dossier, A Performance Piece, A Little-Understood Emotion. In: Tendencies. New York: Duke U P, pp. 215–251. DOI: https://doi.org/10.2307/j. ctv11hpmxs.15 i ctv11hpmxs.15 Sobchack, V., 2000. What My Fingers Knew: The Cinesthetic Subject, or Vision in the Flesh. Senses of Cinema 5. Spence, C., 2020. Scent and the Cinema. i-Perception 11.6, pp. 1–22. DOI: https://doi. org/10.1177/2041669520969710 Spencer, B. S., 2004. Using the Sense of Smell in Telemedical Environments. Studies in Heal Technology and Informatics 103, pp. 135–142. Spencer, B. S., 2006. Incorporating the Sense of Smell into Patient and Haptic Surgical Simulators. IEEE Transactions on Information Technology in Biomedicine 10.1, pp. 168–173. DOI: https://doi. org/10.1109/TITB.2005.856851 17 Tinkcom, M., 2002. ‘Beyond the Critics’ Reach’: John Waters and the Trash Aesthetic. In: Working Like a Homosexual: Camp, Capital, and Cinema. Durham: Duke U P, pp. 155–188. DOI: https://doi. org/10.1215/9780822383772-005 Wells, W. A., 2000. Sniffing for Success. Chemistry & Biology 7.7, pp. R172–R173. DOI: https://doi. org/10.1016/S1074-5521(00)00141-1 Willemen, P., 1991. Towards an Analysis of the Sirkian System. In: Imitation of Life: Douglas Sirk, Director. New Burnswick: Rutgers U P, pp. 273–278. Williams, L., 1991. Film Bodies: Gender, Genre, and Excess. Film Quarterly 44.4, pp. 2–13. DOI: https://doi.org/10.2307/1212758 Williams, L., 1991. Film Bodies: Gender, Genre, and Excess. Film Quarterly 44.4, pp. 2–13. DOI: https://doi.org/10.2307/1212758 Filmography A Dirty Shame, 2004. [Film] John Waters. USA: Killer Films. A Dirty Shame, 2004. [Film] John Waters. USA: Killer Films. All That Heaven Allows, 1955. [Film] Douglas Sirk. USA: Universal Pictures. Behind the Great Wall, 1959. [Film] Carlo Lizzani. Italy: Astra Cinematografica. Cecil B. Demented, 2000. [Film] John Waters. USA: Polar Entertainment. Cry-Baby, 1990. [Film] John Waters. USA: Imagine Entertainment. Desperate Living, 1977. [Film] John Waters. USA: Dreamland. Female Trouble, 1974. [Film] John Waters. USA: Dreamland. Hairspray, 1988. [Film] John Waters. USA: New Line Cinema. I am Divine, 2013. [Film] Jeffrey Schwarz. USA: Automat Pictures. In Bad Taste, 1999. [Film] Steve Yeager. USA: Independent Film Channel. Mondo Trasho, 1969. [Film] John Waters. USA: Dreamland. Multiple Maniacs, 1970. [Film] John Waters. USA: Dreamland. Multiple Maniacs, 1970. [Film] John Waters. USA: Dreamland. Now, Voyager, 1942. [Film] Irving Rapper. USA: Warner Bros. Pecker, 1998. [Film] John Waters. USA: Polar Entertainment. Pink Flamingos, 1972. [Film] John Waters. USA: Dreamland. Polyester, 1981. [Film] John Waters. USA: New Line Cinema. Scent of Mystery, 1960. [Film] Jack Cardiff. USA: Michael Todd. Serial Mom, 1994. [Film] John Waters. USA: Polar Entertainment. Stella Dallas, 1937. [Film] King Vidor. USA: Samuel Goldwyn Productions. Tab Hunter Confidential, 2015. [Film] Jeffrey Schwarz. USA: Automat Pictures. The Girl Can’t Help It, 1956. [Film] Frank Tashlin. USA: Twentieth Century Fox. Written on the Wind, 1956. [Film] Douglas Sirk. USA: Universal Pictures.
https://openalex.org/W4392456737
https://publications.eai.eu/index.php/sis/article/download/4559/2943
English
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Research of IoT Technology Based Online Status Monitoring on Hydropower Station Equipment
ICST transactions on scalable information systems
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Abstract The rapid proliferation of Internet of Things (IoT) has revolutionized the field of status monitoring for electrical equipment such as hydropower station equipment, offering enhanced efficiency and reliability in maintenance and operations. In this paper, we investigate the utilization of IoT transmission technology enhanced by multiple relays, denoted as M relays, to augment the monitoring of hydropower station equipment. To optimize the performance of the system, we employ partial relay selection, commonly referred to as selection combining. This study delves into the analysis of the system performance by deriving an analytical data rate, with a focus on quantifying the benefits of employing partial relay selection in IoT transmission for electrical equipment status monitoring. Our analytical approach enables a comprehensive evaluation of system efficiency, considering factors such as data rate, reliability, and power consumption. Through our analysis, we aim to provide valuable insights into the trade-offs and advantages of incorporating partial relay selection into IoT systems. By examining the impact of M relays and partial relay selection on IoT transmission technology, the work in this paper can help enhance the reliability of status monitoring for electrical equipment, ultimately advancing the capabilities of IoT-based solutions in the context of electrical systems and equipment maintenance. Received on 08 December 2023; accepted on 03 March 2023; published on 05 March 2024 Keywords: IoT, online status monitoring, relay selection, analytical data rate. Copyright © 2023 Y. Ma et al., licensed to EAI. This is an open access article distributed under the terms of the Creative Commons Attribution license, which permits unlimited use, distribution and reproduction in any medium so long as the original work is properly cited. doi:10.4108/eetsis.4559 Received on 08 December 2023; accepted on 03 March 2023; published on 05 March 2024 ; p ; p 5 Keywords: IoT, online status monitoring, relay selection, analytical data rate. Keywords: IoT, online status monitoring, relay selection, analytical data rate. EAI Endorsed Transactions on Scalable Information Systems EAI Endorsed Transactions on Scalable Information Systems Research Article ∗Corresponding author. Email: cchen11@126.com. Research of IoT Technology Based Online Status Monitoring on Hydropower Station Equipment Yuanjiang Ma1, Xudong Lu1, Liang Hong1, Xuan He1, Mianqian Qiu1, Mingliang Tang1, and Lei Chen1,∗ 1Yingxiuwan Hydropower General Plant of State Grid Sichuan Electric Power Company, Chengdu, 611800, Sichuan, China ∗Corresponding author. Email: cchen11@126.com. 1. Introduction seamless integration of sensors, data analytics, and net- work connectivity, IIoT networks empower industries to proactively monitor and manage their assets, minimiz- ing downtime and maximizing productivity [9–12]. As industries continue to embrace the potential of IoT, the development of robust and reliable status monitoring solutions remains at the forefront of innovation, driving advancements in industrial automation and optimiza- tion. Internet of Things (IoT) networks have ushered in a new era of connectivity and automation, offering trans- formative capabilities across a wide spectrum of appli- cations [1–4]. Among these, status monitoring within industrial IoT (IIoT) networks stands out as a pivotal domain. Industrial IoT networks provide the infrastruc- ture for real-time, data-driven insights and control over machinery, equipment, and processes within various industrial sectors [5–8]. This includes predictive main- tenance in manufacturing plants, remote monitoring of critical infrastructure, and efficient asset management in smart cities. These applications not only enhance operational efficiency but also contribute to cost reduc- tion, improved safety, and sustainability. Through the Relaying techniques have emerged as a crucial strat- egy for enhancing the performance of IoT networks, with a profound impact on critical metrics such as data rate, outage probability, and symbol error rate [13–16]. By employing multiple relay nodes strate- gically within the network architecture, data can be transmitted more reliably and efficiently over extended ∗Corresponding author. Email: cchen11@126.com. EAI Endorsed Transactions on Scalable Information Systems Online First EAI Endorsed Transactions on Scalable Information Systems Online First 1 Y. Ma et.al Figure 1. System model of channel-noise-assisted cooperative IoT networks with one transmit source S, M relays, and one destination D. distances. These relays act as intermediaries, ampli- fying and forwarding data, mitigating signal degra- dation, and reducing the probability of data loss or communication interruptions. Consequently, data rates are substantially improved, enabling IoT networks to support bandwidth-intensive applications and services [17–21]. Moreover, the use of relaying techniques can help reduce the outage probability, ensuring that data transmission remains robust even in challenging envi- ronments. Symbol error rates are also investigated as relay nodes enable signal regeneration and error cor- rection, resulting in higher data integrity. This synergy between relaying techniques and IoT networks plays a pivotal role in realizing the full potential of IoT applications, particularly in scenarios where reliability, data rates, and error rates are of paramount importance, such as in critical infrastructure monitoring and health- care systems [22–26]. Figure 1. 1. Introduction System model of channel-noise-assisted cooperative IoT networks with one transmit source S, M relays, and one destination D. with the incorporation of partial relay selection into IoT systems, ultimately aiding in the development of more efficient solutions for electrical equipment status monitoring. By assessing the impact of M relays and partial relay selection on IoT transmission technology, this research contributes to ongoing efforts aimed at enhancing the reliability and resilience of status mon- itoring for electrical equipment, thereby advancing the capabilities of IoT-based solutions in the realm of elec- trical systems and equipment maintenance. Partial relay selection is a key optimization technique within relaying networks, offering significant improve- ments in data rate, outage probability, and symbol error rate. By carefully selecting a subset of relay nodes from the available pool, this approach intelligently leverages the best-performing relays to amplify and forward data, thereby boosting the data rate. This selective relay strat- egy not only conserves network resources but also mini- mizes the interference, resulting in a higher throughput for data transmission. Moreover, partial relay selection reduces the outage probability by dynamically choosing relays that maximize signal quality, ensuring reliable communication, even in challenging conditions. Sym- bol error rates are also markedly improved through the judicious selection of relays, as only the most favorable relay nodes are employed, reducing the likelihood of symbol errors and enhancing overall data integrity. In essence, partial relay selection is a powerful tool for optimizing the performance of relaying networks, offer- ing an elegant balance between the data rate enhance- ment, outage probability reduction, and symbol error rate minimization, thereby rendering it indispensable for various communication applications, from wireless sensor networks to next-generation wireless systems. 2 EAI Endorsed Transactions on Scalable Information Systems Online First 2. System model Then, the probability density functions (PDFs) of ωSRη and ωRηD are respectively expressed as EωSRη n ln(1 + ωSRη) o as EωSRη {ln(1 + ωSRη)} = Z ∞ 0 1 α1 e−ω α1 ln(1 + ω)dr (7) = Z ∞ 0 −ln(1 + ω)de−ω α1 (8) = e 1 α1 Z ∞ 1 α1 e−u 1 u du (9) = e 1 α1 E1( 1 α1 ), (10) fωSRη (ω) = 1 α1 e−ω α1 V(ω), (2) fωRη D(ω) = M α2 M−1 X n=0 M −1 n ! (−1)ne−(n+1)ω α 2V(ω), (3) (2) (8) (10) where V(·) is the unit step function, and α1 and α2 are the average SNR of ωSRη and ωRηD, respectively. where E1(x) = R ∞ x 1 xe−xdx is the exponential integral function. We further utilize the PDF of ωSRη in eq. (3), and derive EωRη D n ln(1 + ωRηD) o as 3. Capacity Analysis 3. Capacity Analysis EωRη D n ln(1 + ωRηD) o (11) = M α2 M−1 X n=0 M −1 n ! (−1)n Z ∝ 0 e−(n+1)ω α2 ln (1 + ω)dω (12) = M M−1 X n=0 M −1 n !(−1)n n + 1 e n+1 α2 Z ∝ n+1 α2 e−u 1 u du (13) = M M−1 X n=0 M −1 n !(−1)n n + 1 e n+1 α2 E1(n + 1 α2 ). (14) EωRη D n ln(1 + ωRηD) o (11) (11) = M α2 M−1 X n 0 M −1 n ! (−1)n Z ∝ 0 e−(n+1)ω α2 ln (1 + ω)dω (12) Capacity analysis in the context of channel-noise- assisted cooperative IoT networks is a fundamental aspect of evaluating the network performance and efficiency. This analysis involves the determination of the maximum achievable data rate, or capacity, under specific network conditions. By taking into account the factors such as the number of relays, channel impairments, signal-to-noise ratios, and modulation schemes, we can assess the network ability to transmit data reliably and efficiently. Capacity analysis aids in understanding the limits of information transfer within the network and provides valuable insights for optimizing the system. It is a critical tool for designing and fine-tuning relaying IoT networks, ensuring that they meet the data rate requirements while maintaining a balance between the spectral efficiency and reliability, ultimately enabling robust and high- performing communication in IoT applications. 2. System model The ergodic capacity of the relaying network with the relay selection is written as = M M−1 X n=0 M −1 n !(−1)n n + 1 e n+1 α2 Z ∝ n+1 α2 e−u 1 u du (13) = M M−1 X n=0 M −1 n !(−1)n n + 1 e n+1 α2 E1(n + 1 α2 ). (14) (13) (14) Now, we turn to derive EωSRη +ωRη D n ln(1 + ωSRη + ωRηD) o of eq. (4). For this, we first derive the pdf of ωSRη + ωRηD. Let ⊗denote the convolutional operation, and then the PDF of ωSRη + ωRηD can be derived as Now, we turn to derive EωSRη +ωRη D n ln(1 + ωSRη + ωRηD) o of eq. (4). For this, we first derive the pdf of ωSRη + ωRηD. Let ⊗denote the convolutional operation, and then the PDF of ωSRη + ωRηD can be derived as Now, Now, we turn to derive EωSRη +ωRη D n ln(1 + ωSRη + ωRηD) o of eq. (4). For this, we first derive the pdf of ωSRη + ωRηD. Let ⊗denote the convolutional operation, and then the PDF of ωSRη + ωRηD can be derived as fωSRη +ωRη D(ω) = fωSRη (ω) ⊗fωRη D(ω) (15) = Z ω 0 fωM(x)fωSRη (r −x)dx (16) = Z ω 0 M α2 M−1 X n=0 M −1 n ! (−1)ne−(n+1)x α2 1 α1 e−ω−x α1 dx (17) = M α1α2 M−1 X n=0 M −1 n ! (−1)ne−ω α1 Z ω 0 e−( n+1 α2 −1 α1 )xdx. (18) fωSRη +ωRη D(ω) = fωSRη (ω) ⊗fωRη D(ω) (15) = Z ω 0 fωM(x)fωSRη (r −x)dx (16) (15) (16) = Z ω 0 M α2 M−1 X n=0 M −1 n ! (−1)ne−(n+1)x α2 1 α1 e−ω−x α1 dx (17) C = 1 2EωRS{log2(1 + ωRS)}. (4) = M α1α2 M−1 X n=0 M −1 n ! (−1)ne−ω α1 Z ω 0 e−( n+1 α2 −1 α1 )xdx. (18) (4) Based on eq. (1), we write eq. (4) as Based on eq. (1), we write eq. 2. System model Fig. 1 provides a comprehensive illustration of the system model for channel-noise-assisted cooperative IoT networks. Within this framework, we have a configuration consisting of a single transmitting source, denoted as S, a set of M relays, and a designated destination labeled as D. This network operates on a dual-hop basis, where the source node S initially dispatches a signal to one of the M relays. Subsequently, the selected relay, denoted as m and belonging to the set M, takes charge of transmitting the signal to the destination D. It is essential to note that, for this specific network, we have employed the amplify-and-forward (AF) relaying protocol. Moreover, the relay selection process hinges on identifying the relay within the set M that exhibits the highest signal-to-noise ratio (SNR) during the second hop of the transmission process. Then, the end-to-end SNR of the cooperative networks with relay selection (RS) is expressed as This paper explores the integration of IoT transmis- sion technology enriched by a set of M relays to enhance the monitoring of electrical equipment. To optimize the system performance, we implement the partial relay selection, often referred to as selection combining. Our investigation delves deep into system performance analysis, primarily focusing on the quantification of the advantages stemming from the integration of partial relay selection in IoT transmission for monitoring the status of electrical equipment. Our analytical approach facilitates a comprehensive assessment of system effi- ciency, taking into account the transmission data rate. Through our analysis, we endeavor to offer valuable insights into the trade-offs and benefits associated ωRS = ωSRηωRηD 1 + ωSRη + ωRηD , (1) (1) where Rη denotes the selected relay of total M relays based on ωη = maxn∈M{ωRnD}, and ωRnD represents the instantaneous SNR of the wireless channel from relay Rn to the destination D. Notation ωSRη denotes the 2 EAI Endorsed Transactions on Scalable Information Systems Online First 2 Research of IoT Technology Based Online Status Monitoring Then, we use the PDF of ωSRη in eq. (2), and derive EωSRη n ln(1 + ωSRη) o as instantaneous SNR from source S to relay Rη. 2. System model (−1)nM " α1e 1 α1 E1( 1 α1 ) − α2 n + 1e n+1 α2 E1(n + 1 α2 ) # + [1 −unm(n −m)] M −1 n !(−1)nM α2 " α1 + (α1 −1)e 1 α1 E1( 1 α1 ) # . (20) EωSRM +ωRη D n ln (1 + ωSRM + ωRηD) o C = 1 2 ln 2e 1 α1 E1( 1 α1 ) + 1 2 ln 2M M−1 X n=0 M −1 n !(−1)n n + 1 e n+1 α2 E1(n + 1 α2 ) + [1 −unm(n −m)] M −1 n !(−1)nM α2 " α1 + (α1 −1)e 1 α1 E1( 1 α1 ) # ) − 1 2 ln 2  M−1 X n=0 unm α1 M −1 n ! (−1)nM " α1e 1 α1 E1( 1 α1 ) − α2 n + 1e n+1 α2 E1(n + 1 α2 ) #  =e 1 α1 E1( 1 α1 ) {Q1} + M−1 X n=0 e n+1 α2 E1(n + 1 α2 ) {Q2} + {Q3} , where C = 1 2 ln 2e 1 α1 E1( 1 α1 ) + 1 2 ln 2M M−1 X n=0 M −1 n !(−1)n n + 1 e n+1 α2 E1(n + 1 α2 ) + [1 −unm(n −m)] M −1 n !(−1)nM α2 " α1 + (α1 −1)e 1 α1 E1( 1 α1 ) # ) − 1 2 ln 2  M−1 X n=0 unm α1 M −1 n ! (−1)nM " α1e 1 α1 E1( 1 α1 ) − α2 n + 1e n+1 α2 E1(n + 1 α2 ) #  M 1 = 1 2 ln 2e 1 α1 E1( 1 α1 ) + 1 2 ln 2M M−1 X n=0 M −1 n !(−1)n n + 1 e n+1 α2 E1(n + 1 α2 ) + [1 −unm(n −m)] M −1 n !(−1)nM α2 " α1 + (α1 − − 1 2 ln 2  M−1 X n=0 unm α1 M −1 n ! 2. System model (−1)nM " α1e 1 α1 E1( 1 α1 ) − α2 n + 1e n+1 α2 E1(n + 1 α2 ) #  =e 1 α1 E1( 1 α1 ) {Q1} + M−1 X n=0 e n+1 α2 E1(n + 1 α2 ) {Q2} + {Q3} , where (21) unm =  1 n−m if n , m 0 if n = m (25) the desired network performance. Key parameters, including the number of relays, transmit power levels, noise characteristics, and relay selection criteria should be carefully configured to strike a balance between data rate, outage probability, and symbol error rate while ensuring efficient and reliable data transmission. These settings play a pivotal role in tailoring the network to meet the specific requirements of the application and address challenges like channel impairments and interference. A well-optimized parameter configuration is essential to harness the full potential of relaying networks and provide robust and high-performing communication solutions for IoT and beyond. (25) Then, fωSRη +ωRη D is obtained by eq. (19). Based on eq. (19), we further derive EωSRη +ωRη D n ln(1 + ωSRη + ωRηD) o , which is expressed in eq. (20). According to eqs. (7), (11), and (20), we derive the ergodic capacity of eq. (4), which is presented in eq. (21). 2. System model (4) as Let m = α1 α2 −1, and unm denotes Let m = α1 α2 −1, and unm denotes Let m = α1 α2 −1, and unm denotes fωSRM +ωRη D(ω) (22) (22) C = 1 2EωSC  log2 (1 + ωRηD)(1 + ωSRη) 1 + ωSRη + ωRηD  (5) = 1 2 ln 2EωSRη n ln(1 + ωSRη) o + 1 2 ln 2EωRη D n ln(1 + ωRηD) o − 1 2 ln 2EωSRη +ωRη D n ln(1 + ωSRη + ωRηD) o . (6) fωSRM +ωRη D(ω) (22) = M α1α2 M−1 X n=0 M −1 n ! (−1)ne−ω α1 Z ω 0 dx (23) = M α1α2 M−1 X n=0 M −1 n ! (−1)ne−ω α1 ω (24) (5) (24) 3 EAI Endorsed Transactions on Scalable Information Systems Online First 3 EAI Endorsed Transactions on Scalable Information Systems Online First Y. Ma et.al fωSRη +ωRη D(ω) = M α1α2  M−1 X n=0 unm(n −m) M −1 n ! (−1)ne−ω α1 Z ω 0 e−( n+1 α2 −1 α1 )xdx +  1 −unm(n −m)  M −1 n ! (−1)ne−ω α1 ω  = M α1α2  M−1 X n=0 unm(n −m) M −1 n ! (−1)ne−ω α1 e−( n+1 α2 −1 α1 )x n+1 α2 −1 α1 |0 ω +  1 −unm(n −m)  M −1 n ! (−1)ne−ω α1 ω  = M α1α2  M−1 X n=0 unm(n −m) M −1 n ! (−1)ne−ω α1 1 −e−( n+1 α2 −1 α1 )ω α1(n+1)−α2 α1α2 +  1 −unm(n −m)  M −1 n ! (−1)ne−ω α1 ω  = M−1 X n=0 unm α1 M −1 n ! (−1)nM  e−ω α1 −e−n+1 α2 ω +  1 −unm(n −m)  M −1 n !(−1)nM α1α2 e−ω α1 ω. (19) (19) (19) EωSRM +ωRη D n ln (1 + ωSRM + ωRηD) o = M−1 X n=0 unm α1 M −1 n ! (−1)nM " α1e 1 α1 E1( 1 α1 ) − α2 n + 1e n+1 α2 E1(n + 1 α2 ) # + [1 −unm(n −m)] M −1 n !(−1)nM α2 " α1 + (α1 −1)e 1 α1 E1( 1 α1 ) # . (20) EωSRM +ωRη D n ln (1 + ωSRM + ωRηD) o = M−1 X n=0 unm α1 M −1 n ! 4. Simulations Results and Discussions α2 (dB) 0 5 10 15 20 Sim M=1 0.1392 0.2331 0.3154 0.3737 0.40 Ana M=1 0.1390 0.2323 0.3165 0.3735 0.40 Sim M=2 0.1870 0.2902 0.3649 0.4041 0.42 Ana M=2 0.1869 0.2894 0.3646 0.4043 0.42 Sim M=3 0.2119 0.3144 0.3814 0.4135 0.42 Ana M=3 0.2122 0.3141 0.3808 0.4122 0.42 f Table 2 Numerical effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. α1 (dB) 0 5 10 15 20 Sim M=1 0.1387 0.2324 0.3164 0.3750 0.40 Ana M=1 0.1390 0.2323 0.3165 0.3735 0.40 Sim M=2 0.1863 0.3170 0.4355 0.5158 0.56 Ana M=2 0.1869 0.3162 0.4351 0.5170 0.56 Sim M=3 0.2118 0.3619 0.5019 0.5981 0.65 Ana M=3 0.2122 0.3618 0.5013 0.5984 0.65 Fig. 2 and Table 1 present the effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. From Fig. 2 and Table 1, we can find that the system capacity increases when α1 increases. This is because that the increasing value of α1 can effectively improve the transmission performance of the first hop, thus increasing the system capacity. Moreover, the system capacity also increases as the value of M increases. This is due to the relay selection, where a larger M means more available relays to be chosen, thereby improving the SNR of the second hop and further increasing the capacity of the whole system. In further, the simulated results match the analytical results very well, which verifies the effectiveness of the derived closed-form expressions of the ergodic capacity of the considered network. table, it is evident that the system capacity exhibits a notable upsurge as α2 increases. This observed phenomenon can be attributed to the enhancement of the transmission capacity in the second hop of the communication process, contributing to an overall boost in the system capacity. Moreover, the system capacity demonstrates growth in proportion to an increase in the number of relays (M) due to the relay selection employed. Additionally, the remarkable agreement between the simulated and analytical results serves as strong validation for the accuracy of the derived expressions governing the system ergodic capacity, further reinforcing the reliability of our analysis and the practical applicability of the findings. 4. Simulations Results and Discussions In the context of relaying networks with partial relay selection, we need to set some parameters for achieving EAI Endorsed Transactions on Scalable Information Systems Online First EAI Endorsed Transactions on Scalable Information Systems Online First 4 Research of IoT Technology Based Online Status Monitoring 0 5 10 15 20 First-hop avarage SNR α1 (dB) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ergodic capacity (bps/Hz) Simulation M=1 Simulation M=2 Simulation M=3 Analysis M=1 Analysis M=2 Analysis M=3 Figure 2. Effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. 0 5 10 15 20 Second-hop avarage SNR α2 (dB) 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Ergodic capacity (bps/Hz) Simulation M=1 Simulation M=2 Simulation M=3 Analysis M=1 Analysis M=2 Analysis M=3 Figure 3. Effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. 0 5 10 15 20 Second-hop avarage SNR α2 (dB) 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Ergodic capacity (bps/Hz) Simulation M=1 Simulation M=2 Simulation M=3 Analysis M=1 Analysis M=2 Analysis M=3 Figure 3. Effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. 0 5 10 15 20 First-hop avarage SNR α1 (dB) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ergodic capacity (bps/Hz) Simulation M=1 Simulation M=2 Simulation M=3 Analysis M=1 Analysis M=2 Analysis M=3 Figure 3. Effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. Figure 2. Effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. f Table 1 Numerical effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. f Table 1 Numerical effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. f Table 2 Numerical effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. f Table 2 Numerical effect of the average SNR α1 on the ergodic capacity of the system, where α2 = 0 dB. 4. Simulations Results and Discussions Table 3 Numerical impact of the number of relays M on the system ergodic capacity under different values of α1, where α2 = 0 dB. M 1 2 3 4 5 Sim α1=0 dB 0.1392 0.1866 0.2117 0.2276 0.23 Ana α1=0 dB 0.1390 0.1869 0.2122 0.2282 0.23 Sim α1=10 dB 0.3155 0.4362 0.5015 0.5439 0.57 Ana α1=10 dB 0.3165 0.4351 0.5013 0.5452 0.57 Sim α1=20 dB 0.4067 0.5620 0.6535 0.7138 0.75 Ana α1=20 dB 0.4048 0.5624 0.6527 0.7137 0.75 1 2 3 4 5 The number of relays M 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Ergodic capacity (bps/Hz) Simulation α1=0 dB Simulation α1=10 dB Simulation α1=20 dB Analysis α1=0 dB Analysis α1=10 dB Analysis α1=20 dB 3 The number of relays M Figure 4. Impact of the number of relays M on the system ergodic capacity under different values of α1, where α2 = 0 dB. f 1 2 3 4 5 The number of relays M 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Ergodic capacity (bps/Hz) Simulation α2=0 dB Simulation α2=10 dB Simulation α2=20 dB Analysis α2=0 dB Analysis α2=10 dB Analysis α2=20 dB Figure 5. Impact of the number of relays M versus the system ergodic capacity under different values of α2, where α1 = 0 dB. f 1 2 3 4 5 The number of relays M 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Ergodic capacity (bps/Hz) Simulation α2=0 dB Simulation α2=10 dB Simulation α2=20 dB Analysis α2=0 dB Analysis α2=10 dB Analysis α2=20 dB Table 4 Numerical impact of the number of relays M versus the system ergodic capacity under different values of α2, where α1 = 0 dB. M 1 2 3 4 5 Sim α2=0 dB 0.1390 0.1875 0.2128 0.2269 0.24 Ana α2=0 dB 0.1390 0.1869 0.2122 0.2282 0.23 Sim α2=10 dB 0.3167 0.3640 0.3810 0.3901 0.39 Ana α2=10 dB 0.3165 0.3646 0.3808 0.3888 0.39 Sim α2=20 dB 0.4045 0.4221 0.4239 0.4271 0.42 Ana α2=20 dB 0.4048 0.4210 0.4241 0.4254 0.42 Table 4 Numerical impact of the number of relays M versus the system ergodic capacity under different values of α2, where α1 = 0 dB. 4. Simulations Results and Discussions This insight underscores the critical role of SNR, relay selection, and the number of relays in shaping the network capacity, which has significant implications for optimizing the performance of cooperative communication systems. Fig. 3 and Table 2 show how α2 affects the ergodic capacity of the system, while holding α1 at a constant value of 0 dB. As depicted in this figure and EAI Endorsed Transactions on Scalable Information Systems Online First EAI Endorsed Transactions on Scalable Information Systems Online First 5 Table 3 Numerical impact of the number of relays M on the system ergodic capacity under different values of α1, where α2 = 0 dB. M 1 2 3 4 5 Sim α1=0 dB 0.1392 0.1866 0.2117 0.2276 0.23 Ana α1=0 dB 0.1390 0.1869 0.2122 0.2282 0.23 Sim α1=10 dB 0.3155 0.4362 0.5015 0.5439 0.57 Ana α1=10 dB 0.3165 0.4351 0.5013 0.5452 0.57 Sim α1=20 dB 0.4067 0.5620 0.6535 0.7138 0.75 Ana α1=20 dB 0.4048 0.5624 0.6527 0.7137 0.75 Table 4 Numerical impact of the number of relays M versus the system ergodic capacity under different values of α2, where α1 = 0 dB. M 1 2 3 4 5 Sim α2=0 dB 0.1390 0.1875 0.2128 0.2269 0.24 Ana α2=0 dB 0.1390 0.1869 0.2122 0.2282 0.23 Sim α2=10 dB 0.3167 0.3640 0.3810 0.3901 0.39 Ana α2=10 dB 0.3165 0.3646 0.3808 0.3888 0.39 Sim α2=20 dB 0.4045 0.4221 0.4239 0.4271 0.42 Ana α2=20 dB 0.4048 0.4210 0.4241 0.4254 0.42 Fig. 5 and Table 4 shows the impact of the number of relays M on the system ergodic capacity under different values of α2, where α1 = 0 dB. It is evident from Fig. 5 and Table 4 that the system ergodic capacity increases with the increasing value of M irrespective of Y. Ma et.al Table 3 Numerical impact of the number of relays M on the system ergodic capacity under different values of α1, where α2 = 0 dB. 1 2 3 4 5 The number of relays M 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Ergodic capacity (bps/Hz) Simulation α1=0 dB Simulation α1=10 dB Simulation α1=20 dB Analysis α1=0 dB Analysis α1=10 dB Analysis α1=20 dB Figure 4. Impact of the number of relays M on the system ergodic capacity under different values of α1, where α2 = 0 dB. 4. Simulations Results and Discussions M 1 2 3 4 5 Sim α2=0 dB 0.1390 0.1875 0.2128 0.2269 0.24 Ana α2=0 dB 0.1390 0.1869 0.2122 0.2282 0.23 Sim α2=10 dB 0.3167 0.3640 0.3810 0.3901 0.39 Ana α2=10 dB 0.3165 0.3646 0.3808 0.3888 0.39 Sim α2=20 dB 0.4045 0.4221 0.4239 0.4271 0.42 Ana α2=20 dB 0.4048 0.4210 0.4241 0.4254 0.42 Table 4 Numerical impact of the number of relays M versus the system ergodic capacity under different values of α2, where α1 = 0 dB. Figure 5. Impact of the number of relays M versus the system ergodic capacity under different values of α2, where α1 = 0 dB. f Fig. 4 and Table 3 present the impact of the number of relays M on the system ergodic capacity under different average SNR α1, where α2 = 0 dB. As described in Fig. 4 and Table 3, we can find that the ergodic capacity increases with the increasing value of M regardless of the value of α1. This is due to the fact that increasing the number of relays M means that more available relays can be chosen in the dual-hop network, which greatly improves the transmission performance and therefore increases the system ergodic capacity. Moreover, the system performance performs better with a higher average SNR α1, as a higher α1 can help improve the capacity of the first-hop. Specifically, when M = 5, the ergodic capacity of α1 = 20 dB is highest, about 28% and 71% higher than those of α1 = 10 dB and α1 = 0 dB. Additionally, the simulated curves match the analytic curves very well, which verifies the effectiveness of the derived closed-form expression of the system ergodic capacity. Fig. 5 and Table 4 shows the impact of the number of relays M on the system ergodic capacity under different values of α2, where α1 = 0 dB. It is evident from Fig. 5 and Table 4 that the system ergodic capacity increases with the increasing value of M irrespective of the specific α2 setting. This is because that increasing the number of relays M means that more available relays can be chosen in the dual-hop network, which leads to substantial improvements in transmission EAI Endorsed Transactions on Scalable Information Systems Online First EAI Endorsed Transactions on Scalable Information Systems Online First 6 Research of IoT Technology Based Online Status Monitoring [4] L. Liu, J. Zhang, S. Song, and K. 4. Simulations Results and Discussions B. Letaief, “Hierarchical federated learning with quantization: Convergence anal- ysis and system design,” IEEE Trans. Wirel. Commun., vol. 22, no. 1, pp. 2–18, 2023. performance and therefore increases the system ergodic capacity. Moreover, it is worth noting that a higher α2 results in a better system performance in ergodic capacity. Specifically, when M = 4, the ergodic capacity of α2 = 20 dB yields the highest ergodic capacity, about 12.5% and 46.2% higher than those of α2 = 10 dB and α2 = 0 dB. Additionally, the simulated curves closely align with the analytic curves, thereby validating the effectiveness of the derived closed-form expressions for the system ergodic capacity. [5] Y. Zheng, C. Wang, R. Yang, L. Yu, F. Lai, J. Huang, R. Feng, C. Wang, C. Li, and Z. Zhong, “Ultra-massive MIMO channel measurements at 5.3 ghz and a general 6g channel model,” IEEE Trans. Veh. Technol., vol. 72, no. 1, pp. 20–34, 2023. [6] F. L. Andrade, M. A. T. Figueiredo, and J. Xavier, “Distributed banach-picard iteration: Application to distributed parameter estimation and PCA,” IEEE Trans. Signal Process., vol. 71, pp. 17–30, 2023. 5. conclusion [7] Q. Wang, S. Cai, Y. Wang, and X. Ma, “Free-ride feedback and superposition retransmission over LDPC coded links,” IEEE Trans. Commun., vol. 71, no. 1, pp. 13–25, 2023. This paper studied the integration of IoT transmission technology with multiple relays, for the enhancement of status monitoring for electrical equipment. Through an analytical analysis on the system data rate, we have demonstrated the advantages of employing partial relay selection in IoT-based electrical equipment monitoring. The results highlight the trade-offs between the system efficiency and performance, shedding light on the benefits of this approach for optimizing data transmission. By quantifying the improvements achieved through partial relay selection and assessing the impact of varying numbers of relays, our research can help reinforce the reliability of status monitoring in electrical systems. This work can serve as a significant step forward in advancing the capabilities of IoT solutions in the field of electrical equipment maintenance, providing a foundation for more efficient and effective monitoring systems in the future. [8] Z. Xie, W. Chen, and H. V. Poor, “A unified framework for pushing in two-tier heterogeneous networks with mmwave hotspots,” IEEE Trans. Wirel. Commun., vol. 22, no. 1, pp. 19–31, 2023. [9] T. Häckel, P. Meyer, F. Korf, and T. C. Schmidt, “Secure time-sensitive software-defined networking in vehicles,” IEEE Trans. Veh. Technol., vol. 72, no. 1, pp. 35–51, 2023. [10] Y. Song, Z. Gong, Y. Chen, and C. Li, “Tensor- based sparse bayesian learning with intra-dimension correlation,” IEEE Trans. Signal Process., vol. 71, pp. 31– 46, 2023. [11] H. Wan and A. Nosratinia, “Short-block length polar- coded modulation for the relay channel,” IEEE Trans. Commun., vol. 71, no. 1, pp. 26–39, 2023. [12] G. Zhang, C. Shen, Q. Shi, B. Ai, and Z. Zhong, “Aoi minimization for WSN data collection with periodic updating scheme,” IEEE Trans. Wirel. Commun., vol. 22, no. 1, pp. 32–46, 2023. 5.1. Copyright The Copyright was licensed to EAI. The Copyright was licensed to EAI. [16] B. Han, V. Sciancalepore, Y. Xu, D. Feng, and H. D. Schotten, “Impatient queuing for intelligent task offloading in multiaccess edge computing,” IEEE Trans. Wirel. Commun., vol. 22, no. 1, pp. 59–72, 2023. Acknowledgment [13] A. Verma and R. Shrestha, “Low computational- complexity soms-algorithm and high-throughput decoder architecture for QC-LDPC codes,” IEEE Trans. Veh. Technol., vol. 72, no. 1, pp. 66–80, 2023. This work was supported by State Grid Sichuan Electric Power Company Science and Technology Project support (521901170004), where the project name is Hydropower Station operation and maintenance assistant decision system project based on real-time data. [14] K. N. Ramamohan, S. P. Chepuri, D. F. Comesaña, and G. Leus, “Self-calibration of acoustic scalar and vector sensor arrays,” IEEE Trans. Signal Process., vol. 71, pp. 61–75, 2023. [15] Q. Lu, S. Li, B. Bai, and J. Yuan, “Spatially-coupled faster- than-nyquist signaling: A joint solution to detection and code design,” IEEE Trans. Commun., vol. 71, no. 1, pp. 52–66, 2023. References [1] Z. Huang, L. Bai, X. Cheng, X. Yin, P. E. Mogensen, and X. Cai, “A non-stationary 6g V2V channel model with continuously arbitrary trajectory,” IEEE Trans. Veh. Technol., vol. 72, no. 1, pp. 4–19, 2023. [1] Z. Huang, L. Bai, X. Cheng, X. Yin, P. E. Mogensen, and X. Cai, “A non-stationary 6g V2V channel model with continuously arbitrary trajectory,” IEEE Trans. Veh. Technol., vol. 72, no. 1, pp. 4–19, 2023. [17] X. Zhou, D. He, M. K. Khan, W. Wu, and K. R. Choo, “An efficient blockchain-based conditional privacy- preserving authentication protocol for vanets,” IEEE Trans. Veh. Technol., vol. 72, no. 1, pp. 81–92, 2023. pp [2] A. E. Haddad and L. Najafizadeh, “The discriminative discrete basis problem: Definitions, algorithms, bench- marking, and application to brain’s functional dynam- ics,” IEEE Trans. Signal Process., vol. 71, pp. 1–16, 2023. [18] D. Malak and M. Médard, “A distributed computation- ally aware quantizer design via hyper binning,” IEEE Trans. Signal Process., vol. 71, pp. 76–91, 2023. g pp [3] R. Gabrys, S. Pattabiraman, and O. Milenkovic, “Recon- struction of sets of strings from prefix/suffix composi- tions,” IEEE Trans. Commun., vol. 71, no. 1, pp. 3–12, 2023. [19] Y. Xiong, S. Sun, L. Liu, Z. Zhang, and N. Wei, “Performance analysis and bit allocation of cell-free massive MIMO network with variable-resolution adcs,” EAI Endorsed Transactions on Scalable Information Systems Online First 7 Y. Ma et.al IEEE Trans. Commun., vol. 71, no. 1, pp. 67–82, 2023. non-homogeneous poisson process,” IEEE Trans. Signal Process., vol. 71, pp. 105–120, 2023. pp [20] J. Shao, Y. Mao, and J. Zhang, “Task-oriented communi- cation for multidevice cooperative edge inference,” IEEE Trans. Wirel. Commun., vol. 22, no. 1, pp. 73–87, 2023. pp [24] F. Hu, Y. Deng, and A. H. Aghvami, “Scalable multi- agent reinforcement learning for dynamic coordinated multipoint clustering,” IEEE Trans. Commun., vol. 71, no. 1, pp. 101–114, 2023. pp [21] H. H. López and G. L. Matthews, “Multivariate goppa codes,” IEEE Trans. Inf. Theory, vol. 69, no. 1, pp. 126– 137, 2023. [25] H. Hui and W. Chen, “Joint scheduling of proactive pushing and on-demand transmission over shared spectrum for profit maximization,” IEEE Trans. Wirel. Commun., vol. 22, no. 1, pp. 107–121, 2023. [22] H. Yao, X. Li, and X. Yang, “Physics-aware learning- based vehicle trajectory prediction of congested traffic in a connected vehicle environment,” IEEE Trans. Veh. Technol., vol. 72, no. 1, pp. EAI Endorsed Transactions on Scalable Information Systems Online First non-homogeneous poisson process,” IEEE Trans. Signal Process., vol. 71, pp. 105–120, 2023. References 102–112, 2023. [26] W. Yu, Y. Xi, X. Wei, and G. Ge, “Balanced set codes with small intersections,” IEEE Trans. Inf. Theory, vol. 69, no. 1, pp. 147–156, 2023. [23] Q. Li, R. Gan, J. Liang, and S. J. Godsill, “An adaptive and scalable multi-object tracker based on the EAI Endorsed Transactions on Scalable Information Systems Online First 8 8
W2914733550.txt
https://www.beilstein-journals.org/bjnano/content/pdf/2190-4286-10-32.pdf
en
Development of an anti-pollution coating process technology for the application of an on-site PV module
Beilstein journal of nanotechnology
2,019
cc-by
2,323
Development of an anti-pollution coating process technology for the application of an on-site PV module Sejin Jung1, Wonseok Choi*1, Jung Hyun Kim2 and Jang Myoun Ko3 Full Research Paper Address: 1Department of Electrical Engineering, Hanbat National University, Dajeon 34158, Republic of Korea, 2Department of Advanced Materials Science and Engineering, Hanbat National University, Daejeon 34158, Republic of Korea and 3Department of Chemical Engineering and Biotechnology, Hanbat National University, Daejeon 34158, Republic of Korea Email: Wonseok Choi* - wschoi@hanbat.ac.kr Open Access Beilstein J. Nanotechnol. 2019, 10, 332–336. doi:10.3762/bjnano.10.32 Received: 21 September 2018 Accepted: 04 January 2019 Published: 01 February 2019 This article is part of the thematic issue "Chemical thin coating methods for functional nanomaterials". Guest Editor: J. Bachmann * Corresponding author Keywords: annealing; anti-pollution; functional film; gas torch; PV module © 2019 Jung et al.; licensee Beilstein-Institut. License and terms: see end of document. Abstract This study aimed to apply annealing processes during the coating of photovoltaic (PV) module glasses to PV modules already installed through an easy and simple procedure. Three types of annealing treatments were applied to PV module glasses, i.e., furnace, rapid thermal annealing (RTA) and torch. Among these, torch annealing, which can be easily carried out at PV module installation sites, was applied to PV module glasses using different numbers of repetition. Light transmittance, contact angle, antipollution characteristics, adhesion and hardness of the functional coating films after using different annealing treatment times and methods were measured, and it was confirmed that these characteristics varied depending on the annealing treatment times and methods. Through this, it was possible to optimize the process conditions that provide excellent anti-pollution characteristics and could be easily utilized at on-site PV modules. Introduction The worldwide consumption of fossil fuels has caused global warming through emitting carbon dioxide (CO2) and greenhouse gases. To address this problem, continuous efforts have been made to reduce CO2 emissions through the Paris Agreement in December 2015. In addition, as fossil fuels are expected to be depleted in approximately 130 years, the development of renewable energy sources that can replace fossil energy is required. Solar energy represents the highest proportion among the renewable energy sources, and it can produce clean electricity without noise or by-products [1,2]. Photovoltaic (PV) modules are installed outdoors and are thus exposed to various external surface pollutants, such as dust, yellow dust, animal excrement and rainfall sediment. These pollutants prevent sunlight from entering the PV modules and thereby degrade the power generation efficiency. Therefore, various studies have been conducted of late to effectively prevent the surface pollution of PV modules [3,4]. The PV module surface coating materials that are currently under research must have anti-pollution 332 Beilstein J. Nanotechnol. 2019, 10, 332–336. functions and must endure severe temperature differences, and harsh physical (e.g., external shock) and chemical environments (e.g., animal excrement). They must also have a light transmittance of 95% or higher [5,6]. Large solar power plants are currently being installed for power generation, and a huge amount of time and cost is required for maintenance, including surface cleaning. Therefore, the introduction of a technology capable of easily removing pollutants using natural water will significantly improve the economic efficiency of the maintenance of solar power generation systems [7]. Although a selfcleaning coating technique using a photocatalyst has been developed, the durability is poor due to low adhesion and hardness. Also, production is very difficult, an energy source that causes catalytic action is needed, and the supply is low. In addition, anti-fogging and anti-condensation technologies have been applied to module production, but their anti-pollution effects are not significant [8,9]. In this study a new, easily usable coating technology is proposed that could be applied to PV modules already installed in the field to effectively improve the self-cleaning of the surfaces of PV modules. This technology should have the advantage of being applicable directly at the PV module installation site, without having to bring the module to the factory for modification. Before applying the coating technology to PV modules, glass substrates for PV modules were coated with a hydrophilic silica-based eco-friendly nanomaterial, and the coating films were thermally annealed using either a furnace, RTA or a torch. The annealing treatment that uses a torch was applied using different periods of time. For the fabricated specimens, the contact angle, anti-pollution characteristics, hardness, and adhesion were measured. The process conditions were optimized by analyzing the measurement results. Experimental The coating solution that was used to improve the anti-pollution characteristics of the PV modules contained silicon dioxide (SiO2), lithium (Li), and potassium (K). The viscosity, density, and specific gravity (referring to the density of water) of the coating solution were 0.01–0.03 kg/m·s, 1.1 g/cm 3 , and 1.13 ± 0.05, respectively. The solution can be used to coat various materials, such as metals, ceramics, and glass [9]. Before coating the glass slide substrates, the substrates were subjected to ultrasonic cleaning for 10 min in, consecutively, trichloroethylene, acetone, methanol and deionized water (DI water). The substrates were coated with the coating solution using a brush. After being dried at room temperature for 20 min, the substrates were thermally annealed using a furnace (L-Series, Jeio Tech Co., South Korea), RTA (RTP-1200, Nextron Co., South Korea), and a gas torch (KT-2211, Kovea Co., South Korea, using butane gas). The length of the flame was about 10 cm, the distance between the flame and the specimen was about 5 cm, and the temperature was about 300 °C. In addition, the annealing treatment that uses a gas torch, which can be easily utilized to surfaces of installed or operating PV modules, was applied from one to five times. The contact angles of the fabricated functional coating films were measured using a contact angle analyzer (Phoenix 300 Touch, S.E.O. Co., South Korea). The anti-pollution characteristics were measured using permanent markers instead of actual pollutants. This method is useful for checking the level of pollutant removal. The hardness was measured using a hardness tester (CT-PC1, CORETECH Co., South Korea) equipped with pencils with hardness values from H to 9H (Mitsubishipencil Co., Ltd., Japan) in accordance with ASTM D3363 of the American Society for Testing and Materials (ASTM). The optical characteristics were measured using the integrating sphere of a UV–visible spectrophotometer (Mega 700, Scinco Co., Ltd., South Korea). Results and Discussion The light transmittance measurement results of the fabricated functional coatings are summarized in Figure 1. The light transmittance of the coating film thermally annealed at 300 °C using a furnace was determined to be 95.4%, and that of the coating film thermally annealed at 300 °C using RTA, 96.5%. When annealing treatment was performed one to five times using a torch, the light transmittance was found to be 91.3, 94.7, 98.3, 98.5, and 98.2%, respectively. The light transmittance increased for the first three times of torch annealing. After the fourth and fifth treatment the transmittance was similar to that after the third annealing. Figure 2 and Figure 3 show the contact angle characteristics according to the annealing treatment method. The contact angle was 15.4° when the coating film was thermally annealed using a furnace, and it was 14.8° when the coating film was thermally annealed using RTA. When annealing treatment was performed one to three times using a torch, the contact angle was 24.3°, 15.5°, and 13.9°, respectively, but the contact angles in the fourth and fifth annealing treatments were similar to that in the third annealing treatment. Figure 4 shows the anti-pollution characteristics after annealing treatment. Black, red, and blue markings were applied on the glass slide substrates coated with the functional coating solution, using oil pens. After the markings were naturally dried and cleaned with water, the specimens thermally annealed using a furnace, RTA, and three to five torch applications showed excellent anti-pollution characteristics. In particular, the speci- 333 Beilstein J. Nanotechnol. 2019, 10, 332–336. Figure 1: Transmittance (a) according to the annealing treatment method and (b) according to the number of annealing treatment using a gas torch. Figure 2: Contact angles of the coating films fabricated using various annealing treatment methods: (a) naturally dried; (b) annealed with RTA; (c) annealed with a furnace; (d) annealed once with a gas torch; (e) annealed two times with a gas torch; (f) annealed three times with a gas torch; (g) annealed four times a with gas torch; and (h) annealed five times with a gas torch. Figure 3: Contact angle by (a) annealing treatment method and (b) annealing treatment times using a gas torch. 334 Beilstein J. Nanotechnol. 2019, 10, 332–336. Figure 4: Anti-pollution characteristics of the coating films fabricated using various annealing treatment methods: (a) naturally dried; (b) annealed with RTA; (c) annealed with a furnace; (d) annealed once with a gas torch; (e) annealed two times with a gas torch; (f) annealed three times with a gas torch; (g) annealed four times with a gas torch; and (h) annealed five times with a gas torch. mens thermally annealed using three to five torch applications exhibited the most excellent characteristics. Figure 5 and Figure 6 show the hardness and adhesion characteristics when annealing treatment was performed on the coating films using a furnace, RTA, and one to five torch applications. For the analysis of the characteristics, the hardness values of the fabricated films were measured using a pencil hardness tester, in accordance with the ASTM D3363 criteria, and the adhesion values were measured in accordance with the ASTM D3359 criteria. All the annealed coating films exhibited 5B adhesion and 9H hardness, while the coating film that had been dried at room temperature exhibited 2B adhesion and 7H hardness. Conclusion A functional coating was applied to glass slide substrates to improve their anti-pollution characteristics for application in PV Figure 5: Adhesion characteristics of the coating films fabricated using various annealing treatment methods: (a-1) naturally dried; (a-2) coating film after the adhesion test; (b-1) annealed; and (b-2) coating film after the adhesion test. Figure 6: Hardness characteristics of the coating films fabricated using various annealing treatment methods: (a-1) naturally dried; (a-2) coating film after the hardness test; (b-1) annealed; and (b-2) coating film after the hardness test. 335 Beilstein J. Nanotechnol. 2019, 10, 332–336. modules. After coating glass slide substrates made of the same material as glass substrates for PV modules with functional films, the results of naturally drying them or thermally annealing them with a furnace, RTA, or a torch were compared. In addition, annealing treatments with a torch were carried out one to five times to observe the changes in the sample characteristics. For the analysis of the characteristics of the fabricated specimens, light transmittance, contact angle, anti-pollution characteristics, hardness, and adhesion were measured. ORCID® iDs Sejin Jung - https://orcid.org/0000-0002-9180-6397 References 1. Tyagi, V. V.; Rahim, N. A. A.; Rahim, N. A.; Selvaraj, J. A. Renewable Sustainable Energy Rev. 2013, 20, 443–461. doi:10.1016/j.rser.2012.09.028 2. Cao, F.; Chen, K.; Zhang, J.; Ye, X.; Li, J.; Zou, S.; Su, X. Sol. Energy Mater. Sol. Cells 2015, 141, 132–138. doi:10.1016/j.solmat.2015.05.030 3. Li, X.; Zhou, Y.; Xue, L.; Huang, L. Technol. Forecast. Soc. Change The thermally annealed specimens showed 91.3% or higher light transmittance. In particular, when annealing using a torch three to four times, high light transmittance values of 98.3 and 98.5% were obtained. For the results of the contact angle analysis, the thermally annealed specimens showed a contact angle of 24.3° or lower. In particular, when annealing using a torch four to five times, the contact angles were 12.9 and 13.2°, respectively, indicating improvements in the hydrophilic characteristics. The analysis of the anti-pollution characteristics revealed that all the thermally annealed specimens exhibited improved anti-pollution characteristics. In particular, it was confirmed that the anti-pollution characteristics improved as the contact angle decreased. As for the measurement results of the hardness and adhesion, which are mechanical characteristics, all the thermally annealed substrates exhibited 9H hardness and 5B adhesion results, but the coating film that had been dried at room temperature showed 7H and 2B values. These results indicate that annealing treatment with a torch produces results similar to or better than those produced by annealing treatment with a furnace or RTA. Therefore, if the surfaces of PV modules installed outdoors are coated and thermally annealed with a torch, the annealing treatment process will be faster and easier. The results of this study confirm that the annealing treatment process using a torch can be applied directly to the installed PV module. On-site PV modules can be immediately processed without having to go to the factory, so it is expected that the amount of maintenance work will be reduced and that the economic efficiency will increase. 2015, 97, 205–222. doi:10.1016/j.techfore.2014.05.007 4. Liu, C.; Zhang, X.; Li, J.; He, Y.; Li, Z.; Li, H.; Guo, W.; Xie, W. Synth. Met. 2015, 204, 65–69. doi:10.1016/j.synthmet.2015.03.012 5. Isshiki, M.; Sichanugrist, P.; Abe, Y.; Oyama, T.; Odaka, H.; Konagai, M. Curr. Appl. Phys. 2014, 14, 1813–1818. doi:10.1016/j.cap.2014.10.021 6. Yu, W.; Shen, L.; Shen, P.; Meng, F.; Long, Y.; Wang, Y.; Lv, T.; Ruan, S.; Chen, G. Sol. Energy Mater. Sol. Cells 2013, 117, 198–202. doi:10.1016/j.solmat.2013.06.002 7. Shan, B.; Kang, H.; Choi, W.; Kim, J. H. Trans. Electr. Electron. Mater. 2017, 18, 148–150. 8. Park, J. T.; Kim, J. H.; Lee, D. Nanoscale 2014, 6, 7362–7368. doi:10.1039/c4nr00919c 9. Joung, Y.-H.; Choi, W. S.; Shin, Y.; Lee, M.; Kim, H.; Song, W. J. Korean Phys. Soc. 2013, 63, 246–250. doi:10.3938/jkps.63.246 License and Terms This is an Open Access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). Please note that the reuse, redistribution and reproduction in particular requires that the authors and source are credited. The license is subject to the Beilstein Journal of Nanotechnology terms and conditions: (https://www.beilstein-journals.org/bjnano) The definitive version of this article is the electronic one which can be found at: doi:10.3762/bjnano.10.32 Supporting Information Supporting Information File 1 Overview. [https://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-10-32-S1.pdf] Acknowledgements This study was supported by Korea Electric Power Corporation (Grant No. R17XA05-01) and Korea Institute of Energy Technology Evaluation and Planning (Grant No. 20184030201900). 336
https://openalex.org/W2989318457
http://dx.doi.org/10.33889/IJMEMS.2020.5.1.009
English
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Time-Based Process Mapping for Lead Time Compression in the Apparel Supply Chain: A Case Study
International journal of mathematical, engineering and management sciences
2,019
cc-by
5,887
Abstract Fashion industries are highly globalized as designing, manufacturing and selling are performed in different countries. As a result, it has a long supply chain and long lead time. Identification of value-added time through-out the supply chain is a key research area in apparel industries. This case study investigated detailed activities of thirty contracts of an apparel supply chain by considering the manufacturer as a coupling point of upstream (suppliers) and downstream (buyer). The contract frequencies were analyzed based on lead time and quantity. Each contract was analyzed using a time-based process mapping (TBPM). TBPMs of all contracts showed that overall 45% were non-value-added activities throughout the chain. It was also identified that the non-value activities were independent of the contract volume. This study recommends time compression methodology to reduce the non-value-added activities by using advanced manufacturing technologies. The methodology and recommended time compression ways of this study can be applied to other supply chains. Keywords- Lead time, Supply chain management, Time to market, TBPM. Time-Based Process Mapping for Lead Time Compression in the Apparel Supply Chain: A Case Study (Received May 17, 2019; Accepted August 30, 2019) International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 1. Introduction The clothing sector is now requiring frequent and trendy product changes in stores, quick response to trendy product development according to customer preferences (Martínez et al., 2015). Contrary, these fashion industries are highly globalized as its operations throughout the supply chain from design to manufacturing, and finally, selling are performed in different countries (Čiarnienė and 96 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 Vienažindienė, 2014). Thus, it has a long supply chain and long lead time. Consequently, this long lead time deals with longer forecast horizon and any longer horizon worsen the forecast (Christopher and Towill, 2002). Whereas, Fashion markets typically show short life-cycles, high volatility, low predictability and high instinct purchasing (Christopher and Towill, 2002). Because of such characteristics, forecasting accuracy is extremely difficult. These market complexities, ferocious competition and fast changes in demand necessitate the companies to be ready to react promptly to customers’ needs (Bhardwaj and Fairhurst, 2010). To do such, apparel manufacturer shifted to Mass-Customization from Mass Production (Moin et al., 2013). Mass customization can be described as manufactures ability to meet market demand with high product variety without substantial trade-offs in cost, delivery and quality (McCarthy, 2004). Mass customization strategies lead the manufacturers to supply an increasingly unsettled marketplace with customized products responding to individual customer requirements with minimal loss of production efficiency (Liu and Deitz, 2011). Two methodologies can be applied to implement Mass customization: i) Processes customization and ii) Advanced manufacturing technologies which include communication and network (Da Silveira et al., 2001). Customers do not care much about how a company gets the order and deliveries of a product (Moin et al., 2018). To satisfy the customers there are three competitive dimensions, i.e. price, product differentiation and delivery time, which depends on the lead time (Magnusson and Simonsson, 2012). This lead time can be divided in many ways. In most common practice, lead-time typically includes two components: Information lead times (i.e., the time it takes to process an order) and Order lead times (i.e., the time it takes to produce and ship the item). Nowadays, time became the most important factor in this competitive business environment. Several strategies are employed by different companies to reduce the lead time (Pine et al., 1993). 1. Introduction 98 short term and long-term solution (Harrison and Hoek, 2011). A methodical framework for implementation of Time-based strategies is shown in Figure 1. Table 1. Lead time reduction strategies depending on their age (Simchi-Levi et al., 2008) Age of Business Time Compression strategies The goal of Time Compression strategies Lead time Customer satisfaction Remarks Age of manufacturing (1900-1960) Setup redesign, Inventory control, Cellular manufacturing, Preventive maintenance, Employee involvement in problem-solving, MRP, MRP II, etc. Reduce production time Long Low Reduce production time Age of distribution (1960-1990) All above + Direct shipment, Warehousing, Cross-docking, Transshipment, Product differentiation, Push-pull boundary, etc. Manufacturing age plus Reduce replenish time Medium Average Reduce production and distribution time Age of information (1990-2010) All above + Concurrent and parallel processing, Electronic Data Interchange, Delayed product differentiation, Supplier relationship, Vendor-managed inventory (VMI), E-commerce, ERP, ERP II, etc. Manufacturing and distribution age plus Reduce ordering time Medium Above average Reduce information & material flow time. Age of the customer (2010-next) All above + Customer Relationship Management, Customer-focused strategy, Demand forecasting from customer behavior, involving the customer in new products development, shortening business life cycle and Introduce customer-centric business, etc. Manufacturing, distribution and information age plus Reduce demand forecasting time Short High Satisfy & Surprise the customers Age of market- on-time (Authors’ perception All above + Demand rise & creation, manufacturing as per demand and fulfillment of the demand. All of the above plus Dominate market like fashion leader and Push sell with demand rise and creation Very short Optimum Satisfy & Surprise the customers and control market Figure 1. Methodology for time-based process improvement (Harrison and Hoek 2011) Mind-set to required changes for improvement Go through and evaluate the process Identify non- value-adding processes Identify the causes of non- value-added processes Improve the process Review the changes in process Table 1. Lead time reduction strategies depending on their age (Simchi-Levi et al., 2008) Age of Business Time Compression strategies The goal of Time Compression strategies Lead time Customer satisfaction Remarks Age of manufacturing (1900-1960) Setup redesign, Inventory control, Cellular manufacturing, Preventive maintenance, Employee involvement in problem-solving, MRP, MRP II, etc. Reduce production time Long Low Reduce production time Age of distribution (1960-1990) All above + Direct shipment, Warehousing, Cross-docking, Transshipment, Product differentiation, Push-pull boundary, etc. 1. Introduction For example, information lead time can be reduced by using very sophisticated and modern communication system while Order lead time can be reduced through efficient supply chain management (Simchi-Levi et al., 2008). Different time compression strategies, their goals and effects on lead time based on the age of the business are summarized in Table 1. One of the effective ways to reduce the lead time is Time-based study. The term time-based study first appeared in the open literature in 1988 (Stalk, 1988). Time-based competition is a strategy by which a firm makes better use of time as a resource to acquire a competitive advantage. It seeks to compress the time required to Propose, Develop, Manufacture, Market and Deliver its products (Qunhui and Yang, 2011). To get such time-based advantages it is necessary to shorten the lead time as much as possible. There are two measures prior to implementing time-based strategies- i) Timeliness-to-market (this includes Fast-to-market, Fast-to-produce and Early withdraw the product from the market) and ii) Measure of ‘P time’ and ‘D time’. P time measures the time needed for all the necessary processes and delivers that product to the customer (generally known as Lead time). D time measures the time for which a customer is willing to wait to have their demand fulfilled (Harrison and Hoek, 2011). For managing timeliness or reducing the gap between P time and D time in the logistics pipeline several strategies are in practiced like make to order (MTO) and assemble to order (ATO) which consider a customer order decoupling point (CODP). MTO is a branch of Lean Manufacturing philosophy where manufacturing starts only after receiving the customer’s order (Kabaivanova, 2015). This is a Pull type production system an oppose to a Push type production system like Make to Stock (MTS). ATO is a similar pull-type production system where assembly starts only after a demand is created (Song and Zipkin, 2003). A Time-Based Process Map (TBPM) identifies the 97 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 , , , https://doi.org/10.33889/IJMEMS.2020.5.1.009 value-adding and non-value adding activities on a supply chain and guides the user for generating short term and long-term solution (Harrison and Hoek, 2011). A methodical framework for implementation of Time-based strategies is shown in Figure 1. 1. Introduction Manufacturing age plus Reduce replenish time Medium Average Reduce production and distribution time Age of information (1990-2010) All above + Concurrent and parallel processing, Electronic Data Interchange, Delayed product differentiation, Supplier relationship, Vendor-managed inventory (VMI), E-commerce, ERP, ERP II, etc. Manufacturing and distribution age plus Reduce ordering time Medium Above average Reduce information & material flow time. Age of the customer (2010-next) All above + Customer Relationship Management, Customer-focused strategy, Demand forecasting from customer behavior, involving the customer in new products development, shortening business life cycle and Introduce customer-centric business, etc. Manufacturing, distribution and information age plus Reduce demand forecasting time Short High Satisfy & Surprise the customers Age of market- on-time (Authors’ perception All above + Demand rise & creation, manufacturing as per demand and fulfillment of the demand. All of the above plus Dominate market like fashion leader and Push sell with demand rise and creation Very short Optimum Satisfy & Surprise the customers and control market Table 1. Lead time reduction strategies depending on their age (Simchi-Levi et al., 2008) ad time reduction strategies depending on their age (Simchi-Levi et al., 2008) Figure 1. Methodology for time-based process improvement (Harrison and Hoek 2011) Mind-set to required changes for improvement Go through and evaluate the process Identify non- value-adding processes Identify the causes of non- value-added processes Improve the process Review the changes in process Mind-set to required changes for improvement Go through and evaluate the process Review the changes in process Identify non- value-adding processes Improve the process Identify the causes of non- value-added processes Figure 1. Methodology for time-based process improvement (Harrison and Hoek 2011) 98 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 Every process mapping including TBPM demonstrates the information flows and the physical flows together (Bicheno and Holweg, 2000). TBPM is one of the seven mapping tools of ‘Seven Value Stream Mapping Tools’ (Gracanin et al., 2014). ‘Seven Value Stream Mapping Tools’ includes: (i) Process activity mapping, (ii) Supply chain response matrix, (iii) Production variety funnel, (iv) Quality filter mapping, (v) Demand amplification mapping, (vi) Decision point analysis and (vii) Physical structure mapping such as Value stream costing, Cost-time and TBPM (Chapman, 2003). 1. Introduction For the application of Physical structure mapping, cost and quality cannot be measured properly due to the poor understanding of costs and quality in reality (Harrison and Hoek, 2011). Whereas time can be measured easily and that everyone understands, even people with minimal training can measure the performance of processes or activities of any large issues in terms of time. Hence TBPM fits well to all kind of process more than a cross-functional toolbox of Seven Value Stream Mapping Tools. The application of TBPM was used to a range of industrial sectors including aerospace and construction, textile supply chain and automotive industries to decrease the lead time in production (Forza and Filippini, 1993). Generally, all participants of a supply chain (manufacturers, suppliers, buyer and retailers) have their own and individual core competencies. When all the partners merge and conjunct their individual competencies, they can achieve their goals. The time-based strategy for Quick Response also becomes a great premium for both the organization and supply chain level. To achieve time- based competitive advantages, this case study adopts time-based process mapping (TBPM), an effective tool to create the visibility of value-adding and non-value adding time across the network. 2. The Supply Chain under Investigation This case study focused on an apparel supply chain, of which upstream, downstream and manufacturer are located in different countries. The manufacturer is performing as a coupling point of upstream (Suppliers) and downstream (Buyers). In this supply chain, the buyer has direct linkages with consumers through his own retail outlet. Hence the buyer develops the forecasted styles through a manufacturer and manufacturer produces those styles by collecting required materials from suppliers. After developing a style, a corresponding contract is placed by the buyer to the manufacturer and the contract is executed through all supply chain participants. The chain structure is shown in Figure 2. It should be noted that no MTO or ATO strategy is applied currently on this supply chain. However, the authors of this manuscript offered an improved supply chain in the recommendation section. Figure 2. Supply chain structure of the current case study Products Products Information Monetary Supplier Manufacturer Buyer & Retailer Materials Materials Monetary Monetary Customer Lead time (P time) Monetary Figure 2. Supply chain structure of the current case study 99 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 3. Methodology gy Thirty contracts for the year 2017 of an apparel supply chain was collected. The activities for forecasting and product development were excused due to business confidentiality. Data on those contracts having different quantity for different product styles and different lead time were collected (Table 2). All contracts were categorized according to quantity and number of days in lead time. In addition to that, each contract was also analyzed through Time Based Process Mapping (TBPM). Table 2. Lead times of 30 contracts of the case study Serial no. Lead time Serial no. Lead time Serial no. Lead time Serial no. Lead time 1 171 9 155 17 148 25 135 2 148 10 153 18 147 26 165 3 149 11 152 19 136 27 148 4 147 12 178 20 151 28 156 5 160 13 179 21 179 29 143 6 151 14 162 22 135 30 139 7 171 15 179 23 149 8 165 16 133 24 174 Table 2. Lead times of 30 contracts of the case study By doing so, the non-value-added items were identified in the supply chain and the lead time was reduced by eliminating the non-value-added items. For example, a TBPM of a contract (no. 19 in Table 2) is shown in Figure 3. Thirty-seven activities were identified through TBPM and divided into five phases. Due to business confidentiality style no., activity start date and end date are not shown in Figure 3. 4. Data Analysis The example shows that the TBPM for contract 1xxxxxx shows that total lead time for the contract was 136 days and among those 85 days for value-added and 51 days were non-value added. To identify value-added activities, the value gaining activities by supply chain participants were considered rather than the value-added to products. As for example, transportation activities are not adding any value to products but when products transport to profitable market helps the supply chain to gain more profits. So, only delayed or waiting were considered as non-value-added activities. Some value-added concurrent activities were also found but for those activities, time was calculated once. Phase wise value-added (VA) and non-value-added (NVA) activity days were calculated from the TBPM of each contract and tabulated in Table 3. 4.1 Contract Variation The quantity of the selected contracts ranges from 2251 to 25264 pieces and those are divided into five groups according to quantity. The following Figure 4 shows the contract frequencies in each group and found that contract frequencies are more for a smaller quantity. The lead time of those contracts spans from 133 to 179 days with an average of 155.23 days and divided into five groups also. Among the contracts, 60% had lead time less than the average of lead time and 40% above the said. This is also a similar phenomenon like the number of contracts, i.e. contract frequencies are more for shorter lead time. 100 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 , g g g Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 Figure 3. Time based process mapping (TBPM) https://doi.org/10.33889/IJMEMS.2020.5.1.009 Figure 3. Time based process mapping (TBPM) 4.2 TBPM Activity wise time-based process mappings for thirty contracts help to identify value-added (VA) and non-value-added (NVA) activities. The following Figure 5 shows the percentages of value- added and non-value added activities against the contract variations: lead time and number of contracts as mentioned in section 4.1. 101 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi org/10 33889/IJMEMS 2020 5 1 009 , , , https://doi.org/10.33889/IJMEMS.2020.5.1.009 Table 3. Phase wise value-added and non-value-added activity in days Serial No. 4.2 TBPM Total lead time (day) Order confirmation phase (OCP) Waiting in OCP Material sourcing phase (MSP) Waiting in MSP Sample confirmation phase (SCP) Waiting in SCP Production phase Waiting in PP Shipping phase (SP) Waiting in SP VA NVA VA NVA VA NVA VA NVA VA NVA 1 171 3 25 38 16 9 6 16 25 23 10 2 148 3 25 36 18 6 1 13 13 23 10 3 149 3 18 37 13 9 10 12 14 23 10 4 147 3 25 34 20 4 2 20 6 23 10 5 160 3 18 32 18 6 7 24 19 23 10 6 151 3 18 34 16 6 3 15 24 22 10 7 171 3 18 38 29 6 18 23 3 23 10 8 165 3 18 32 31 6 1 19 22 23 10 9 155 3 25 38 16 4 0 11 25 23 10 10 153 3 18 31 19 5 3 21 19 24 10 11 152 3 25 35 19 6 8 22 2 22 10 12 178 3 26 36 13 10 39 19 0 22 10 13 179 3 26 46 49 2 4 12 6 22 9 14 162 3 26 43 10 10 8 27 1 24 10 15 179 3 13 35 19 11 36 22 7 23 10 16 133 3 13 34 20 5 1 14 11 22 10 17 148 3 21 33 13 7 17 17 4 23 10 18 147 3 20 30 14 10 23 11 4 22 10 19 136 3 20 34 10 7 11 19 0 22 10 20 151 3 20 39 25 6 5 17 3 23 10 21 179 3 13 31 23 10 49 16 2 23 9 22 135 3 21 29 17 4 3 16 8 24 10 23 149 3 21 32 22 5 2 30 1 23 10 24 174 3 10 35 24 12 31 20 5 24 10 25 134 3 10 36 29 3 0 16 4 23 10 26 165 3 27 41 24 5 0 21 12 22 10 27 148 3 24 39 18 6 6 17 3 22 10 28 156 3 20 36 26 4 6 19 8 24 10 29 143 3 20 33 21 6 2 16 10 22 10 30 139 3 11 35 31 2 6 14 5 22 10 Table 3. 4.2 TBPM Phase wise value-added and non-value-added activity in days From Figure 5, it is found that the average non-value-added activities of the process in the supply chain range from 42% to 49% for led time contract variation and 43% to 48% for product quantity contract variation. The average value-added time of 30 contracts is found 56% according to the lead time. The non-value added (NVA) and value-added (VA) activities of each category (lead time vs quantity) are very similar. So, it can be said that the length of lead time and quantity in each contact does not influence the non-value added and value-added activities percentages. From Figure 5, it is found that the average non-value-added activities of the process in the supply chain range from 42% to 49% for led time contract variation and 43% to 48% for product quantity contract variation. The average value-added time of 30 contracts is found 56% according to the lead time. The non-value added (NVA) and value-added (VA) activities of each category (lead time vs quantity) are very similar. So, it can be said that the length of lead time and quantity in each contact does not influence the non-value added and value-added activities percentages. From Figure 5, it is found that the average non-value-added activities of the process in the supply chain range from 42% to 49% for led time contract variation and 43% to 48% for product quantity contract variation. The average value-added time of 30 contracts is found 56% according to the lead time. The non-value added (NVA) and value-added (VA) activities of each category (lead time vs quantity) are very similar. So, it can be said that the length of lead time and quantity in each contact does not influence the non-value added and value-added activities percentages. 102 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 Figure 4. Variation on contracts depending on (a) quantity and (b) lead time Vol. 5, No. 1, 96 107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 Figure 4. Variation on contracts depending on (a) quantity and (b) lead time Figure 4. Variation on contracts depending on (a) quantity and (b) lead time TBPM is usually done for individual products and activity-wise. To get the time compression scenario it was analyzed phase-wise. 4.2 TBPM The average percentage of VA activities was also analyzed according to the 5 phases (Order confirmation, Materials sourcing, Sample confirmation, Production, and Shipping) shown in Figure 3. The average percentages of value-added and non- value-added times according to phases are shown in Figure 6a. Figure 5. Non-value-added and value-added activities depending on (a) quantity and (b) lead time Figure 5. Non-value-added and value-added activities depending on (a) quantity and (b) lead time Among the five phases, Order confirmation phase and Material sourcing phase have long waiting time and in both cases, it is 13% of the total lead time. It is found that overall of 44% of lead time was lost in non-value-added activities. The percentage of non-value activities against each phase was also studied and shown in Figure 6b. Around 60% of the NVA happened during the order confirmation and material sourcing phase. 5. Discussion and Recommendations In the first phase (order confirmation phase), monetary issues slower the process and TBPM in Figure 3 show that 20 days (around 15 % of the total lead time) are non-value-added time. For all thirty contracts, NVA is 13% of the total lead time and 29% of the total non-value-added time (Figure 6). The second phase (material sourcing) is the longest period where 11 out of 37 activities took place. Major activities in this phase are fabric production and shipment of fabric to apparel manufacturer. Figure 3 shows that these major activities conceive 31 days (around 23 % of the total 103 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 lead time), other value-added activities conceive only 04 days and non-value-added activities conceive time 10 days. Sample confirmation phase is complicated to distinguish because of parallel activities within the phase and with other phases (predecessor and successor). Figure 3 shows that this is the shortest one and non-value-added time (11 days) is greater than the value-added time (07 days). For all thirty contracts, it is found similar as shown in Figure 6. For the apparel production phase, all the items were found to be value-added activities without having a waiting time (Figure 3). However, overall 6% NVA was identified for this phase (Figure 6) when considering all the thirty contracts. Figure 3 also shows the last one is shipping phase where transportation to destination time is 22 days (around 16% of the total lead time) and waiting time is 10 days (around 7 % of the total lead time). A very similar result was also found when considering all thirty contracts as shown in Figure 6. lead time), other value-added activities conceive only 04 days and non-value-added activities conceive time 10 days. Sample confirmation phase is complicated to distinguish because of parallel activities within the phase and with other phases (predecessor and successor). Figure 3 shows that this is the shortest one and non-value-added time (11 days) is greater than the value-added time (07 days). For all thirty contracts, it is found similar as shown in Figure 6. 5. Discussion and Recommendations For the apparel production phase, all the items were found to be value-added activities without having a waiting time (Figure 3). However, overall 6% NVA was identified for this phase (Figure 6) when considering all the thirty contracts. Figure 3 also shows the last one is shipping phase where transportation to destination time is 22 days (around 16% of the total lead time) and waiting time is 10 days (around 7 % of the total lead time). A very similar result was also found when considering all thirty contracts as shown in Figure 6. Figure 6. (a) Phase wise value/non-value added activities, (b) % of non-value against total time Figure 6. (a) Phase wise value/non-value added activities, (b) % of non-value against total time Contract frequencies are higher for the smaller quantity with comparatively shorter lead time i.e. the supply chain under investigation faces the volatile market behavior regarding high product variety and shorter lead time. Additionally, the buyer follows a long forecast period throughout the chain. The longer forecast always increases the risk of forecasting error which may result in high stocks in-store as well as stock out from the market. Higher inventory in the store will decrease the business performance due to an increase in inventory holding cost and a decrease in capital flow. Stockout and late replenishment of products also decrease business performance as a result of losing the market share and valuable customer. A short supply chain is recommended for the apparel industry under investigation as the fashion market is volatile and fashion product lifecycle is short. With shorter lead time, the forecasting period can be shorter which ultimately reduces the risk of forecasting error. Which ultimately increases the buyers’ capacity to replenish quickly. 104 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 It is also recommended that the supply chain follow true MTO strategy where the D time (the time a customer is willing to wait) is equal to the P time (total lead time). In every phase, there are scopes to reduce P time. By using advanced information technology and long-term relationship with corresponding financial institutes the non-value-added time in order confirmation phase can be eliminated. 5. Discussion and Recommendations Again, the commitment and continuous long-term relationship among the supply chain participants may establish a trustworthy business relationship which will also help the chain to minimize the non-value-added time in the order confirmation phase. The P time for sample confirmation phase can be shortened with the empowerment of all participants in the supply chain. The participation of all the parties (through organizational learning) within the supply chain in forecasting and product development will reduce forecasting error. Organizational leanings will develop the capability of fabric producers to develop a new fabric during the order confirmation phase before the material sourcing phase as a concurrent activity. In this way, the waiting time (NVA) for the material sourcing phase can be eliminated and the lead time (P time) of this phase can be shortened. Additionally, vendor managed inventory system and empowerment of vendor will help this supply chain to shorten lead time. A modified supply chain as opposed to the current supply chain (Figure 2) by introducing a customer order decoupling point (CODP) to the manufacturer is recommended as shown in Figure 7. As a result, the P time will become equal to the D time. Figure 7. The modified supply chain structure of the current case study with the introduction of CODP Improved Lead time (P time) CODP D time Information Products Products Supplier Manufacturer Buyer & Retailer Materials Materials Customer Supplier gure 7. The modified supply chain structure of the current case study with the introduction of CODP A further improvement of the supply chain can be achieved by introducing scope for quick replenishment of an old style with a new one through a concurrent and parallel production of various styles as shown in Figure 8. In this way, the performance of timeliness-to-market will be increased by fast-to-market and early withdraw a product from the market. When style -1 entered the market earlier it can be withdrawn earlier. A high-profit margin can be achieved as well as the invested capital becomes free earlier. This free capital can be re-inverted for the new style-n. Freeing the capital and gaining the high profit motivates the supply chain participants to develop a new product. In the shipping phase, the non-value-added time can be also minimized with proper planning for transportation schedule, which will reduce waiting time in various stations. 105 International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 5. Discussion and Recommendations 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 Figure 8. Concurrent and parallel production of various styles …… Lead time (P time) Information Products Products Supplier Manufacturer Buyer & Retailer Materials Materials Customer Style-1 Style-2 Style-3 Style-(n-1) Style- n CODP International Journal of Mathematical, Engineering and Management Sciences Vol. 5, No. 1, 96-107, 2020 https://doi.org/10.33889/IJMEMS.2020.5.1.009 Figure 8. Concurrent and parallel production of various styles …… Lead time (P time) Information Products Products Supplier Manufacturer Buyer & Retailer Materials Materials Customer Style-1 Style-2 Style-3 Style-(n-1) Style- n CODP Information Products Buyer & Retailer Figure 8. Concurrent and parallel production of various styles Conflict of Interest Conflict of Interest The authors declare that there is no conflict of interest for this publication. The authors declare that there is no conflict of interest for this publication. Acknowledgments The authors appreciate the effort from editors and reviewers. This paper did not receive any financial assistance/grants from any funding agencies. 6. Conclusion The apparel supply chain facing tough and global competition for their products even in case of basic items. The competitive issues are price, quality and availability. These three issues are always related to cost and time. Utilization of time along with other resources determines the supply chain performance. The Time-based process mapping is an effective tool to diagnose the process which able the supply chain to take initiatives for better utilization of time. The study reveals that from ordering to retailing there are 44% to 45% non-value-added time. The study recommends minimizing this non-value-added time with the introduction of CODP by following an MTO strategy. This MTO strategy can be achieved by the use of advanced information, manufacturing and operational technology in planning, manufacturing, integration and collaboration of different participants of the supply chain. 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The use of positron emission tomography/computed tomography imaging in radiation therapy: a phantom study for setting internal target volume of biological target volume
Radiation oncology
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* Correspondence: rt01@mattohp.com 1Department of Radiological Technology, Public Central Hospital of Matto Ishikawa, 3-8, Kuramitsu, Hakusan City, Ishikawa Pref 924-8588, Japan 2Department of Quantum Medical Technology, Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan Full list of author information is available at the end of the article Open Access Open Access Open Access The use of positron emission tomography/ computed tomography imaging in radiation therapy: a phantom study for setting internal target volume of biological target volume Wataru Kawakami1,2*, Akihiro Takemura2, Kunihiko Yokoyama3, Kenichi Nakajima4, Syoichi Yokoyama1 and Kichiro Koshida2 © 2014 Kawakami et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kawakami et al. Radiation Oncology (2015) 10:1 DOI 10.1186/s13014-014-0315-2 Background contained six hollow spheres with internal diameters of 10, 13, 17, 22, 28, and 37 mm, inside a simulated body cavity that may be filled with water. The phantom background was filled with either water or air. The phantom background was filled with water to simulate a human head-and-neck, abdominal and pelvic tumour. In this series of experiments, all spheres were filled with 2-[fluorine-18] fluoro-2-deoxy-D-glucose (18F-FDG) so- lution that was measured in a dose calibrator and di- luted with water to obtain a radioisotope concentration (RC) of 16–64 kBq/mL. The total weight of the filled phantom was 12.4 kg. The phantom background was filled with air to simulate a human lung tumour. In this series of experiments, all spheres were filled with 18F-FDG solution diluted with water to obtain an RC of 64–256 kBq/mL. The total weight of the phantom in this situation was 2.4 kg. Positron emission tomography/computed tomography (PET/CT) has become an important and commonly used imaging method to detect tumours, plan radiotherapy treatment, and evaluate responses to therapy, as it allows for simultaneous functional and anatomical imaging. During treatment planning for radiation therapy, gross tumour volume (GTV) is calculated based on CT im- ages. Moreover, it has been reported that when planning treatment for tumours of the head-and-neck, inter- and intra-observer differences in GTV may be reduced by using PET images [1]. There are many issues that arise when determining GTV with only PET imaging, such as accounting for respiratory movement and distinguishing inflammation. PET imaging also involves limited spatial resolution and the partial volume effect (PVE). Moreover, it has been reported that extraction volume differs according to the standardized uptake values (SUV) of normal tissue and tumour [2]. Thus, it is not suitable to automatically ex- tract GTV using the SUV from PET images. Realistically, it is preferable that the radiotherapist uses the PET images as a reference [3]. The phantom background was filled with water or air, yielding sphere-to-background (S/B) ratios of approxi- mately 4, 8, and 16 to 1 or null. To achieve the same S/B ratio, each given dose was adjusted by the difference of the background. The RC values of the spheres and back- ground and the S/B ratios were chosen to simulate the ranges obtained from clinical conditions based on the tumour concentration of 18F-FDG and the SUV. The SUV is commonly used as a relative measure of FDG uptake. Imaging CT transmission (for AC) and PET scans were performed using a PET/CT scanner (Discovery PET/CT 600 Motion; General Electric Medical Systems [GEMS], Milwaukee, WI, USA) with a stationary or moving phantom. Scans of the moving phantom were acquired without gating. Slow CT scanning (SCS; 120 kV, 45 mA, and 4.0 s/rotation) or the helical CT scanning (HCS; 120 kV, 360 mA, 0.5 s/rota- tion, and pitch 0.938) imaging data, followed by PET emission data, were acquired for 300 s from one field of Abstract Background: Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) is an important method for detecting tumours, planning radiotherapy treatment, and evaluating treatment responses. However, using the standardized uptake value (SUV) threshold with PET imaging may be suitable not to determine gross tumour volume but to determine biological target volume (BTV). The aim of this study was to extract internal target volume of BTV from PET images. Methods: Three spherical densities of 18F-FDG were employed in a phantom with an air or water background with repetitive motion amplitudes of 0–30 mm. The PET data were reconstructed with attenuation correction (AC) based on CT images obtained by slow CT scanning (SCS) or helical CT scanning (HCS). The errors in measured SUVmax and volumes calculated using SUV threshold values based on SUVmax (THmax) in experiments performed with varying extents of respiratory motion and AC were analysed. Results: A partial volume effect (PVE) was not observed in spheres with diameters of ≥28 mm. When calculating SUVmax and THmax, using SCS for AC yielded smaller variance than using HCS (p < 0.05). For spheres of 37- and 28-mm diameters in the phantom with either an air or water background, significant differences were observed when mean THmax of 30-, 20-, or 10-mm amplitude were compared with the stationary conditions (p < 0.05). The average THmax values for 37-mm and 28-mm spheres with an air background were 0.362 and 0.352 in non-motion, respectively, and the mean THmax values for 37-mm and 28-mm spheres with a water background were 0.404 and 0.387 in non-motion and 0.244 and 0.263 in motion, respectively. When the phantom background was air, dl f h i i TH d d l i li d egardless of sphere concentration or size, THmax was dependent only on motion amplitude. Conclusions: We found that there was no PVE for spheres with ≥28-mm diameters, and differences between SUVmax and THmax were reduced by using SCS for AC. In the head-and-neck and the abdomen, the standard values of THmax were 0.25 and 0.40 with and without respiratory movement, respectively. In the lungs, the value of THmax became the approximate expression depending on motion amplitude. Keywords: PET/CT, BTV, Respiratory motion, ITV, Attenuation correction Page 2 of 9 Page 2 of 9 Kawakami et al. Radiation Oncology (2015) 10:1 Page 2 of 9 Background The basic formula for SUV was as follows: Given that the PET image was a function image, the extracted volume is thought to be biological target vol- ume (BTV). BTV often indicates the part with espe- cially high tumour revitalization in GTV [4]. It became possible to irradiate GTV and BTV by a separate dose by the development of the radiation therapy machine and the plan device. Therefore, BTV is being used for radiation therapy [5]. SUV ¼ C A=W ð Þ ; ð1Þ ð1Þ where C was the RC (kBq/mL) measured by the PET scanner within the region of interest, A was the amount of injected activity (kBq), and W was the weight of the patient (g). Several research reports suggest extracting BTV from PET images [6-12]. However, a concrete extraction con- dition that tempers with various situations has not been established to date. The purpose of this study was to ob- tain conditions to automatically extract internal target volume of BTV (ITV-b) from phantom experiments using SUV threshold values based on SUVmax (THmax). The items that should be considered for extracting ITV-b using THmax are as follows: (1) tumour size (PVE), (2) mo- tion amplitude, (3) tumour site (lung or head-and-neck, abdomen), and (4) attenuation correction (AC). We exam- ined tumour sizes for which a PVE was not observed [13,14] in addition to changes in SUVmax and THmax ac- cording to the degree of motion amplitude [15-17], the position of the tumour [18], and the method of attenu- ation correction [19-21]. A motion system (QRP-01; Qualita, Ageo City, Saitama Prefecture, Japan) was used in this study to simulate re- spiratory motion. The NEMA/IEC phantom was placed on a moving table. The moving table was oscillated with a displacement of 10, 20, and 30 mm in the cranio-caudal direction at a frequency of 15 cycles/min. These param- eters were selected to simulate displacements and re- spiratory cycles typically observed in normal respiratory motion. Analysis of SUVmax and THmax Effects of sphere size, motion amplitude, background medium, and attenuation correction on measured SUVmax and THmax were examined. The spheres with 37-mm and 28-mm diameters were used for this evaluation. The phantom, with backgrounds of both air and water, were scanned with the SCS and HCS, as described above. Dur- ing scanning, the phantom was moved with amplitudes of 0, 10, 20, or 30 mm. To evaluate the influence of all com- bined S/B ratios on SUVmax, the ratios of SUVmax (SUVr) were calculated using the average SUVmax for the three S/B ratios in scans performed with no phantom motion. Image analysis was performed using PET VCAR™soft- ware and the Advantage Workstation (GE Healthcare, Milwaukee, WI, USA). This software is designed to measure SUV and extracted volume for any PET-defined region with detected activity. SUVmax values were mea- sured in spheres with 144 image acquisitions. The SUV threshold values based on SUVmax (THmax) were calcu- lated so that the volume extracted by the THmax matched the ideal volume of the spherical tumours in the phantom, taking into consideration the motion amplitude. Thus, an ideal phantom volume becomes a total of the volume of spheres and the volume of the column corresponding to the amount of the amplitude (Figure 1). The SUVr was calculated as follows: SUVr ¼ Cj=Ca; ð3Þ ð3Þ SUVr ¼ Cj=Ca; Methods The National Electrical Manufacturers Association (NEMA) 2001 International Electrotechnical Commission (IEC) Phantom In this study, we imaged a PET image quality phantom (NEMA/IEC phantom; Data Spectrum Corp.). This phantom Kawakami et al. Radiation Oncology (2015) 10:1 Page 3 of 9 Figure 1 Diagram depicting the volumes of stationary and moving spherical phantoms. The ideal phantom volumes were divided into a sphere and a column, and each volume was calculated. d, motion amplitude. view. Both PET scan types (stationary and motion) were acquired in 3-dimensional (3D) mode. The 3D data were first Fourier rebinned, and then all emission scans were reconstructed into a 256 matrix using ordered-subset expectation maximization algorithms. The numbers of iterations and subsets were 2 and 32, respectively. The PET-reconstructed slice thickness was 3.75 mm. Phantom object h The NEMA/IEC phantom, which contained six spheres ranging from 10 to 37 mm in diameter, was filled with 18F-FDG. PET/CT data acquired with the stationary phantom was used as a standard (non-motion). Respira- tory motion was simulated by a motor-driven plastic platform that moved the phantom with a displacement of 10, 20, and 30 mm in the cranio-caudal direction at a frequency of 15 cycles/min. A 4-s period of oscillation was selected based on the average respiratory period measured in 50 patients with lung tumours at our centre using a respiratory gating system (Real-time Positioning Management System; Varian, Palo Alto, CA, USA). With the phantom in motion, PET data were acquired in the static mode, wherein data were collected for a definite time period according to the width of the detector’s body axis instead of continuous movement like in CT. Acquisitions using three types of S/B ratios, four different motion amplitudes, two different background media, and two types of CT scans were repeated three times; for a total of 144 image acquisitions performed. Figure 1 Diagram depicting the volumes of stationary and moving spherical phantoms. The ideal phantom volumes were divided into a sphere and a column, and each volume was calculated. d, motion amplitude. where Ci was the maximum radioactivity of each sphere at each S/B ratio, and Cr was the maximum radioactivity of the 37-mm sphere at each S/B ratio in non-motion, both measured in scans with no phantom motion. Evalu- ation was performed using two types of background (air and water) and attenuation corrections (SCS and HCS). An analysis of variance (ANOVA) was used to check for significant differences in the sphere diameter. Next, multiple comparisons between six types of spheres of (10–37 mm) were performed using Dunnett’s method and the size without the influence of PVE was then examined. p p p g p p o, recovery coefficient; SCS, slow computed tomography scanning; HCS, helical computed tomography scanning; SD, standard deviation Partial volume effects Table 1 shows the RCo values measured in our study. A significant difference was observed between RCo values measured in spheres of different sizes with phantoms using either air or water as background, scanned with SCS or HCS. When evaluating all RCo values measured using each of the CT methods and both of the back- ground media used, no PVE influence on RCo was ob- served in spheres with diameters of 28 mm or larger. Conversely, we demonstrated that the spheres with diame- ters of 22 mm or less might be affected by PVE. Therefore, we only analysed 28- and 37-mm spheres in subsequent experiments. Evaluation of PVE To correctly assess the influence of respiratory move- ment, it is necessary to use a volume that will not be affected by the PVE. Therefore, the PVE was evaluated using the data from scans with the stationary phan- tom, with the recovery coefficient (RCo) calculated as follows: where Cj was the average of SUVmax values obtained for all S/B ratios with a particular sphere size, and Ca was the average of SUVmax values obtained for all S/B ratios with the same sphere size and no phantom motion. The variances in SUVr were compared between the attenu- ation corrections using the F-test. The differences be- tween the mean SUVr obtained with different motion ð2Þ RCo ¼ Ci=Cr; Page 4 of 9 Kawakami et al. Radiation Oncology (2015) 10:1 The effect of motion amplitude on SUVr was then investigated. There was a significant difference be- tween SUVr values obtained with different amplitudes. Conversely, the 10-mm motion amplitude did not affect the SUVr. With motion amplitudes of 20 mm and above, the decrease in SUVr was found to be relative to the amplitude of phantom motion. The largest observed SUVr difference, compared with the SUVr obtained under stationary conditions, was 30.9%, calculated for the 28-mm sphere in the phantom with water background at 30-mm motion amplitude (Figure 3b). amplitudes were evaluated using ANOVA with Dunnett’s multiple comparisons. amplitudes were evaluated using ANOVA with Dunnett’s multiple comparisons. The variances of THmax were compared with the attenu- ation corrections using the F-test. The variations of the mean THmax with different amplitudes were evaluated by ANOVA and Dunnett’s multiple comparisons. **indicates p < 0.05 compared with the 37-mm sphere using Dunnett’s multiple comparisons. THmax THmax values obtained in the phantom with an air back- ground using 37- and 28-mm spheres are presented as box plots in Figure 4a and b, respectively. The variance of THmax values obtained using the SCS technique was significantly smaller than that obtained using the HCS approach. Figure 5a and b present box plots of the THmax calculated for 37- and 28-mm spheres, respectively, using measurements obtained in a phantom with a water back- ground. For the 37- and 28-mm spheres, the variance of THmax did not differ significantly between SCS and HCS. When we consider the results of THmax for the phantoms with a water or air background together, the use of the SCS method for AC of PET data resulted in less variance, compared with the HCS approach. Therefore, in subse- quent experiments we only analysed the THmax values de- rived from PET data with AC utilizing the SCS method. **indicates p < 0.05 compared with the 37-mm sphere using Dunnett’s multiple comparisons. SUVmax (a) The variance of SUVr with slow computed Figure 2 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with air as the phantom background. (a) The variance of SUVr with slow computed Figure 3 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with water as the phantom background. (a) The variance of SUVr with slow computed Figure 3 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with water as the phantom background. (a) The variance of SUVr with slow computed tomography (CT) scanning (SCS) was significantly smaller than that of SUVr with helical CT scanning (HCS) for a 37-mm sphere with 10- and 20-mm amplitudes (F-test: p < 0.05). A significant difference was observed when comparing the mean SUVr of 30- and 20-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). (b) The variance of SUVr with SCS was significantly smaller than that of SUVr with HCS for a 28-mm sphere with 30-mm amplitude (F-test: p < 0.05). Significant differences were observed when comparing the mean SUVr of 30- and 20-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). Figure 2 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with air as the phantom background. (a) The variance of SUVr with slow computed tomography (CT) scanning (SCS) was significantly smaller than that of SUVr with helical CT scanning (HCS) for a 37-mm sphere with 20- and 30-mm amplitude (F-test: p < 0.05). A significant difference was observed when comparing the mean SUVr between 30-mm amplitude and stationary conditions (Dunnett’s test: p < 0.05). (b) The variance of SUVr with SCS was significantly smaller than that of SUVr with HCS for a sphere of 28 mm with 10-, 20-, and 30-mm motion amplitudes (F-test: p < 0.05). Significant differences were observed when comparing the mean SUVr of 30- and 20-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). only on the motion amplitude. Therefore, it may be pos- sible to approximate THmax using the following formula (Figure 6): significant differences were individually compared with the stationary conditions (Figure 5a and b). The average THmax value for 37-mm and 28-mm spheres were 0.404 and 0.387 in non-motion, respectively. Moreover, the average THmax value for 37-mm and 28-mm spheres were 0.244 and 0.263 in motion, respectively. SUVmax The SUVr measured in the phantom with an air back- ground and spheres sized 37 and 28 mm are presented as box plots in Figure 2a and b, respectively. The variance of SUVr using the SCS CT technique was significantly smaller than that of SUVr obtained using the HCS ap- proach. Figure 3a and b show box plots of the SUVr of spheres of 37 and 28-mm diameters, respectively, measured in a phantom with water background. The variance in SUVr calculated using the SCS CT technique was signifi- cantly smaller than that of SUVr obtained by HCS. Consid- ering SUVr calculated in phantoms with air and water backgrounds together, the SCS CT technique was found to be a more suitable approach for AC, compared with HCS. Consequently, we only analysed SUVr calculated using the SCS CT technique in subsequent experiments. The effect of motion amplitude on THmax was then investigated. We observed a significant difference be- tween THmax values obtained at different amplitudes. For spheres of 37- and 28-mm diameters in the phantom with an air background, significant differences were com- pared with the stationary conditions (Figure 4a and b). The average THmax values for 37-mm and 28-mm spheres were 0.362 and 0.352 in non-motion, respectively. For 37- and 28-mm spheres with the water background phantom, Table 1 Recovery coefficients by sphere size Sphere size (mm) Background Air Water SCS HCS SCS HCS Mean ± SD Mean ± SD Mean ± SD Mean ± SD Rco 37 1.00 ± 0.01 1.00 ± 0.01 1.00 ± 0.03 1.00 ± 0.02 28 0.99 ± 0.01 0.99 ± 0.01 1.00 ± 0.04 0.98 ± 0.02 22 0.98 ± 0.01** 0.98 ± 0.01** 0.95 ± 0.03 0.93 ± 0.04** 17 0.91 ± 0.01** 0.90 ± 0.01** 0.91 ± 0.06** 0.87 ± 0.06** 13 0.80 ± 0.02** 0.79 ± 0.01** 0.74 ± 0.06** 0.76 ± 0.04** 10 0.56 ± 0.02** 0.56 ± 0.02** 0.57 ± 0.04** 0.56 ± 0.05** **indicates p < 0.05 compared with the 37-mm sphere using Dunnett’s multiple comparisons. Rco, recovery coefficient; SCS, slow computed tomography scanning; HCS, helical computed tomography scanning; SD, standard deviation. Table 1 Recovery coefficients by sphere size Page 5 of 9 Kawakami et al. Radiation Oncology (2015) 10:1 significant differences were individually compared with Figure 2 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with air as the phantom background. SUVmax (a) The variance of SUVr with slow computed tomography (CT) scanning (SCS) was significantly smaller than that of SUVr with helical CT scanning (HCS) for a 37-mm sphere with 20- and 30-mm amplitude (F-test: p < 0.05). A significant difference was observed when comparing the mean SUVr between 30-mm amplitude and stationary conditions (Dunnett’s test: p < 0.05). (b) The variance of SUVr with SCS was significantly smaller than that of SUVr with HCS for a sphere of 28 mm with 10-, 20-, and 30-mm motion amplitudes (F-test: p < 0.05). Significant differences were observed when comparing the mean SUVr of 30- and 20-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). Figure 3 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with water as the phantom background. (a) The variance of SUVr with slow computed tomography (CT) scanning (SCS) was significantly smaller than that of SUVr with helical CT scanning (HCS) for a 37-mm sphere with 10- and 20-mm amplitudes (F-test: p < 0.05). A significant difference was observed when comparing the mean SUVr of 30- and 20-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). (b) The variance of SUVr with SCS was significantly smaller than that of SUVr with HCS for a 28-mm sphere with 30-mm amplitude (F-test: p < 0.05). Significant differences were observed when comparing the mean SUVr of 30- and 20-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). Figure 2 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with air as the phantom background. (a) The variance of SUVr with slow computed tomography (CT) scanning (SCS) was significantly smaller than that of SUVr with helical CT scanning (HCS) for a 37-mm sphere with 20- and 30-mm amplitude (F-test: p < 0.05). A significant difference was observed when comparing the mean SUVr between 30-mm amplitude and stationary conditions (Dunnett’s test: p < 0.05). (b) The variance of SUVr with SCS was significantly smaller than that of SUVr with HCS for a sphere of 28 mm with 10-, 20-, and 30-mm motion amplitudes (F-test: p < 0.05). Significant differences were observed when comparing the mean SUVr of 30- and 20-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). Figure 2 Effect of motion amplitude on maximum standard uptake value ratio (SUVr), evaluated with air as the phantom background. SUVmax (a) For a 37-mm sphere, the variance of TH did not differ significantly between slow computed Figure 5 Effect of motion amplitude on threshold values based on maximum standard uptake values (THmax), evaluated with water as the phantom background. (a) For a 37-mm sphere, the variance of THmax did not differ significantly between slow computed tomography (CT) scanning (SCS) and helical CT scanning (HCS). A significant difference was observed between the mean THmax of 30-, 20-, and 10-mm motion amplitudes compared with stationary conditions (Dunnett’s test: p < 0.05). The average THmax values for non-motion and motion were 0.404 and 0.244, respectively. (b) For a 28-mm sphere, the variance of THmax did not differ significantly between SCS and HCS. A significant difference was observed between the mean THmax of 30-, 20-, and 10-mm, motion amplitudes compared with stationary conditions (Dunnett’s test: p < 0.05). The average THmax values for non-motion and motion were 0.387 and 0.263, respectively. Figure 4 Effect of motion amplitude on threshold values based on maximum standard uptake values (THmax), evaluated with air as the phantom background. (a) The variance of THmax with slow computed tomography (CT) scanning (SCS) was significantly smaller than that of THmax with helical CT scanning (HCS) for a 37-mm sphere with 10-, 20- and 30-mm amplitudes (F-test: p < 0.05). A significant difference was observed comparing the mean THmax of 30-, 20-, and 10-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). The average THmax value for non-motion was 0.362. (b) The variance of THmax with SCS was significantly smaller than that of THmax with HCS for a 28-mm sphere with 30-mm amplitude (F-test: p < 0.05). A significant difference was observed when comparing the mean THmax of 30-, 20-, and 10-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). The average THmax value for non-motion was 0.352. greater than 10 mm, THmax was reduced in comparison to the stationary conditions (Figure 5a and b). The largest THmax deviation from the stationary con- ditions was 82.0%, calculated for the 37-mm diameter sphere with an air background phantom and 30-mm motion amplitude (Figure 4a). When the phantom back- ground was water, no relation between the decrease in THmax and amplitude was observed. At amplitudes SUVmax When the phantom background was filled with air, regardless of the sphere concentration or size, THmax was dependent y ¼ 0:08 þ 0:28 exp −0:14x ð Þ; ð4Þ ð4Þ where y is the approximate value of THmax, and x is the motion amplitude. where y is the approximate value of THmax, and x is the motion amplitude. Page 6 of 9 Kawakami et al. Radiation Oncology (2015) 10:1 Figure 5 Effect of motion amplitude on threshold values based on maximum standard uptake values (THmax), evaluated with water as the phantom background. (a) For a 37-mm sphere, the variance of THmax did not differ significantly between slow computed tomography (CT) scanning (SCS) and helical CT scanning (HCS). A significant difference was observed between the mean THmax of 30-, 20-, and 10-mm motion amplitudes compared with stationary conditions (Dunnett’s test: p < 0.05). The average THmax values for non-motion and motion were 0.404 and 0.244, respectively. (b) For a 28-mm sphere, the variance of THmax did not differ significantly between SCS and HCS. A significant difference was observed between the mean THmax of 30-, 20-, and 10-mm, motion amplitudes compared with stationary conditions (Dunnett’s test: p < 0.05). The average THmax values for non-motion and motion were 0.387 and 0.263, respectively. Figure 4 Effect of motion amplitude on threshold values based on maximum standard uptake values (THmax), evaluated with air as the phantom background. (a) The variance of THmax with slow computed tomography (CT) scanning (SCS) was significantly smaller than that of THmax with helical CT scanning (HCS) for a 37-mm sphere with 10-, 20- and 30-mm amplitudes (F-test: p < 0.05). A significant difference was observed comparing the mean THmax of 30-, 20-, and 10-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). The average THmax value for non-motion was 0.362. (b) The variance of THmax with SCS was significantly smaller than that of THmax with HCS for a 28-mm sphere with 30-mm amplitude (F-test: p < 0.05). A significant difference was observed when comparing the mean THmax of 30-, 20-, and 10-mm motion amplitudes with stationary conditions (Dunnett’s test: p < 0.05). The average THmax Figure 5 Effect of motion amplitude on threshold values based on maximum standard uptake values (THmax), evaluated with water as the phantom background. Discussion In this study, we aimed to extract ITV-b from PET images using calculations from phantom studies. The results of the current study have demonstrated that tumour size, motion amplitude, tumour site (lung or head-and-neck, Kawakami et al. Radiation Oncology (2015) 10:1 Page 7 of 9 Figure 6 Threshold values (THmax) obtained at a range of motion amplitudes measured in a phantom with an air background. Regardless of concentration or size, THmax was dependent only on amplitude. Because the data fit well (R2 = 0.982) with an exponential function, it may be possible to use the function to approximate THmax from motion amplitude. Figure 6 Threshold values (THmax) obtained at a range of motion amplitudes measured in a phantom with an air background. Regardless of concentration or size, THmax was dependent only on amplitude. Because the data fit well (R2 = 0.982) with an exponential function, it may be possible to use the function to approximate THmax from motion amplitude. abdomen), and AC can have a significant impact on PET/CT imaging outcomes, including SUVmax and THmax. These four factors are individually discussed below. be expected to be more susceptible to errors arising from AC differences because of large density differences between sphere phantoms and air. Using air as the phan- tom background, regardless of RC or size, THmax was dependent only on amplitude. Therefore, in this context, it was possible to approximate THmax using the equation derived from the cubic equation fit to the data (Eq. 4). Using water as the background for the phantom, THmax was dependent on RC, size, and amplitude. When the average threshold of width in the value is selected, the error margin is included in the extraction capacity. How- ever, we believe that this will become the standard for determining ITV-b. In considering tumour size, we found no influence of PVE in the experiments with 28- and 37-mm spheres. In spheres with a diameter of 22 mm or less, reduced SUV estimates can result from PVE. Because the underesti- mation of SUV is exacerbated by respiratory movement, one should be careful when evaluating targets smaller than 28 mm by estimating the SUV. In cases with volumes larger than spheres of 37-mm diameter, the potential in- fluence of PVE remains to be determined. When evaluating the effect of target motion amplitude, we observed no SUVr underestimation for 10-mm re- spiratory motion. Discussion In the case of amplitudes of 20 mm or more, SUVr decreased in an amplitude-dependent manner. Moreover, differences in background affected calculated volumes. When the background was air, a significant amplitude-dependent difference in THmax was observed. When the background was water, there was no significant effect of amplitude on THmax. With amplitudes greater than 10 mm, the THmax was re- duced compared with the stationary condition. THmax was found to be more strongly affected by motion than SUVmax. Because SUVmax is the value of a certain point in an ideal phantom volume, the impact of movement on it is less severe than the impact that movement has on THmax. When we consider differences between ACs, the vari- ance of SUVr and THmax with SCS was not significantly different than that of SUVr and THmax with HCS with water as the phantom background. However, when the phantom background was air, the variance of SUVr and THmax with SCS was significantly smaller than that of SUVr and THmax with HCS. In evaluating the response to therapy, or for planning radiotherapy treatment using PET images, SUV and extracted volumes must not change between scans. Our data suggest that the SCS method provides superior AC in comparison to HCS. Past phantom experiments indicate variation in the value of THmax, although they have been reported 40-50% in the non-motion and 25-35% in the moving phantoms [6-9]. THmax for tumours in different areas has also been reported. They are 15-50% for lung tumours [22,23], 40-50% for head-and-neck tumours [24], and 40% pelvic tumours [25]. In summary, when ITV-b is extracted, the approximate expression based on motion amplitude can be used in the lung field. The approximate expression can be used by understanding the amount of the breath When considering the differences between simulated tumour sites (lung or head-and-neck, abdomen), head- and-neck and abdominal lesions were less susceptible to error caused by AC because the attenuation factors were uniform throughout water. In contrast, lung lesions may Page 8 of 9 Page 8 of 9 Kawakami et al. Radiation Oncology (2015) 10:1 Received: 15 May 2014 Accepted: 18 December 2014 Received: 15 May 2014 Accepted: 18 December 2014 Acknowledgements g This study was partly supported by the Foundation for Promotion of Hokkoku Cancer Research. 18. Park SJ, Ionascu D, Killoran J, Mamede M, Gerbaudo VH, Chin L, et al. Evaluation of the combined effects of target size, respiratory motion and background activity on 3D and 4D PET/CT images. Phys Med Biol. 2008;53:3661–79. This study was partly supported by the Foundation for Promotion of Hokkoku Cancer Research. References R errors in tumor quantification and delineation in CT atten PET images: effects of tumor size, tumor location, and res simulation study using the 4D XCAT phantom. Mol Imag 2013;15:655–65. 15. Nehmeh SA, Erdi YE. Respiratory motion in positron emiss computed tomography: a review. Semin Nucl Med. 2008;3 16. Kasuya T, Tateishi U, Suzuki K, Daisaki H, Nishiyama Y, Hata respiratory-gated PET/CT for pancreatic tumors: a prelimin Radiol. 2013;82:69–74. 17. Callahan J, Kron T, Schneider-Kolsky M, Hicks RJ. The clinic management of lesion motion due to respiration during P Cancer Imaging. 2011;11:224–36. 18. Park SJ, Ionascu D, Killoran J, Mamede M, Gerbaudo VH, C Evaluation of the combined effects of target size, respirato background activity on 3D and 4D PET/CT images. Phys M 2008;53:3661–79. 19. Didierlaurent D, Ribes S, Batatia H, Jaudet C, Dierickx LO, Z The retrospective binning method improves the consisten binning in respiratory-gated PET/CT. Phys Med Biol. 2012;5 20. Lamare F, Ledesma Carbayo MJ, Cresson T, Kontaxakis G, S CC, et al. List-mode-based reconstruction for respiratory m in PET using non-rigid body transformations. Phys Med Bi 2007;52:5187–204. 1. Yu H, Caldwell C, Mah K, Poon I, Balogh J, MacKenzie R. Automated radiation targeting in head-and-neck cancer using region-based texture analysis of PET and CT images. Int J Radiat Oncol Biol Phys. 2009;75:618–25. 2. Le Maitre A, Hatt M, Pradier O, Cheze-le Rest C, Visvikis D. Impact of the accuracy of automatic tumour functional volume delineation on radiotherapy treatment planning. Phys Med Biol. 2012;57:5381–97. 3. MacManus M, Nestle U, Rosenzweig KE, Carrio I, Messa C, Belohlavek O, et al. Use of PET and PET/CT for radiation therapy planning: IAEA expert report 2006–2007. Radiother Oncol. 2009;91:85–94. 4. Jingu K, Takeda K, Metoki T, Ogawa Y, Ariga H, Yamada S, et al. The difference between gross tumor volume and biological target volume in postoperative local recurrent rectal cancer; a preliminary study of PET/CT radiation therapy planning. J Jpn Soc Ther Radiol Oncol. 2008;20:127–31. 4. Jingu K, Takeda K, Metoki T, Ogawa Y, Ariga H, Yamada S, et al. The difference between gross tumor volume and biological target volume in postoperative local recurrent rectal cancer; a preliminary study of PET/CT radiation therapy planning. J Jpn Soc Ther Radiol Oncol. 2008;20:127–31. Competing interests h h d l h The authors declare that they have no competing interests. Abbreviations PET P i PET: Positron emission tomography; CT: Computed tomography; GTV: Gross tumour volume; PVE: Partial volume effect; SUV: Standardized uptake value; BTV: Biological target volume; ITV: Internal target volume; AC: Attenuation correction; RC: Radioisotope concentration; Rco: Recovery coefficient; SUVmax: Maximum standardized uptake value; SUVr: Ratios of SUVmax; THmax: Threshold values based on SUVmax; NEMA: National Electrical Manufacturers Association; IEC: International Electrotechnical Commission; 18F-FDG: 2-[fluorine-18] fluoro-2-deoxy-D-glucose; SCS: Slow computed tomography scanning; HCS: Helical computed tomography scanning; ANOVA: Analysis of variance. 11. Toya R, Murakami R, Tashiro K, Yoshida M, Sakamoto F, Kawanaka K, et al. FDG-PET/CT-based gross tumor volume contouring for radiation therapy planning: an experimental phantom study. J Radiat Res. 2012;53:338–41. 12. Niyazi M, Landrock S, Elsner A, Manapov F, Hacker M, Belka C, et al. Automated biological target volume delineation for radiotherapy treatment planning using FDG-PET/CT. Radiat Oncol. 2013;8:180. 13. Liu C, Pierce 2nd LA, Alessio AM, Kinahan PE. The impact of respiratory motion on tumor quantification and delineation in static PET/CT imaging. Phys Med Biol. 2009;54:7345–62. 14. Geramifar P, Zafarghandi MS, Ghafarian P, Rahmim A, Ay MR. Respiratory-induced errors in tumor quantification and delineation in CT attenuation-corrected PET images: effects of tumor size, tumor location, and respiratory trace: a simulation study using the 4D XCAT phantom. Mol Imaging Biol. 2013;15:655–65. Received: 15 May 2014 Accepted: 18 December 2014 movement by the fluoroscope image. Because THmax has a width with the same amplitude in the head-and-neck and the abdomen, it is difficult to compute a concrete threshold value. However, we believe that extracting BTV using the mean value of THmax may aid in treatment plan- ning. As for the value of THmax, 0.40 is the standard value in head-and-neck and lower abdominal areas without re- spiratory movement. Moreover, 0.25 is the standard value in upper abdominal areas with respiratory movement. However, these threshold values resemble those reported previously and become the first choice, it will be necessary to check the extracted volume. Authors’ contributions d f d h 15. Nehmeh SA, Erdi YE. Respiratory motion in positron emission tomography/ computed tomography: a review. Semin Nucl Med. 2008;38:167–76. WK drafted the manuscript and performed all mathematical analyses. SY participated in the sequence alignment. KY, KN, and KK participated in the design of the study and performed the statistical analysis. AT conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript. 16. Kasuya T, Tateishi U, Suzuki K, Daisaki H, Nishiyama Y, Hata M, et al. Role of respiratory-gated PET/CT for pancreatic tumors: a preliminary result. Eur J Radiol. 2013;82:69–74. 17. Callahan J, Kron T, Schneider-Kolsky M, Hicks RJ. The clinical significance and management of lesion motion due to respiration during PET/CT scanning. Cancer Imaging. 2011;11:224–36. Conclusions 5. Devic S, Tomic N, Faria S, Dean G, Lisbona R, Parker W, et al. Impact of 18FDG-PET/CT on biological target volume (BTV) definition for treatment planning for non-small cell lung cancer patients. Nucl Instrum Methods Phys Res Section A. 2007;571:89–92. In this study, we demonstrate concrete conditions to set ITV-b. There was no PVE with tumour sizes ≥28 mm, and the variation between SUVmax and THmax may be reduced by using SCS for AC. There was no decrease in respiratory movement from approximately 10 mm for SUVmax. As for the value of THmax, 0.40 became the standard value in areas without respiratory movement in the head-and-neck and the lower abdominal area, and 0.25 became the standard value in upper abdominal areas with respiratory movement. The approximate ex- pression based on the motion amplitude may be adjusted in the lung area. 6. Erdi YE, Mawlawi O, Larson SM, Imbriaco M, Yeung H, Finn R, et al. Segmentation of lung lesion volume by adaptive positron emission tomography image thresholding. Cancer. 1997;80:2505–9. 7. Nagel CC, Bosmans G, Dekker AL, Ollers MC, De Ruysscher DK, Lambin P, et al. Phased attenuation correction in respiration correlated computed tomography/positron emitted tomography. Med Phys. 2006;33:1840–7. 7. Nagel CC, Bosmans G, Dekker AL, Ollers MC, De Ruysscher DK, Lambin P, et al. Phased attenuation correction in respiration correlated computed tomography/positron emitted tomography. Med Phys. 2006;33:1840–7. 8. Okubo M, Nishimura Y, Nakamatsu K, Okumura M, Shibata T, Kanamori S, et al. Static and moving phantom studies for radiation treatment planning in a positron emission tomography and computed tomography (PET/CT) system. Ann Nucl Med. 2008;22:579–86. 8. Okubo M, Nishimura Y, Nakamatsu K, Okumura M, Shibata T, Kanamori S, et al. Static and moving phantom studies for radiation treatment planning in a positron emission tomography and computed tomography (PET/CT) system. Ann Nucl Med. 2008;22:579–86. 9. Yaremko B, Riauka T, Robinson D, Murray B, Alexander A, McEwan A, et al. Thresholding in PET images of static and moving targets. Phys Med Biol. 2005;50:5969–82. 9. Yaremko B, Riauka T, Robinson D, Murray B, Alexander A, McEwan A, et al. Thresholding in PET images of static and moving targets. Phys Med Biol. 2005;50:5969–82. 10. Sakaguchi Y, Mitsumoto T, Zhang T, Mitsumoto K, Tachiya Y, Ohya N, et al. 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Devic S, Tomic N, Faria S, Dean G, Lisbona R, Parker W, et 18FDG-PET/CT on biological target volume (BTV) definition planning for non-small cell lung cancer patients. Nucl Inst Phys Res Section A. 2007;571:89–92. 6. Erdi YE, Mawlawi O, Larson SM, Imbriaco M, Yeung H, Finn Segmentation of lung lesion volume by adaptive positron tomography image thresholding. Cancer. 1997;80:2505–9. 7. Nagel CC, Bosmans G, Dekker AL, Ollers MC, De Ruysscher et al. Phased attenuation correction in respiration correlat tomography/positron emitted tomography. Med Phys. 200 8. Okubo M, Nishimura Y, Nakamatsu K, Okumura M, Shibata et al. Static and moving phantom studies for radiation tre in a positron emission tomography and computed tomog system. Ann Nucl Med. 2008;22:579–86. 9. Yaremko B, Riauka T, Robinson D, Murray B, Alexander A, M Thresholding in PET images of static and moving targets. 2005;50:5969–82. 10. 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Author details 1 f 1Department of Radiological Technology, Public Central Hospital of Matto Ishikawa, 3-8, Kuramitsu, Hakusan City, Ishikawa Pref 924-8588, Japan. 2Department of Quantum Medical Technology, Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan. 3PET Imaging Center, Public Central Hospital of Matto Ishikawa, Ishikawa, Japan. 4Department of Nuclear Medicine, Kanazawa University Hospital, Ishikawa, Japan. 19. Didierlaurent D, Ribes S, Batatia H, Jaudet C, Dierickx LO, Zerdoud S, et al. The retrospective binning method improves the consistency of phase binning in respiratory-gated PET/CT. Phys Med Biol. 2012;57:7829–41. 20. Lamare F, Ledesma Carbayo MJ, Cresson T, Kontaxakis G, Santos A, Le Rest CC, et al. List-mode-based reconstruction for respiratory motion correction in PET using non-rigid body transformations. Phys Med Biol. 2007;52:5187–204. Page 9 of 9 Page 9 of 9 Kawakami et al. Radiation Oncology (2015) 10:1 21. 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